Note: Descriptions are shown in the official language in which they were submitted.
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TRANSGENIC ANIMA)L.S
FIELD OF THE INVENTION
The present invention relates to improved methods for the generation of
transgenic
non-human animals. In particular, the present invention relates to the
introduction of
retrovirai particles into the perivitelline space of gametes, zygotes and
early stage embryos to
allow the insertion of genetic material into the genome of the recipient
gamete or embryo.
BACKGROUND
The ability to alter the genetic make-up of animals, such as domesticated
mammals
such as cows, pigs and sheep, allows a number of commercial applications.
These
applications include the production of animals which express large quantities
of exogenous
proteins in an easily harvested form (eg., expression into the milk), the
production of animals
which are resistant to infection by specific microorganisms and the production
of animals
having enhanced growth rates or reproductive performance. Animals which
contain
exogenous DNA sequences in their genome are referred to as transgenic animals.
The most widely used method for the production of transgenie animals is the
microinjection of DNA into the pronuclei of fertilized embryos. This method is
efficient for
the production of traasgenic mice but is.much less efficient for the
production of transgenic
animals using large mammals such as cows and sheep. For example, it has been
reported that
1,000 to 2,000 bovine embryos at the pronuclear stage must be microinjected to
produce a
single transgenic cow at an estimated cost of more than $500,000 (Wall et al.,
J. Cell.
Biochem. 49:113 [1992]). Furthermore, microinjection of pronuclei is more
difficult when
embryos from domestic livestock (e.g., cattle, sheep, pigs) is employed as the
pronuclei are
often obscured by yolk material. While techniques for the visualization of the
pronuclei are
known (i.e., centrifugation of the embryo to sediment the yolk), the injection
of pronuclei is
an invasive technique which requires a high degree of operator skill.
Alternative methods for the production include the infection of embryos with
retroviruses or with retroviral vectors. Infection of both pre- and post-
implantation mouse
embryos with either wild-type or recombinant retroviruses has been reported
(Janenich, Proc.
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Natl. Aced. Sci. USA 73:1260 [1976]; Janenich et al., Cell 24:519 [1981];
Stuhlmann et al.,
Proc. Natl. Aced. Sci. USA 81:7151 [1984]; Jahner et al., Proc. Natl. Aced
Sci. USA 82:6927
[1985]; Van der Putten et al., Proc. Natl. Aced Sci. USA 82:6148-6152 [1985];
Stewart et a1,
EMBO J. 6:383-388 [1987]). The resulting transgenic animals are typically
mosaic for the
transgene since incorporation occurs only in a subset of cells which form the
transgenic
animal. The consequences of mosaic incorporation of retroviral sequences (i.
e., the transgene)
include lack of transmission of the transgene to progeny due to failure of the
retrovirus to
integrate into the germ line, difficulty in detecting the presence of viral
sequences in the
founder mice in those cases where the infected cell contributes to only a
small part of the
fetus and difficulty in assessing the effect of the genes carried on the
retrovirus.
In addition to the production of mosaic founder animals, infection of embryos
with
retrovirus (which is typically performed using embryos at the 8 cell stage or
later) often
results in the production of founder animals containing multiple copies of the
retroviral
provirus at different positions in the genome which generally will segregate
in the offspring.
Infection of early mouse embryos.by co-culturing early embryos with cells
producing
retroviruses requires enzymatic treatment to remove the zone peUucida (Hogan
et al:, In
Manipulating the Mouse Embryro: A Laboratory Mam~al, 2nd P.d., Cold Spring
Harbor
Laboratory Press, Cold Spring Harbor, NY, [1994], pp. 2~1 252). In contrast to
mouse
embryos, bovine embryos dissociate when removed from the zone pellucida.
Therefore,
infection protocols which remove the zone pellucida cannot be employed for the
production of
transgenic cattle or other animals whose embryos dissociate or suffer a
significant decrease in
viability upon removal of the zone pellucida (e.g., ovine embryos).
An alternative means for infecting embryos with retroviruses is the injection
of virus
or virus-producing cells into the blastocoele of mouse embryos (Jahner, D. et
al., Nature
298:623 [1982]). As is the case for infection of eight cell stage embryos,
most of the
founders produced by injection into the blastocoele will be mosaic. The
introduction of
transgenes into the germline of mice has been reported using intrauterine
retroviral infection
of the midgestation mouse embryo (Jahner et al., supra [1982]). This technique
suffers from
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a low efficiency of generation of transgenic animals and in addition produces
animals which
are mosaic for the transgene.
Infection of bovine and ovine embryos with retroviruses or retroviral vectors
to create
transgenic animals has been reported. These protocols involve the micro-
injection of
retroviral particles or growth arrested (i. e-, mitomycin C-treated) cells
which shed retroviral
particles into the perivitelline space of fertilized eggs or early embryos
(PCT International
Application WO 90/08832 [1990]; and Haskell and Bowers, Mol. Reprod. Dev.,
40:386
[1995]). PCT International Application WO 90/08832 describes the injection of
wild-type
feline leukemia virus B into the perivitelline space of sheep embryos at the 2
to 8 cell stage.
Fetuses derived from injected embryos were shown to contain multiple sites of
integration.
The efficiency of producing transgenic sheep was low (efficiency is defined as
the number of
iransgcnics produced compared to the number of embryos manipulated); only 4.2%
of the
injected embryos were found to be transgenia
Haskell and Bowers (supra) describe the micro-injection of mitomycin C-treated
cells
producing retrovirus into the perivitelline space of 1 to 4 cell bovine
embryos. The use of
virus-producing cells precludes the, delivery of a controlled amount of viral
particles per
embryo. The resulting fetuses contained betvvecn 2 and 12 provinces and were
shown to be
mosaic for proviral integration~sites, the'presence of pmvin~s, or both. The
efficiency of
producing transgenic bovine embryos was low; only 7% of the injected embryos
were found
to be transgenic.
The art needs improved methods for the production of transgenic animals,
particularly
for the production of transgenics using large domestic livestock animals. The
ideal method
would be simple to perform and less invasive than pronuclear injection,
efficient, would
produce mosaic transgenic founder animals at a low frequency and would result
in the
integration of a defined number of copies of the introduced sequences into the
genome of the
transgenic animal.
SUMMARY OF THE INVENTION
The present invention provides improved methods and compositions for the
production
of transgenic non-human animals. In one embodiment, the present invention
provides a
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composition comprising a non-human unfertilized oocyte comprising a
heterologous
oligonucleotide (i.e., a heterologous polynucleotide) integrated into the
genome of the oocyte.
In a preferred embodiment the unfertilized oocyte is a pre-maturation oocyte.
In another
preferred embodiment the unfertilized oocyte is a pre-fertilization oocyce.
The present
invention is not limited by the nature of the heterologous oligonucleotide
contained within the
genome of the oocyte. In a preferred embodiment, the heterologous
oligonucleotide is the
proviral form of a retroviral vector.
The invention is not limited by the nature of the retroviral vector employed.
Retroviral vectors containing a variety of genes may be employed. For example,
the
IO retroviral vector may contain sequences encoding proteins which modify
growth rate, size
and/or carcass composition (e.g., bovine growth hormone or other growth
hormones) or
foreign proteins of commercial value that are expressed in, and harvested
from, a particular
tissue component (e.g., blood or milk). 'The retroviral vector may contain
genes that confer
resistance to viruses or other microorganisms, including DNA sequences that
are
15 transcribed into RNA sequences that catalYac~ly cleave specific RNAs (i.
e., ribozymes) such
as viral RNAs and DNA sequences that are transcribed into anti-sense RNA of an
essential
gene of a pathogenic microorganism. 'The, above, protein-encoding genes and
DNA sequences
are examples of "genes of inte~st."
The compositions of the present invention are not limited by the nature of the
non-
20 human animal employed to provide oocytes. In a preferred embodiment, the
non-htmnan
animal is a mammal (e.g., cows, pigs, sheep, goats, rabbits, rats, mice,
etc.). In a particularly
preferred embodiment, the non-human animal is a cow.
The present invention further provides a method for introducing a heterologous
polynucleotide into the genome of a non-human unfertilized oocyte, comprising:
a)
25 providing: i) a non-human unfertilized egg comprising an oocyte having a
plasma membrane
and a zona pellucida, the plasma membrane and the zona pellucida defining a
perivitelline
space; ii) an aqueous solution comprising a heterologous polynucleotide; and
b) introducing
the solution comprising the heterologous polynucleotide into the perivitelline
space under
conditions which permit the introduction of the heterologous polynucleotide
into the genome
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of the oocyte. The method of the present invention is not limited by the
nature of the
heterologous polynucleotide employed. In a preferred embodiment, the
heterologous
polynucleotide encodes a protein of interest. In a particularly preferred
embodiment, the
heterologous polynucleotide is contained within genome of a recombinant
retrovirus.
The method of the present invention may be practiced using unfertilized eggs
comprising a pre-maturation oocyte. Alternatively, the method of the present
invention may
employ pre-fertilization oocytes as the unfertilized egg.
When a recombinant retrovirus is employed infectious retroviral particles
comprising
the heterologous polynucleotide are preferentially employed. The method of the
present
IO invention is not limited by the nature of the infectious retrovirus
employed to deliver nucleic
acid sequences to an oocyte. Any retr°~ which is capable of infecting
the species of
oocyte to be injected may be employed. In a preferred embodiment, the
infectious retrovirus
comprises a heterologous membrane-associated protein. In a preferred
embodiment, the
hetemlogous .membrano-associated protein is ~a CI glycoprotein selected from a
virus within the
15 ~ family Rhabdoviridae. In another preferred embodiment, the heterologous
membrano-
associated protein is selected fmm the group consisting of the C3.glycoprotein
of vesicular
stomatitis virus, Pity ~ p~ ~' S, virennia of carp. vines and Mokola virus.,
In a particularly preferred embodiment, the heterologous membrane-associated
protein is the' Cl
glycoprotein of vesicular stomatitis virus-
20 The method of the present invention is not limited by the nature of the non-
human
animal employed to provide oocytes. In a preferred embodiment, the non-human
animal is a
mammal (e.g., cows, pigs, sheep, goats, rabbits, rats, mice, etc.). In a
particularly preferred
embodiment, the non-human animal is a cow.
The present invention further provides a method for the production of a
transgenic
25 non-human animal comprising: a) providing: e) an unfertilized egg.
comprising an oocyte
having a plasma membrane and a zona pellucida, the plasma membrane and the
zona
pellucida defining a perivitelline space; ii) an aqueous solution containing
infectious
retrovirus; b) introducing the solution containing infectious retrovirus into
the perivitelline
space under conditions which permit the infection of the oocyte; and c)
contacting the infected
30 oocyte with sperm under conditions which permit the fertilization of the
infected oocyte to
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produce an embryo. In a preferred embodiment, the method of the present
invention further
comprises, following the fertilization of the infected oocyte, the step of
transferring the
embryo into a hormonally synchronized non-human recipient animal (i.e., a
female animal
hormonally synchronized to stimulate early pregnancy). In another preferred
embodiment, the
method comprises the step of allowing the transferred embryo to develop to
term. In still
another referred embodiment, at least one transgenic offspring is identified
from the offspring
allowed to develop to term.
The method of the present invention may be practiced using unfertilized eggs
comprising a pre-maturation oocyte. Alteratively, the method of the present
invention may
employ pre-fertilization oocytes as the unfertilized egg.
When pre-maturation oocytes are employed in the method of the present
invention, the
metbod may further comprise, following the introduction of the solution
containing infectious
retrovirus into the pre-maturation oocyte, the further step of culturing the
infected pre-
maturatioa oocyte under conditions which permit the matiu'ation of the pre-
maturation oocyte.
The art. is well aware of culture conditions which permit the In vitro
maturation of pre-
maturation oocytes .from a variety of mammalian species.
'The method Qf the present invention ~ not limitod liy the nature of the
infectious
rebrovirus employed to deliver nucleic acid sequences to ~an oocyte. Any
retrovirus ~wbich is
capable of infecting the species of oocyte to be injected may be employed. In
a preferred
embodiment, the infectious retrovirus comprises a heterologous membrane-
associated protein.
In a preferred embodiment, the heterologous membrane-associated protein is a G
~glycoprotein
selected from a virus within the family Rhabdoviridae. In another preferred
embodiment, the
heterologous membrane-associated protein is selected from the group consisting
of the G
glycoprotein of vesicular stomatitis virus, Piry virus, Chandipura virus,
Spring viremia of carp
virus and Mokola virus. In a particularly preferred embodiment, the
heterologous membrane-
associated protein is the G glycoprotein of vesicular stomatitis virus.
The method of the present invention is not limited by the nature of the non-
human
animal employed to provide oocytes. In a preferred embodiment, the non-human
animal is a
mammal (e.g., cows, pigs, sheep, goats, rabbits, rats, mice, etc.). In a
particularly preferred
embodiment, the non-human animal is a bovine.
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PCT/US99/26848
The present invention further provides compositions comprising a stably
maintained
recombinant mammalian zygote, wherein the zygote comprises a polynucleotide
containing the
proviral form of a retroviral vector integrated into the genome of the zygote.
In particularly
preferred embodiments, the mammalian zygote is a bovine zygote, while in other
preferred
embodiments, the zygote is any mammalian zygote. Indeed, it is not intended
that the present
invention be limited to any particular animal species. In still other
embodiments, the proviral
form of the retroviral vector encodes a protein of interest. In yet further
preferred
embodiments, the recombinant retroviral vector comprises Moloney murine
leukemia virus
LTR.. However, it is not intended that the present invention be Limited to any
particular
retroviral LTR. Indeed, it is contemplated that other retroviral LTRs,
including, but not
Limited, to mouse mt~or'nrus LTR, "~11 find use in the present invention.
The present invention also provides methods for introducing a polynucleodde
~n~ned within the genome of a recombinant retrovirus into the genome of a
mammalian
zygote, comprising: a) providing: i) a mammalian Zygote having a p1membrane
and a .
Zona~ pellucida, wherein the plasma membrane and zona pellucida define a
periviteLLine space;
i17 an aqueous solution comprising a polynucleotide~ contained within the
genome of a
recombinant retrovirns; and.b) introducing the solution comprising the
poLynucleotide
contained within the genome of a recombinant retrovirus into the pcrivitelline
space, under
conditions which permit the introduction of the poLynucleotide contained
within the genome
of the recombinant retrovirus into the genome of the zygote, such that the
polynucleotide is
stabl maintained. In particularly preferred embodiments of the method, the
efficiency of the
y
introduction of the polynucleotide into the genome of the zygote is at least
twenty percent. In
still other embodiments, the efficiency ranges from approximately twenty
percent to one
heed percent. In yet other preferred embodiments, the polynucleotide contained
within the
genome of the recombinant retrovirus encodes a protein of interest. In further
embodiments,
the method further comprises the step of transferring the zygote into a
mammalian female
recipient that is hormonally synchronized to simulate early pregnancy, thereby
giving a
transferred embryo. In other particularly preferred embodiments, the method
further
comprises the step of allowing the transferred embryo to develop to term. In
further
embodiments,. the method comprises the additional step of identifying at least
one transgenic
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offspring. In other particularly preferred embodiments, the present invention
provides
transgenic animals produced according to the above methods. In particularly
preferred
embodiments, the mammalian zygote is a bovine zygote, while in other preferred
embodiments, the zygote is any other mammalian zygote. Indeed, is not intended
that the
present invention be limited to any particular animal species.
In still other embodiments of the above methods and transgenic animals, the
recombinant retrovirus comprises Moloney marine leukemia virus long terminal
repeat.
However, it is not intended that the present invention be limited to any
particular retroviral
LTR. Indeed, it is contemplated that other retroviral LTRs, including, but not
limited to
mouse mammary ~°r ~ LTA ~18nd use in the present invention. In
particularly
preferred embodiments, the protein of interest is expressed by the transgenic
offspring. In
some embodiments, the protein of interest is expressed in at least one body
fluid of the
transgenic offspring. In some parttcularlY Preferred embodiments, the
expression of the
protein of interest is preferentially mammary-specific ~Pression.
. In further embodied of the above methods and transgenic animals, the
recombinant
retmvirus comprises a heterologous, membrane-associated protein In some
embodiments, the
hetcrologous membrano-associated protein is =a G glycoprotcin selected from a
virus within the
family Rhabdoviridae. In other embodiments, the G glYcoProtem is selected from
the group
comprising the G glycoprotein of vesicular stomatitis virus, Pity virus,
C1'umdipura virus,
Spring viremia of carp virus, Rabies virus, and Mokola virus.
The present invention also provides methods for producing transgenic non-human
animals, wherein the genome of the transgenic non-human animal comprises a
polynucleotide
encoding a recombinant retrovirus and at least one protein of interest,
comprising the steps of:
a) providing: i) a non-human mammalian zygote having a plasma membrane and a
zona
pellucida, wherein the plasma membrane and the zona pellucida define a
perivitelline space;
ii) an aqueous solution comprising a polynucleotide contained within the
genome of a
recombinant retrovirus; b) introducing the solution comprising the
polynucleotide contained
within the genome of a recombinant retrovirus into the perivitelline space
under conditions
which permit the introduction of the polynucleotide contained within the
genome of a
recombinant retrovirus into the genome of the zygote, such that the
polynucleotide is stably
_g_
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maintained in a recombinant zygote; c) transferring the recombinant zygote
into a non-human
female mammalian recipient that is hormonally synchronized to simulate early
pregnancy,
thereby giving a transferred embryo; d) allowing the transferred embryo to
develop to term to
produce a transgcnic animal. In some particularly embodiments, at least one
protein of
interest is expressed by the transgenic animal. In other preferred
embodiments, the
recombinant retrovirus comprises Moloney marine leukemia virus long terminal
repeat.
However, it is not intended that the present invention be limited to any
particular retroviral
LTR. Indeed, it is contemplated that other retroviral LTRs, including, but not
limited to
mop m~ary tumor virus LTR, will find use in the present invention.
' In still other embodiments of the above methods, the efficiency of the
introduction of
the polynucleotide is at least twenty percent. In still other embodiments, the
efficiency ranges
from approximately twenty percent to one hundred percent. In further
particularly preferred
embodiments, the expression of the polynucleotide is preferentially mammary-
specific
expression. In other embodiments, the methods comprise the further step of
mating the
transgenic animal to a non-transgenic animal under conditions such that
transgenic offspring
are produced. In particularly preferred embodiments, the transgenic offspring
express the
polynucleotide: -In other particularly preferred embodiments, the oxPr~on of
the
polynucleotide is mammary-specific exp~sion' .In yet other particularly
preferred
embodiments, the mammalian zYg°~ is a bovine zygote, while in other
preferred
embodiments, the zygote is any other mammalian zygote. Indeed, is not intended
that the
present invention be limited to any particular animal species.
The present invention also provides methods for expressing a protein of
interest,
wherein the protein of interest is encoded by a polynucleotide contained
within the genome of
a recombinant retrovirus, comprising the steps of: a) providing: i) a non-
human mammalian
zygote having a plasma membrane and a zona pellucida, wherein the plasma
membrane and
the zona pellucida define a perivitelline space; ii) an aqueous solution
comprising a
polynucleotide encoding a protein of interest contained within the genome of a
recombinant
retrovirus; and b) introducing the solution comprising the polynucleotide
encoding a protein of
interest contained within the genome of a recori~binant retrovirus into the
perivitelline space,
under conditions which permit the introduction of the polynucleotide contained
within the
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genome of a recombinant retrovirus into the genome of the zygote, such that
the
polynucleotide is stably maintained; and c) allowing the zygote to develop
into viable non-
human animal, under conditions such that the protein of interest is expressed
by the non-
human animal.
In some preferred embodiments of the above methods, the recombinant retrovirus
comprises Moloney marine leukemia virus long terminal repeat. However, it is
not intended
that the present invention be limited to any particular retroviral LTR Indeed,
it is
contemplated that other retroviral LTRs, including, but not limited, to mouse
mammary tumor
virus LTR, will find use in the present invention. In yet other preferred
embodiments,
introduction of the polynucleotide into the genome of the zygote is at least
twenty percent. In
still other embodiments, the effciency ranges from approximately twenty
percent to one
hundred percent. In yet other embodiments, the polynucleotide contained within
the genome
of a recombinant retrovirus encodes a viral protein. In other embodiments,
viral protein is
hepatitis B surface antigen In still other embodiments, the present invention
provides protein
produced according to the.above methods. In yet other embodiments, the method
further
comprises the step of harvesting the expressed protein of interest. In further
embodiments,
the expressed protein is ~ ~ the body,fluids of the noirhtunan animal- Ia
pearly
preferred embodiments, body fluids are selected from the group consisting of
blood, mills,
semen, and urine. In particularly Preferred embodiments, the mammalian zygote
is a bovine
,20 zygote, while in other preferred embodiments, the zygote is any mammalian
zygote. Indeed,
it is not intended that the present invention be limited to any particular
animal species.
The present invention also provides methods for expressing a protein of
interest
wherein the protein of interest is encoded by a polynucleotide contained
within the genome of
a recombinant retrovirus, and the polynucleotide is integrated into the genome
of a
mammalian unfertilized oocyte, comprising the steps of: a) providing: i) an
unfertilized
mammalian egg comprising an oocyte having a plasma membrane and a zona
pellucida,
wherein the plasma membrane and the zona pellucida define a perivitelline
space; ii) an
aqueous solution containing recombinant retrovirus, wherein the recombinant
retrovirus
comprises a polynucleotide encoding a protein of interest; b) introducing the
solution
containing recombinant retrovirus into the perivitelline space under
conditions which permit
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the infection of the oocyte to provide an infected oocyte; c) contacting the
infected oocyte
with sperm under conditions which permit the fertilization of the infected
oocyte to produce
an embryo; d) transferring the embryo into a hormonally synchronized mammalian
recipient
animal; e) allowing the embryo to develop into at least one viable transgenic
mammalian
animal, under conditions such that the protein of interest is expressed by the
transgenic
mammalian animal.
In some preferred embodiments, the unfertilized oocyte is a pre-maturation
oocyte. In
other embodiments, following the introduction of the solution containing
infectious retrovirus
into the pre-maturation oocyte, the method comprises the further step of
culturing the infected
pre-maturation oocyte under conditions which permit the maturation of the pre-
maturation
oocyte. In other preferred embodiments, the unfertilized oocyte is a pre-
fertilization oocyte.
In still other preferred embodiments, the method further comprises the step of
identifying at least one iransgenic offspring. In particularly preferred
embodiments, the
mammal .is a bovine. However, it is not intended that the present invention be
limited to any
particular animal species.
In further preferred embodiments, the recombinant retrovirus comprises Moloney
marine leukemiavirus long terminal . rep~i ~Iovirever, , it is not intendal,
that the present
invention be limited to any particular retroviral LTR- Indeed, it is
contemplated that other
retroviral LTRs, including, but not limited, to mouse mammary tumor virus LTR,
will find
use in the present invention. In yet other preferred embodiments, the
expression of the
protein of interest is preferentially mammary specific expression. In some
particularly
preferred embodiments of the method, the introduction of the polynucleotide
into the genome
of the infected oocyte, is greater than twenty percent. In still other
embodiments, the
efficiency ranges from approximately twenty percent to one hundred percent. In
some
preferred embodiments, the polynucleotide contained within the genome of a
recombinant
retrovirus encodes a viral protein. In some particularly preferred embodiments
the viral
protein is hepatitis B surface antigen. In alternative particularly preferred
embodiments, the
expressed protein is expressed in the body fluids of the mammalian animal. In
some
particularly preferred embodiments, the body fluids are selected from the
group consisting of
blood, milk, semen, and urine. In still other embodiments, the methods further
comprise the
CA 02351553 2006-06-07
7~E667-188
step of f) harvesting the expressed protein of interest.
Tree present invention also provides a protein of interest
e~cpressed using the above methods.
In yet other embodiments of the methods, the
recombinant retrovirus comprises a heterologous membrane-
a~~sociated protein. In some embodiments, the heterologous
membrane-associated protein is a G glycoprotein selected
from a virus within the family Rhabdoviridae. In yet other
embodiments, the G glycoprotein is selected from the group
comprising the G glycoprotein of vesicular stomatitis virus,
Piry virus, Chandipura virus, Spring viremia of carp virus
and Mokola virus.
In another embodiment, the present invention
pz-ovides a method of producing a heterologous protein in the
mammary gland of a bovine, comprising the steps of: a)
pz-oviding a transgenic bovine, wherein said transgenic
bovine has at least one mammary gland and wherein the germ
cells and somatic cells of said transgenic bovine contain at
least one Moloney Murine Leukemia Virus 5' long terminal
repeat and at least one polynucleotide encoding a
heterologous protein, said Moloney Murine Leukemia Virus
long terminal repeat and said polynucleotide encoding a
heterologous protein being operably linked; b) expressing
said protein in said mammary gland of said transgenic
bovine, under conditions such that said protein is
px-eferentially expressed in said mammary gland of said
tx-ansgenic bovine as compared to other tissues; and c)
px-oducing milk from said bovine, wherein said milk comprises
s~iid heterologous protein.
In another embodiment, the present invention
provides a method for producing a transgenic bovine, wherein
said bovine preferentially expresses a heterologous protein
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in its milk, and further wherein the genome of said
transgenic bovine comprises a recombinant retrovirus
comprising a polynucleotide encoding at least one
hEaerologous protein, comprising the steps of: a) providing:
i) a bovine zygote having a plasma membrane and a zona
pe~llucida, said plasma membrane and said zona pellucida
defining a perivitelline space; ii) an aqueous solution
comprising a polynucleotide encoding a heterologous protein
contained within the genome of a recombinant retrovirus,
wherein said retrovirus comprises the Moloney Murine
Leukemia Virus 5' long terminal repeat; b) introducing said
aqueous solution comprising said polynucleotide contained
within the genome of a recombinant retrovirus into said
perivitelline space of said bovine zygote, under conditions
such that said polynucleotide contained within the genome of
a recombinant retrovirus is introduced into the genome of
said zygote, and said polynucleotide is stably maintained in
a recombinant zygote; c) transferring said recombinant
zygote into a bovine female recipient hormonally
s~Tnchronized to simulate early pregnancy, thereby providing
a transferred embryo; d) allowing said transferred embryo to
develop to term to produce the transgenic bovine, wherein
tree mammary gland of said transgenic bovine is capable of
px-eferentially expressing said heterologous protein as
compared to other tissues; and e) producing milk from said
bovine, wherein said milk comprises said heterologous
protein .
In another embodiment, the present invention
provides a method for expressing a heterologous protein
wherein said heterologous protein is encoded by a
polynucleotide contained within the genome of a recombinant
re~trovirus, and said polynucleotide is integrated into the
genome of a bovine unfertilized oocyte, comprising the steps
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of: a) providing: i) an unfertilized bovine egg comprising
or.. oocyte having a plasma membrane and a zona pellucida,
said plasma membrane and said zona pellucida defining a
pe:rivitelline space; ii) an aqueous solution containing
recombinant retrovirus, wherein said recombinant retrovirus
comprises a polynucleotide encoding the heterologous protein
ar..d a Moloney Murine Leukemia Virus 5' long terminal repeat;
b) introducing said solution into said perivitelline space
under conditions such that said oocyte is infected with said
recombinant retrovirus to provide an infected oocyte; c)
contacting said infected oocyte with sperm under conditions
such that said infected oocyte is fertilized to produce an
erribryo; d) transferring said embryo into a hormonally-
synchronized bovine female recipient; e) allowing said
embryo to develop into at least one viable transgenic
be>vine, under conditions such that said protein is
preferentially expressed in the mammary gland of said
transgenic bovine as compared to other tissues; and f)
producing milk from said bovine, wherein said milk comprises
s~~id heterologous protein.
DESCRIPTION OF THE DRAWINGS
Figure 1 provides a schematic showing the
production of pre-maturation oocytes, pre-fertilization
oocytes and fertilized oocytes (zygotes).
Figure 2 shows an autoradiogram of a Southern blot
of: genomic DNA isolated from the skin (A) and blood (B) of
calves derived from pre-fertilization oocytes and zygotes
which where injected with pseudotyped LSRNL retrovirus.
Figure 2C shows a 1.6 kb fragment generated from the
digestion of calf DNA with HindIII.
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Figure 3 shows an ethidium bromide stained agarose
gE:l containing electrophoresed PCR products which were
amplified using neo gene primers (A) or HBsAg primers (B)
from the blood and skin of calves derived from pre-
y fertilization oocytes and zygotes injected with pseudotyped
L~>RNL retrovirus.
Figure 4 shows an ethidium bromide stained agarose
gel containing electrophoresed PCR products amplified using
the neo gene primers (A) or HBsAg primers (B) from skin
samples obtained from twin calves, who were offspring of a
transgenic bull.
Definitions
To facilitate understanding of the invention, a
number of terms are defined below.
As used herein, the term "egg", when used in
reference to a mammalian egg, means an oocyte surrounded by
a zona pellucida and a mass of cumulus cells (follicle
cells) with their associated proteoglycan. The term "egg"
i~> used in reference to eggs recovered from antral follicles
in an ovary (these eggs comprise pre-maturation oocytes) as
well as to eggs which have been released from an antral
follicle (a ruptured follicle).
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As used herein, the term "oocyte" refers to a female gamete cell and includes
primary
oocytes, secondary oocytes and mature, unfertilized ovum. An oocyte is a large
cell having a
large nucleus (i.e., the germinal vesicle) surrounded by ooplasm. The ooplasm
contains non-
nuclear cytoplasmic contents including mRNA, ribosomes, mitochondria, yolk
proteins, etc.
The membrane of the oocyte is referred to herein as the "plasma membrane."
The term "pre-maturation oocyte," as used herein refers to a female gamete
cell
following the oogonia stage (i.e., mitotic proliferation has occurred) that is
isolated from an
ovary (e.g., by aspiration) but which has not been exposed to maturation
medium in vitro.
Those of skill in the art know that the process of aspiration causes oocytes
to begin the
maturation process but that completion of the maturation process (i. e.,
formation of a
secondary oocyte which has extruded the first polar body) in vitro requires
the exposure of the
aspirated oocytes to maturation medium. Pre-maturation oocytes will generally
be arrested at
the first anaphase of meiosis...
The term "pre-fertilization oocyte" as used herein, refers to a female gamete
cell such
as a pre-maturation oocyte following exposure to maturation medium in vitro
but prior to
exposure to sperm (i.e., matured but not fertilized). The pre-fertilization
oocyte has
completed the first meiotic division, has released the first. polar body and
lacks a nuclear
membrane (the nuclear membrane will not reform until fertilization occurs;
after fertilization,
the second meiotic division occurs along with the extrusion of the second
polar body and the
formation of the male and female pronuclei). Pre-fertilization oocytes may
also be referred to
as matured oocytes at metaphase II of the second meiosis.
The terms "unfertilized egg" or "unfertilized oocyte" as used herein, refers
to any
female gamete cell which has not been fertilized and these terms encompass
both pre-
maturation and pre-fertilization oocytes.
The term "zygote" as used herein, refers to a fertilized oocyte that has not
yet
undergone the first cleavage step in the development of an embryo (i.e., it is
at the single-cell
stage).
The term "perivitelline space" refers to the space located between the zona
pellucida
and the plasma membrane of a mammalian egg or oocyte.
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As used herein, the term "traps" is used in reference to the positioning of
genes of
interest on the different strands of nucleic acid (e.g., alleles present on
the two chromosomes
of a chromosomal pair). The term "traps-acting" is used in reference to the
controlling effect
of a regulatory gene on a gene present on a different chromosome. In contrast
to promoters,
repressors are not limited in their binding to the DNA molecule that includes
their genetic
information. Therefore, repressors are sometimes referred to as traps-acting
control elements.
The term "traps-activation" as used herein refers to the activation of gene
sequences by
factors encoded by a regulatory gene which is not necessarily contiguous with
the gene
sequences which it binds to and activates.
As used herein, the term "cis" is used in reference to the presence of genes
on the
same chromosome. The term "cis-acting" is used in reference to the controlling
effect of a
regulatory gene on a gene present on the same chromosome. For example,
promoters, which
ailiect the synthesis of downstream mRNA are cis-acting control elements.
As used herein, the term "retrovirus" is used in reference to RNA viruses
which utilize
reverse transcriptase during their replication cycle (i.e., retroviruses are
incapable of
replication; rather, these are useful RNA sequences that are packaged with at
least two
enzymes that are required foi the insertion of the RNA. sequences into the
host cell genome).
The retroviral genomic RNA is converted into double=stranded DNA by reverse
transcriptase.
This double-stranded DNA form of the virus integrates into the chromosome of
the infected
cell and is referred to as a "provirus." In preferred embodiments of the
present invention, the
term "proviral" is used in reference to constructs that are similar to
"retrotransposons." These
are integrated genes that are bracketed by LTRs in the host cell genome.
However, in
preferred embodiments, the proviral constructs cannot replicate. In contrast,
in wild-type
viruses, the provirus serves as a template for RNA polymerase II and directs
the expression of
RNA molecules which encode the structural proteins and enzymes needed to
produce new
viral particles. At each end of the provirus are structures called "long
terminal repeats" or
"LTRs". The LTR contains numerous regulatory signals including transcriptional
control
elements, polyadenylation signals and sequences needed for replication and
integration of the
viral genome. The viral LTR is divided into three regions called U3, R and U5.
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The U3 region contains the enhancer and promoter elements. The US region
contains
the polyadenylation signals. The R (repeat) region separates the U3 and US
regions and
transcribed sequences of the R region appear at both the 5' and 3' ends of the
viral RNA
As used herein, the term "provirus" is used in reference to a virus that is
integrated
into a host cell chromosome (or genome), and is transmitted from one cell
generation to the
next, without causing lysis or destruction of the host cell. The term is also
used in reference
to a duplex DNA sequence present in an eulcaryotic chromosome, which
corresponds to the
genome of an RNA retrovirus.
As used herein, the term "endogenous virus" is used in reference to an
inactive virus
which is integrated into the chromosome of its host cell (often in multiple
copies), and can
thereby exhibit vertical transmission. Endogenous viruses can spontaneously
express
themselves and may result in malignancies.
As used herein, the terms "amphotrope" and "amphotropic" are used in reference
to
endogenous viruses that readily multiply in cells of the species in which they
were induced, as
well as cells of other species.
As used herein, the term "ecotrope" and "ecotropic" are uscd in reference ~to
endogenous viruses that multiply readily in cells of the species in-which they
were induced,
but cannot multiply in cells of other species.
As used herein, the term "xenotrope" and "xenotropic" are used in reference to
endogenous viruses that cannot infect cells of the species in which they were
induced, but can
infect and multiply in cells of other species.
The term "infectious retrovirus" refers to a retroviral particle which is
capable of
entering a cell (i.e., the particle contains a membrane-associated protein
such as an envelope
protein or a viral G glycoprotein which can bind to the host cell surface and
facilitate entry of
the viral particle into the cytoplasm of the host cell) and integrating the
retroviral genome (as
a double-stranded provirus) into the genome of the host cell.
As used herein, the term "retroviral vector" is used in reference to
retroviruses which
have been modified so as to serve as vectors for introduction of nucleic acid
into cells.
As used herein, the term "vector" is used in reference to nucleic acid
molecules that
transfer DNA segments) from one cell to another. Retroviral vectors transfer
RNA, which is
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then reverse transcribed into DNA. The term "vehicle" is sometimes used
interchangeably _.
with "vector."
The term "expression vector" as used heiein refers to a recombinant molecule
containing a desired coding sequence and appropriate nucleic acid sequences
necessary for the
expression of the operably linked coding sequence in a particular host
organism. Nucleic acid
sequences necessary for expression in prokaryotes. usually include a promoter,
an operator
(optional), and a ribosome binding site, often along with other sequences.
Eukaryotic cells
are known to utilize promoters, enhancers, and termination and polyadenylation
signals.
The terms "in operable combination," "in operable order," and "operably
linked;' as
used herein refer to the linkage of nucleic acid sequences in such a manner
that a nucleic acid
molecule capable of directing the transcription of a given gene and/or the
synthesis of a
desired protein molecule is produced. The term also refers to the linkage of
amino acid
sequences in such a manner so that a functional protein is produced. -
As used herein, the term "protein of interest" refers to any protein for which
expression is desired. For example, the term encompasses any recombinant forms
of a protein
that is desired. The term "gene of interest" refers to any gene that is
desired. In particularly
preferred embodiments, the gene .of.interest .encodes at least a portion of a
protein of interest.
The term "genetic cassette" as used herein refers to a fragment or segment of
nucleic
acid containing a particular grouping of genetic elements. The cassette can be
removed and
inserted into a vector or plasmid as a single unit.
As used herein, the term "long terminal repeat (LTR)" is used in reference to
domains
of base pairs located at the ends of retroviral DNA's. These LTRs may be
several hundred
base pairs in length. LTR's often provide functions fundamental to the
expression of most
eukaryotic genes (e.g., promotion, initiation and polyadenylation of
transcripts).
Retroviral vectors can be used to transfer genes efficiently into host cells
by exploiting
the viral infectious process. Foreign or heterologous genes cloned (i. e.,
inserted using
molecular biological techniques) into the retroviral genome can be delivered
efficiently to host
cells which are susceptible to infection by the retrovirus. Through well known
genetic
manipulations, the replicative capacity of the retroviral genome can be
destroyed. The
resulting replication-defective vectors can be used to introduce new genetic
material to a cell
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but they are unable to replicate. A helper virus or packaging cell line can be
used to permit
vector particle assembly and egress from the cell.
The terms "vector particle" or "retroviral particle" refer to viral-like
particles that are
capable of introducing nucleic acid into a cell through a viral-like entry
mechanism.
The host range of a retroviral vector (i. e., the range of cells that these
vectors can
infect) can be altered by including an envelope protein from another closely
related virus.
As used herein, the term "packaging signal" or "packaging sequence" refers to
non
coding sequences located within the retroviral genome which are required for
insertion of the
viral RNA into the viral eapsid or particle. Several retroviral vectors use
the minimal
packaging signal (also referred to as the psi sequence) needed for
encapsidation of the viral
genome. This minimal packaging signal encompasses bases 212 to 563 of the Mo-
MuLV
genome (Mann et al., Cell 33:153 [1983]).
As used herein, the term "extended packaging signal" or "extended packaging
sequence" refers to the use of sequences around the psi sequence with further
extension into
the gag gene. In Mo-MuLV, this extended. packaging sequence corresponds to the
region
encompassing base 1039 to base 1906 (Akagi et a~, Gene.106:235 [1991])., The
frequently
used M MuLV vector, pLNL6 (Bender et al.;. J. Virol., 61:1639 [1987]),
contains the entire 5'
region of the genome including an extended packaging signal from bases 206 to
1039 of the
Moloney marine sarcoma virus genome (numbering from Supplements and Appendices
in
RNA Tumor Viruses, 2nd Ed. [1985] pp. 986-988). The inclusion of these
additional
packaging sequences increases the efficiency of insertion of vector RNA into
viral particles.
As used herein, the term "packaging cell lines" is used in reference to cell
lines that express
viral structural proteins (e.g., gag, pol and envy, but do not contain a
packaging signal.
When retroviral vector DNA is transfected into the cells, it becomes
integrated into the
chromosomal DNA and is transcribed, thereby producing full-length retroviral
vector RNA
that has a psi~ sequence. Under these concunons, onry lne vccwmv~ew m yawagcu
uiav u...
viral capsid structures These complete, yet replication-defective, virus
particles can then be
used to deliver the retroviral vector to target cells with relatively high
efficiency.
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The term "transfection" as used herein refers to the introduction of foreign
DNA into
eukaryotic cells. Transfection may be accomplished by a variety of means known
in the art
including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated
transfection,
polybrene-mediated transfection, electroporation, microinjection, liposome
fusion, lipofection,
protoplast fusion, retroviral infection, and biolistics. In contrast, as used
herein, the term
"transduction" refers to the delivery of a genes) using a retroviral vector by
means of
infection rather than by transfection.
The term "membrane-associated protein" refers to a protein (e.g., a viral
envelope
glycoprotein or the G proteins of viruses in the Rhabdoviridae family such as
VSV, Piry,
Chandipura and Mokola) which are associated with the membrane surrounding a
viral particle;
these membrane-associated proteins mediate the entry of the viral particle
into the host cell.
The membrane associated protein may bind to specific cell surface protein
receptors, as is the
case for retroviral envelope proteins or the membrane-associated protein may
interact with a
phospholipid component of the plasma membrane of the host cell, as is the case
for the G
1 S proteins derived from members of the Rhabdoviridae family. .
The term "heterologous membrane-associated protein" refers to a membrane-
associated
pmtein which is derived from _a virus which is not a member of the same viral
class or family
as that from which the nucleocapsid protein of the vector particle is,
derived. "Viral class or
family" refers to the taxonomic rank of class or family, as assigned by the
International
Committee on Taxonomy of Viruses.
The term "Rhabdoviridae" refers to a family of enveloped RNA viruses that
infect
animals, including humans, and plants. The Rhabdoviridae family encompasses
the genus
Vesiculovirus which includes vesicular stomatitis virus (VSV), Cocal virus,
Piry virus,
Chandipura virus, and Spring viremia of carp virus (sequences encoding the
Spring viremia of
carp virus are available under GenBank accession number U 18101 ). The G
proteins of
viruses in the Vesiculovirus genera are virally-encoded integral membrane
proteins that form
externally projecting homotrimeric spike glycoproteins complexes that are
required for
receptor binding and membrane fusion. The G proteins of viruses in the
Vesiculovirus genera
have a covalently bound palmitic acid (C,6) moiety. The amino acid sequences
of,the G
proteins from the Vesiculoviruses are fairly well conserved. For example, the
Piry virus G
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protein share about 38% identity and about 55% similarity with the VSV G
proteins (several
strains .of VSV are known, e.g., Indiana, New Jersey, Orsay, San Juan, etc.,
and their G
proteins are highly homologous). The Chandipura virus G protein and the VSV G
proteins
share about 37% identity and 52% similarity. Given the high degree of
conservation (amino
acid sequence) and the related functional characteristics (e.g., binding of
the virus to the host
cell and fusion of membranes, including syncytia formation) of the G proteins
of the
Vesiculoviruses, the G proteins from non-VSV Vesiculoviruses may be used in
place of the
VSV G protein for the pseudotyping of viral particles. The G proteins of the
Lyssa viruses
(another genera within the Rhabdoviridae family) also share a fair degree of
conservation with
the VSV G proteins and function in a similar manner (e.g., mediate fusion of
membranes) and
therefore may be used in place of the VSV G protein for the pseudotyping of
viral particles.
The Lyssa viruses include the Mokola virus and the Rabies viruses (several
strains of Rabies
virus are known and their G proteins have been cloned and sequenced). The
Mokola virus G
protein shares stretches of homology (particularly over the cxtracellular and
transmembrane
domains) with the VSV G proteins which show about 31% identity and 48%
similarity with
the VSV G proteins. Preferred G proteins share at least 25% identity,
preferably at least 30%
identity and most prcf~avbly ai least 35% identity with the VSV G proteins.
The VSV G
protein from which New Jersey strain (the sequence of this G protein is
provided in GenBank
accession numbers M27165 and M21557) is employed as the reference VSV G
protein.
The term "conditions which permit the maturation of a pre-maturation oocyte"
refers to
conditions of in vitro cell culture which permit the maturation of a pre-
maturation oocyte to a
mature ovum (e.g., a pre-fertilization oocyte). These culture conditions
permit and induce the
events which are associated with maturation of the pre-maturation oocyte
including
stimulation of the first and second meiotic divisions. In vitro culture
conditions which permit
the maturation of pre-maturation oocytes from a variety of mammalian species
(e.g., cattle,
hamster, pigs and goats) are well know to the ari (See e.g., Parrish et al.,
Theriogenol.,
24:537 [ 1985]; Rosenkrans and First, J. Anim. Sci., 72:434 [ 1994]; Bavister
and Yanagimachi,
Biol. Reprod., 16:228 [1977]; Bavister et al., Biol. Reprod., 28:235 (1983];
Leibfried and
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Bavister, J. Reprod. Fert., 66:87 [1982]; Keskintepe et al., Zygote 2:97
[1994]; Funahashi et
al., J. Reprod. Fert., 101:159 [1994]; and Funahashi et al., Biol. Reprod
50:1072 [1994].
As used herein, the term "remedial gene" refers to a gene whose expression is
desired
in a cell to correct an error in cellular metabolism, to inactivate a pathogen
or to kil! a
cancerous cell.
As used herein, the term ,"selectable marker" refers to the use of a gene
which encodes
an enzymatic activity.that confers resistance to an antibiotic or drug upon
the cell in which
the selectable marker is expressed. Selectable markers may be "dominant"; a
dominant
selectable marker encodes an enzymatic activity which can be detected in any
eukaryotic cell
line. Examples of dominant selectable markers include the bacterial
aminoglycoside 3'
phosphotransferase gene (also referred to as the neo gene) which confers
resistance to the
drug 6418 in mammalian cells, the bacterial hygromycin G phosphotransferase
(hyg) gene
which confers resistance to the antibiotic hygromycin and the bacterial
xanthine-guanine
phosphoribosyl transferase gene (also referred to as the gpt gene) which
confers the ability to
grow in the presence of mycophenolic acid. Other selectable markers are not
dominant in that
there use must be in conjunction with a cell line that lacks the relevant
enzyme activity.
Examples of non-dominant selectable markers include the thymidine kinase (tk)
'gene which is
used in conjunction with tk cell lines, the CAD gene which is used in
conjunction with CAD-
deficient cells and the mammalian hypoxanthine-guanine phosphoribosyl
transferase (hprt)
gene which is used in conjunction with hprt cell lines. A review of the use of
selectable
markers in mammalian cell lines is provided in Sambrook, J. et al., Molecular
Cloning: A
Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York
(1989)
pp.16.9-16.15.
As used herein, the terms "complementary" or "complementarily" are used in
reference
to polynucleotides (i.e., a sequence of nucleotides) related by the base-
pairing rules. For
example, for the sequence "A-G-T," is complementary to the sequence "T-C-A."
Complementarily may be "partial," in which only some of the nucleic acids'
bases are
matched according to the base pairing rules. Or, there may be "complete" or
"total"
complementarily between the nucleic acids. The degree of complementarily
between nucleic
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acid strands has significant effects on the efficiency and strength of
hybridization between
nucleic acid strands. This is of particular importance in amplification
reactions, as well as
detection methods which depend upon binding between nucleic acids.
As used herein, the term "hybridization" is used in reference to the pairing
of
S complementary nucleic acids. Hybridization and the strength of hybridization
(i.e., the
strength of the association between the nucleic acids) is impacted by such
factors as the
degree of complementary between the nucleic acids, stringency of the
conditions involved, the
Tm of the formed hybrid, and the G:C ratio within the nucleic acids.
As used herein, the term "T,"" is used in reference to the "melting
temperature." The
melting temperature is the temperature at which a population of double-
stranded nucleic acid
molecules becomes half dissociated into single strands. The equation for
calculating the Tm of
nucleic acids is well known in the art. As indicated by standard references, a
simple estimate
of the T" value may be calculated by the equation: T- = 81.5 + 0.41 (% G + C),
when a
nucleic acid is in aqueous solution at 1 M NaCI (See e.g., Anderson and Young,
Quantitative
Filter Hybridization, in Nucleic Acid Hybridization (1985). Other. references
include more
sophisticated computations which take structural as well as sequence
characteristics into
account for the calculation of T":.
As used heiein the term "stringency" is used in reference to the conditions of
temperature, ionic strength, and the presence of other compounds such as
organic solvents,
under which nucleic acid hybridizations are conducted. With "high stringency"
conditions,
nucleic acid base pairing will occur only between nucleic acid fragments that
have a high
frequency of complementary base sequences. Thus, conditions of "weak" or "low"
stringency
are often required with nucleic acids that are derived from organisms that are
genetically
diverse, as the frequency of complementary sequences is usually less.
As used herein, the term "amplifiable nucleic acid". is used in reference to
nucleic
acids which may be amplified by any amplification method. It is contemplated
that
"amplifiable nucleic acid" will usually comprise "sample template."
As used herein, the term "sample template" refers to nucleic acid originating
from a
sample which is analyzed for the presence of "target" (defined below). In
contrast,
"background template" is used in reference to nucleic acid other than sample
template which
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may or may not be present in a sample. .Background template is most often
inadvertent. It
may be the result of carryover, or it may be due to the presence of nucleic
acid contaminants
sought to be purified away from the sample. For example, nucleic acids from
organisms
other than those to be detected may be present as background in a test sample.
As used herein, the term "primer" refers to an oligonucleotide, whether
occurring
naturally as in a purified restriction digest or produced synthetically, which
is capable of
acting as a point of initiation of synthesis when placed under conditions in
which synthesis of
a primer extension product which is complementary to a nucleic acid strand is
induced, (i.e.,
in the presence of nucleotides and an inducing agent such as DNA polymerase
and at a
suitable temperature and pI-~. The primer is preferably single stranded for
maximum
efficiency in amplification, but may alternatively be double stranded. If
double stranded, the
primer is first treated to separate its strands before being used to prepare
extension products.
Preferably, the primer is an oligodeoxyribonucleotide. The primer must be
suflaciently long
to prime the synthesis of extension products in the presence of the inducing
agent. The exact
lengths of the primers will depend on many factors, including temperature,
source of primer
and the use of the method.
As used herein, the term "probe": refers to an oligonucleotide (i:e., a
sequence of
nucleotides), whether occurring naturally as in a ~purified.res4riction digest
or produced
synthetically, which is capable of hybridizing to another oligonucleotide of
interest Probes
are useful in the detection, identification and isolation of particular gene
sequences. It is
contemplated that any probe used in the present invention will be labelled
with any "reporter
molecule," so that is detectable in any detection system, including, but not
limited to enzyme
(e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent,
radioactive, and
luminescent systems. It is further contemplated that the oligonucleotide of
interest (i.e., to be
detected) will be labelled with a reporter molecule. It is also contemplated
that both the
probe and oligonucleotide of interest will be labelled. It is not intended
that the present
invention be limited to any particular detection system or label.
As used herein, the term "target" refers to the region of nucleic acid bounded
by the
primers used for polymerase chain reaction. Thus, the "target" is sought to be
sorted out from
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other nucleic acid sequences. A "segment" is defined as a region of nucleic
acid within the
target sequence.
As used herein, the term "polymerise chain reaction" ("PCR") refers to the
methods of
U.S. Patent Nos. 4,683,195, 4,683,202, and 4,965,188,
directed to methods for increasing the concentration of a segment of a target
sequence in a mixture of genomic DNA without cloning or purification. This
process for
amplifying the target sequence consists of introducing a large excess of two
oligonucleotide
primers to the DNA mixture containing the desired target sequence, followed by
a precise
sequence of thermal cycling in the presence of a DNA polymerise. The two
primers are
complementary to their respxtive strands of the double stranded target sequ~.
To effect
amplification, the mixture is denatured and the primers then annealed to their
complem~tary
sequences within the target molecule. Following annealing, the primers are
extended with a
polymerise so as to form a new pair of complementary strands. The steps of
denaturation,
primer annealing and polymerise extension can be repeated many times ~.e.,
denaturation,
annealing and extension constitute one "cycle"; there can be numerous "cycles'
to obtain a
high concentration of an ampli5ed segment of the desired target sequence. The
length of the
amplified scgmart of t1u desired target seqn~x is dctamiaed by the relative
positions of the
primers with respect to each other, and therefore, this length is a
controllable paramet~x. By
virtue of the repeating aspect of the process, the method is referred to as
the "polymerise
chain reaction" (hereinaRer "PCR'~. Because the desired amplified segments of
the target
sequence become the predominant sequences (in terms of concentration) in the
mixture, they
are the to be "PCR amplified".
With PCR, it is possible to amplify a single copy of a specific target
sequence in
genomic DNA to a level delectable by several different methodologies (e.g.,
hybridization
with a labeled probe; incorporation of biotinylated primers followed by avidin-
enzyme
conjugate detection; incorporation of 3~P-labeled deoxynucleotide
triphosphates, such as dCTP
or dATP, into the amplified segment). In addition to genomic DNA, any
oligonucleotide
sequence can be amplified with the appropriate set of primer molecules. In
particular, the
amplified segments created by the PCR process itself are, themselves,
efficient templates for
subsequent PCR amplifications.
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OM i T~'E c~
D ~l-C
s
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As used herein, the term "nested primers" refers to primers that anneal to the
target
sequence in an area that is inside the annealing boundaries used to start PCR
(Mullis, et al.,
Cold Spring Harbor Symposia, Vol. II, pp.263-273 [1986]). Because the nested
primers
anneal to the target inside the annealing boundaries of the starting primers,
the predominant
PCR-amplified product of the starting primers is necessarily a longer
sequence, than that
defined by the annealing boundaries of the nested primers. The PCR-amplified
product of the
nested primers is an amplified segment of the target sequence that cannot,
therefore, anneal
with the starting primers. Advantages to the use of nested primers include the
large degree of
specificity, as well as the fact that a smaller sample portion may be used and
yet obtain
specific and efficient amplification.
As used herein, the term "amplification reagents" refers to those reagents
(deoxyribonucleoside triphosphates, buffer, etc.), needed for amplification
except for primers,
nucleic acid template and the amplification enzyme. Typically, amplification
reagents along
with other reaction components are placed and contained in a reaction vessel
(test tube,
IS microwell, etc.).
As used herein, the terms "restriction endonucleases" and "restriction
enzymes" refer to
bacterial enzymes, each of which cut double-stranded DNA at or near a specific
nucleotide
As used herein, the term "recombinant DNA molecule" as used herein refers to a
DNA
molecule which is comprised of segments of DNA joined together by means of
molecular
biological techniques.
DNA molecules are said to have "5' ends" and "3' ends" because mononucleotides
are
reacted to make oligonucleotides in a manner such that the 5' phosphate of one
mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in
one direction via a
phosphodiester linkage. Therefore, an end of an oligonucleotides referred to
as the "S' end" if
its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose
ring and as the
"3' end" if its 3' oxygen is not linked to a 5' phosphate of a subsequent
mononucleotide
pentose ring. As used herein, a nucleic acid sequence, even if internal to a
larger
oligonucleotide, also may be said to have 5' and 3' ends. In either a linear
or circular DNA
molecule, discrete elements are referred to as being "upstream" or 5' of the
"downstream" or
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3' elements. This terminology reflects the fact that transcription proceeds in
a 5' to 3'
fashion along the DNA strand. The promoter and enhancer elements which direct
transcription of a linked gene are generally located 5' or upstream of the
coding region
However, enhancer elements can exert their effect even when located 3' of the
promoter
element and the coding region. Transcription termination and polyadenylation
signals are
located 3' or downstream of the coding region.
As used herein, the term "an oligonucleotide having a nucleotide sequence
encoding a
gene" means a DNA sequence comprising the coding region of a gene or in other
words the
DNA sequence which encodes a gene product. The coding region may be present in
either a
cDNA or genomic DNA form. Suitable control elements such as
enhancers/promoters, splice
junctions, polyadenylation signals, etc. may be placed in close proximity to
the coding region
of the gene if needed to permit proper initiation of transcription and/or
correct processing of
the primary RNA transcript. Alternatively, the coding region utilized in the
expression
vectors of the present invention may contain endogenous enhaacers/promoters,
splice
junctions, intervening sequences, polyadenylation signals, etc. or a
combination of both
endogenous and exogenous control elements.
As used herein, the term "transcription unit" refers to the: segment, of DNA
between the
sites of initiation and termination of tr~ansctiption and the regulatory
elements necessary for
the efficient initiation and termination. For example, a segment of DNA
comprising an
enhancer/promoter, a coding region and a termination and polyadenylation
sequence comprises
a transcription unit.
As used herein, the term "regulatory element" refers to a genetic element
which
controls some aspect of the expression of nucleic acid sequences. For example,
a promoter is
a regulatory element which facilitates the initiation of transcription of an
operably linked
coding region. Other regulatory elements are splicing signals, polyadenylation
signals,
termination signals, etc. (defined infra).
Transcriptional control signals in eukaryotes comprise "promoter" and
"enhancer"
elements. Promoters and enhancers consist of short arrays of DNA sequences
that interact
specifically with cellular proteins involved in transcription (Maniatis et
al., Science 236:1237
[ 1987]). Promoter and enhancer elements have been isolated from a variety of
eukaryotic
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sources including genes in yeast, insect and mammalian cells and viruses
(analogous control
elements, i.e., promoters, are also found in prokaryotes). The selection of a
particular
promoter and enhancer depends on what cell type is to be used to express the
protein of
interest. Some eukaryotic promoters and enhancers have a broad host range
while others are
functional in a limited subset of cell types (for review see Voss et al.,
Trends Biochem. Sci.,
11:287 [1986]; and Maniatis et al., supra [1987]). For example, the SV40 early
gene
enhancer is very active in a wide variety of cell types from many mammalian
species and has
been widely used for the expression of proteins in mammalian cells (Dijkema et
al., EMBO
J., 4:761 [1985]). Two other examples of promoter/enhancer elements active in
a broad range
of mammalian cell types are those from the human elongation factor la gene
(tJetsuki et al.,
J. Biol. Chem., 264:5791 [1989]; Kim et al., Gene 91:217 [1990]; and Mizushima
and
Nagata, Nuc. Acids. Res., 18:5322 [1990]) and the long terminal repeats of the
Rous sarcoma
virus (Gorman et al., Proc. Natl. Acad. Sci. USA 79:6777 [1982]) and the human
cytomegalovirus (Boshart et al., Cell 41:521 [1985p.
As used herein, the .terns "promoter/enhancer" denotes a segment of DNA which
contains sequences capable of,providing both promoter and enhancer functions
(~e., the
functions provided by a pmmote= element and an enhancer eleme~, see above for
a .
discussion of these functions). For example; the long terminal repeats of
retroviruses contain
both promoter and enhancer functions. The enhancer/promoter may be
"endogenous" or
"exogenous" or "heterologous." An "endogenous" enhancer/promoter is one which
is naturally
linked with a given gene in the genome. An "exogenous" or "heterologous"
enhancerlpromoter is one which is placed in juxtaposition to a gene by means
of genetic
manipulation (s. e., molecular biological techniques) such that transcription
of that gene is
directed by the linked enhancer/promoter.
The term "factor" refers to a protein or group of proteins necessary for the
transcription or replication of a DNA sequence. For example, SV40 T antigen is
a replication
factor which is necessary for the replication of DNA sequences containing the
SV40 origin of
replication. Transcription factors are proteins which bind to regulatory
elements such as
promoters and enhancers and facilitate the initiation of transcription of a
gene.
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Promoters and enhancers may bind to specific factors which increase the rate
of
activity from the promoter or enhancer. These factors may be present in all
cell types or may
be expressed in a tissue-specific manner or in virus infected cells. In the
absence of such a
factor the promoter may be inactive or may produce a low level of
transcriptional activity.
Such a low level of activity is referred to as a baseline or "basal" rate of
activity.
Additionally, viral promoter and enhancers may bind to factors encoded by the
virus such that
the viral promoter or enhancer is "activated" in the presence of the viral
factor (in a virus
infected cell or in a cell expressing the viral factor). The level of activity
in the presence of
the factor (i.e., activity "induced" by the factor) will be higher than the
basal rate.
l0 Different promoters may havc different levels of basal activity in the same
or different
cell types. When two different promoters are compared in a given cell type in
the absence of
any inducing factors, if one promoter expresses at a higher level than the
other it is said to
have a higher basal activity.
The activity of a promoter and/or enhancer is measured by detecting directly
or
indirectly'the level of transcription from the element(s). Direct detection
involves quantitating
the level of the RNA transcripts produced from that promoter andJor enhancer.
Indirect
detection involves quantitation. .of the level of a protein;.often an enzyme,
produced from
RNA transcn'bed from the promoter and/or enhancer. . An commonly employed
assay for
promoter or enhancer activity utilizes the chloramphenicol acetyltransferase
(CAT) gene. A
promoter and/or enhancer is inserted upstream from the coding region for the
CAT gene on a
plasmid; the plasmid is introduced into a cell line. The levels of CAT enzyme
are measured.
The level of enzymatic activity is proportional to the amount of CAT RNA
transcribed by the
cell line. This CAT assay therefore allows a comparison to be made of the
relative strength
of different promoters or enhancers in a given cell line. When a promoter is
said to express
at "high" or "low" levels in a cell line this refers to the level of activity
relative to another
promoter which is used as a reference or standard of promoter activity.
The presence of "splicing signals" on an expression vector often results in
higher levels
of expression of the recombinant transcript. Splicing signals mediate the
removal of introns
from the primary RNA transcript and consist of a splice donor and acceptor
site (See e.g.,
Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold
Spring Harbor
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Laboratory Press, New York [1989], pp. 16.7-16.8). A commonly used splice
donor and
acceptor site is the splice junction from the 16S RNA of SV40.
Efficient expression of recombinant DNA sequences in eukaryotic cells requires
expression of signals directing the efficient termination and polyadenylation
of the resulting
$ transcript. Transcription termination signals are generally found downstream
of the
polyadenylation signal and are a few hundred nucleotides in length. The term
"poly A site"
or "poly A sequence" as used herein denotes a DNA sequence which directs both
the
termination and polyadenylation of the nascent RNA transcript. Efficient
polyadenylation of
the recombinant transcript is desirable as transcripts lacking a poly A tail
are unstable and are
rapidly degraded. The poly A signal utilized in an expression vector may be
"heterologous" or
"endogenous." An endogenous poly A signal is one that is found naturally at
the 3' end of
the coding region of a given gene in the genome. A heterologous poly A signal
is one which
is one which is isolated from one gene and placed 3' of another gene. A
commonly used
heterologous poly A signal is the SV40 poly A signal. The SV40 poly A signal
is contained
on a 237 by Bam HIIBcI I restriction fragment and directs both termination and
polyadenylation (Sambrook, J., supra, at 16.6-16.7). '
Eukaryotic cacpression vectors may also contain "viral replicons "or "viral
origins of
replication." Viral replicons are viral DNA sequences which allow for the
extrachromosomal
replication of a vector in a host cell expressing the appropriate replication
factors. Vectors
which contain either the SV40 or polyoma virus origin of replication replicate
to high copy
number (up to 10~ copies/cell} in cells that express the appropriate viral
antigen. Vectors
which contain the replicons from bovine papillomavirus or Epstein-Barr virus
replicate
extrachromosomally at low copy number 0100 copies/cell).
The term "stable transfection" or "stably transfected" refers to the
introduction and
integration of foreign DNA into the genome of the transfected cell. The term
"stable
transfectant" refers to a cell which has stably integrated foreign DNA into
the genomic DNA.
As used herein, the term "stably maintained" refers to characteristics of
recombinant
(i.e., transgenic) animals that maintain at least one of their recombinant
elements (i.e., the
element that is desired) through multiple generations. For example, it is
intended that the
term encompass the characteristics of transgenic animals that are capable of
passing the
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transgene to their offspring, such that the offspring are capable of
maintaining the expression
and/or transcription of the transgene. It is not intended that the term be
limited to any
particular organism or any specific recombinant element.
The term "transient transfection" or "transiently transfected" refers to the
introduction
of foreign DNA into a cell where the foreign DNA fails to integrate into the
genome of the
transfected cell. The foreign DNA persists in the nucleus of the transfected
cell for several
days. During this time the foreign DNA is subject to the regulatory controls
that govern the
expression of endogenous genes in the chromosomes. The term "transient
transfectant" refers
to cells which have taken up foreign DNA but have failed to integrate this
DNA.
As used herein, the term "gene of interest" refers to the gene inserted into
the
polylinker of an expression vector. When the gene of interest encodes a gene
which provides
a therapeutic function, the gene of interest may be alternatively called a
remedial gene.
As used herein, the terms "nucleic acid molecule encoding," "DNA sequence
encoding," and "DNA encoding" refer to the order or sequence of
deoxyribonucleotides along
a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides
determines the
order of amino acids along the polypeptide (protein) chain. The DNA sequence
thus codes
for the amino acid sequence.
As used herein, the term "adoptive transfer" is used in reference to the
transfer of one
function to another cell or organism. For example, in "adoptive immunity,"
transfer of an
immune function is made from one organism to another through the transfer of
immunologically competent cells.
DESCRIPTION OF THE INVENTION
The present invention provides improved methods for the production of
transgenic
animals. The methods of the present invention provide, for the first time, the
production of
transgenic animals by the introduction of exogenous DNA into pre-maturation
oocytes and
mature, unfertilized oocytes (i. e., pre-fertilization oocytes) using
retroviral vectors which
transduce dividing cells (e.g., vectors derived from marine leukemia virus
(MLV]). In
addition, the present invention provides methods and compositions for
cytomegalovirus
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promoter-driven, as well as mouse mammary tumor LTR expression of various
recombinant
proteins. .
For example, the human cytomegalovirus (CMV) promoter has been developed for
use
in retroviral vectors for driving the expression of various recombinant
proteins, and cell lines
have been infected with these vectors, with resultant recombinant protein
expression. In
addition, the mouse mammary tumor virus (IvBvITV) LTR has been previously
shown to
control expression of a recombinant protein in transgenic mice (Yom et al.,
Animal Biotech.,
4:89-107 [1993]). In these mouse lines, expression was predominately observed
in the
mammary gland and milk, but low expression was also observed in the salivary
gland, spleen,
lung and kidney. The transgenic mice used in this experiment were produced
using typical
microinjection techniques. In contrast, the present invention provides methods
and
compositions for the use of MMTV LTR driven expression which avoids the need
for
microinjection techniques. For example, the MMT'V LTR has been developed for
use in
retroviral vectors for driving the expression of various recombinant proteins,
and cell lines
have been infected with these vectors, with resultant recombinant protein
expression.
The following Description of the Invention is divided into the following
sections: I.
lZetrovin~ses and Retrovi=al Vectors; II. Integration. of Retmviral DNA; III.
Introduction of
Retroviral Vectors Into Gametes Before the Last Meiotic Division; IV.
Detection of the
Retrovirus Following Injection Into Oocytes or Embryos; and V. Expression of
Foreign
Proteins in Transgenic Animals.
I. Retroviruses and Retroviral Vectors
Retroviruses (family Retroviridae) are divided into three groups: the
spumaviruses
(e.g., human foamy virus); the lentiviruses (e.g., human immunodeficiency
virus and sheep
visna virus) and the oncoviruses (e.g., MLV, Rous sarcoma virus).
Retroviruses are enveloped (i.e., surrounded by a host cell-derived lipid
bilayer
membrane) single-stranded RNA viruses which infect animal cells. When a
retrovirus infects
a cell, its RNA genome is converted into a double-stranded linear DNA form
(i.e., it is
reverse transcribed). The DNA form of the virus is then integrated into the
host cell genome
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as a provirus. The provirus serves as a template for the production of
additional viral
genomes and viral mRNAs. Mature viral particles containing two copies of
genomic RNA
bud from the surface of the infected cell. The viral particle comprises the
genomic RNA,
reverse transcriptase and other pot gene products inside the viral capsid
(which contains the
viral gag gene products) which is surrounded by a lipid bilayer membrane
derived from the
host cell containing the viral envelope glycoproteins (also referred to as
membrane-associated
proteins).
The organization of the genomes of numerous retroviruses is well known in the
art and
this has allowed the adaptation of the retroviral genome to produce retroviral
vectors. The
production of a recombinant retroviral vector carrying a gene of interest is
typically achieved
in two stages. First, the gene of interest is inserted into a retroviral
vector which contains the
sequences necessary for the efficient expression of the gene of interest
(including promoter
and/or enhancer elements which may be provided by the viral long terminal
repeats [LTRs] or
by an internal promoter/enhancer and relevant splicing signals), sequences
required for the
efficient packaging of the viral RNA into infectious virions (e.g., the
packaging signal [Psi],
the tRNA primer binding site [-PBS], the 3' regulatory sequences required for
reverse
transcription t+PBS] and the viral LTRs). The LTRs contain sequences required
for the
association of viral genomic RNA, reverse transcriptase and integrase
functions, and sequences
involved in directing the expression of the genomic RNA to be packaged in
viral particles.
For safety reasons, many recombinant retroviral vectors lack functional copies
of the genes
which are essential for viral replication (these essential genes are either
deleted or disabled);
the resulting virus is said to be replication defective.
Second, following the construction of the recombinant vector, the vector DNA
is
introduced into a packaging cell line. Packaging cell lines provide viral.
proteins required in
traps for the packaging of the viral genomic RNA into viral panicles having
the desired host
range (i.e., the viral-encoded gag, pol and env proteins). The host range is
controlled, in part,
by the type of envelope gene product expressed on the surface of the viral
particle.
Packaging cell lines may express ecotrophic, amphotropic or xenotropic
envelope gene
products. Alternatively, the packaging cell line may lack sequences encoding a
viral envelope
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(env) protein. In this case the packaging cell line will package the viral
genome into particles
which lack a membrane-associated protein (e.g., an env protein). In order to
produce viral
particles containing a membrane associated protein which will permit entry of
the virus into a
cell, the packaging cell line containing the retroviral sequences is
transfected with sequences
encoding a membrane-associated protein (e.g., the G protein of vesicular
stomatitis virus
[VSV]). The transfected packaging cell will..then produce viral particles
which contain the
membrane-associated protein expressed by the transfected packaging cell line;
these viral
particles which contain viral genomic RNA derived from one virus encapsidated
by the
envelope proteins of another virus are said to be pseudotyped virus particles.
Viral vectors, including recombinant retroviral vectors, provide a more
efficient means
of transferring genes into cells as compared to other techniques such as
calcium phosphate-
DNA co-precipitation or DEAF-dextran-mediated transfection, electroporation or
microinjection of nucleic acids. It is believed that the efficiency of viral
transfer is due in
part to the fact that the transfer of nucleic acid is a receptor-mediated
process (~e., the virus
binds to a specific receptor protein on the surface of the cell to be
infected). In addition, the
virally transferred nucleic acid once inside a cell integrates in controlled
manner in contrast to
the integration of nucleic acids which are not virally transferred; nucleic
acids transferred by
other means such as calcium phosphate-DNA ~co-precipitation are subject to
rearrangement
and degradation.
The most commonly used recombinant retroviral vectors are derived from the
amphotropic Moloney marine leukemia virus (MoMLV) (Miller and Baltimore, Mol.
Cell.
Biol., 6:2895 [1986]). The MoMLV system has several advantages: 1} this
specific retrovirus
can infect many different cell types, 2) established packaging cell lines are
available for the
production of recombinant MoMLV viral particles and 3} the transferred genes
are
permanently integrated into the target cell chromosome. The established MoMLV
vector
systems comprise a DNA vector containing a small portion of the retroviral
sequence (the
viral long terminal repeat or "LTR" and the packaging or "psi" signal) and a
packaging cell
line. The gene to be transferred is inserted into the DNA vector. The viral
sequences present
on the DNA vector provide the signals necessary for the insertion or packaging
of the vector
RNA into the viral particle and for the expression of the inserted gene. The
packaging cell
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line provides the viral proteins required for panicle assembly (Markowitz et
al., J. Virol.,
62:1120 [1988]).
Despite these advantages, existing retroviral vectors based upon MoMLV are
limited
by several intrinsic problems: 1) they do not infect non-dividing cells
(Miller et al., Mol.
Cell. Biol., 10:4239 [1992]), 2) they produce low titers of the recombinant
virus (Miller and
Rosman, BioTechn., 7: 980 [1989]; and Miller, Nature 357: 455 [1992]) and 3)
they infect
certain cell types (e.g., human lymphocytes) with low efficiency (Adams et
al., Proc. Natl.
Acad. Sci. USA 89:8981 [1992]). The low titers associated with MoMLV-based
vectors has
been attributed, at least in part, to the instability of the virus-encoded
envelope protein.
Concentration of retrovirus stocks by physical means (e.g.,
ultracentrifugation and
ultrafiltration) leads to a severe loss of infectious virus.
The low titer and inefficient infection of certain cell types by MoMLV-based
vectors
has been overcome by the use of pseudotyped retroviral vectors which contain
the G protein
of VSV as the membrane associated protein. Unlike retroviral envelope proteins
which bind
to a specific cell surface protein receptor to gain entry into a cell, the VSV
G protein interacts
with~a phospholipid component of the plasma membrane (Mastromarino et al., J.
Gen. Virol.,
68:2359 [1977]). Because entry of VSV into a cell is not dependent upon the
presence of
specific protein receptors, VSV has an extremely broad host range.
~Pseudotyped retroviral
vectors bearing the VSV G protein have an altered host range characteristic of
VSV (i.e., they
can infect almost all species of vertebrate, invertebrate and insect cells).
Importantly, VSV G-
pseudotyped retroviral vectors can be concentrated 2000-fold or more by
ultracentrifugation
without significant loss of infectivity (Bums et al., Proc. Natl. Acad. Sci.
USA 90:8033
[1993]).
The VSV G protein has also been used to pseudotype retroviral vectors based
upon the
2S human immunodeficiency virus (HIV) (Naldini et al., Science 272:263
[1996]). Thus, the
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VSV G protein may be used to generate a variety of pseudotyped retroviral
vectors and is not
limited to vectors based on MoMLV.
The present invention is not limited to the use of the VSV G protein when a
viral G
protein is employed as the heterologous membrane-associated protein within a
viral particle.
The G proteins of viruses in the Vesiculovirus genera other than VSV, such as
the Piry and
Chandipura viruses, that are highly homologous to the VSV G protein and, like
the VSV G
protein, contain covalently linked palmitic acid (Bran et al., Intervirol.,
38:274 [1995]; and
Masters et al., Virol., 171:285 [1990]). Thus, the G protein of the Piry and
Chandipura
viruses can be used in place of the VSV G protein for the pseudotyping of
viral particles. In
addition, the VSV G proteins of viruses within the Lyssa virus genera such as
Rabies and
Mokola viruses show a high degree of conservation (amino acid sequence as well
as
functional conservation) with the VSV G proteins. For example, the Mokola
virus G protein
has been shown to function in a manner similar to the VSV G protein (i.e., to
mediate
membrane fusion) and therefore may be used in place of the VSV G protein for
the
' 15 pseudotyping of viral particles (Mebatsion et al., J. Virol., 69:1444
[1995]). The nucleotide
sequence encoding the Piry G protein is provided in SEQ ID NO:S and the amino
acid
sequence.of the Piry G protein is provided in SEQ ID N0:6, The nucleotide
sequence
encoding the Chandipura G protein is provided in SEQ ID N0:7 and the amino
acid sequence
of the Chandipura G protein is provided in SEQ ID N0:8. The nucleotide
sequence encoding
the Mokola G protein is provided in SEQ ID N0:9 and the amino acid sequence of
the
Mokola G protein is provided in SEQ ID NO:10. Viral particles may be
pseudotyped using
either the Piry, Chandipura or hiokola G protein as described in Example 2
with the exception
that a plasmid containing sequences encoding either the Piry, Chandipura or
Mokola G protein
under the transcriptional control of a suitable promoter element (e.g., the
CMV intermediate-
early promoter; numerous expression vectors containing the CMV IE promoter are
available,
such as the pcDNA3.1 vectors [Invitrogen]) is used in place of pHCMV-G.
Sequences
encoding other G proteins derived from other members of the Rhabdoviridae
family may be
used; sequences encoding numerous rhabdoviral G proteins are available from
the GenBank
database.
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II. Integration of Retroviral DNA
The majority of retroviruses can transfer or integrate a double-stranded
linear form of
the virus (the provirus) into the genome of the recipient cell only if the
recipient cell is
cycling (i. e., dividing) at the time of infection. Retroviruses which have
been shown to infect
dividing cells exclusively, or more efficiently, include MLV, spleen necrosis
virus, Rous
sarcoma virus and human immunodeficiency virus (HIV; while HIV infects
dividing cells
more efficiently, HIV can infect non-dividing cells).
It has been shown that the integration of MLV virus DNA depends upon the host
cell's progression through mitosis and it has been postulated that the
dependence upon mitosis
reflects a requirement for the breakdown of the nuclear envelope in order for
the viral
integration complex to gain entry into the nucleus (Roe et al., EMBO J.,
12:2099 [1993]).
However, as integration does not occur in cells arrested in metaphase, the
breakdown of the
nuclear envelope alone may not be sufficient to permit viral integration;
there may be
additional requirements such as the state of condensation of the genomic DNA
(Roe et al.,
sr~pro).
III,. Introanction of Retroviral Vectors Into Gametes
Before the Last Meiotic Division
The nuclear envelope of a cell breaks down during meiosis as well as during
mitosis.
Meiosis occurs only during the final stages of gametogenesis. The methods of
the present
invention exploit the breakdown of the nuclear envelope during meiosis to
permit the
integration of recombinant retroviral DNA and permit for the first time the
use of unfertilized
oocytes (i. e., pre-fertilization and pre-maturation oocytes) as the recipient
cell for retroviral
gene transfer for the production of transgenic animals. Because infection of
unfertilized
oocytes permits the integration of the recombinant provirus prior to the
division of the one
cell embryo, all cells in the embryo will contain the proviral sequences.
Oocytes which have not undergone the final stages of gametogenesis are
infected with
the retroviral vector. The injected oocytes are then permitted to complete
maturation with the
accompanying meiotic divisions. The breakdown of the nuclear envelope during
meiosis
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permits the integration of the proviral form of the retrovirus vector into the
genome of the
oocyte. When pre-maturation oocytes are used, the injected oocytes are then
cultured in vitro
under conditions which permit maturation of the oocyte prior to fertilization
in vitro.
Conditions for the maturation of oocytes from a number of mammalian species
(e.g., bovine,
ovine, porcine, marine, caprine) are well known to the art. In general, the
base medium used
herein for the in vitro maturation of bovine oocytes, TC-M199 medium, may be
used for the
in vitro maturation of other mammalian oocytes. TC-M199 medium is supplemented
with
hormones (e.g., luteinizing hormone and estradiol) from the appropriate
mammalian species.
The amount of time a pre-maturation oocyte must be exposed to maturation
medium to permit
maturation varies between mammalian species as is known to the art. For
example, an
exposure of about 24 hours is sufficient to permit maturation of bovine
oocytes while porcine
oocytes require about 44-48 hours.
pocytes may be matured in vivo and employed in place of oocytes matured in
vitro in
the practice of the present invention For example, when porcine oocytes are to
be employed
in the methods of the present invention, matured pre-fertilization oocytes may
~ l~es<ed
directly from pigs that are induced to superovulate as is known to the art.
Briefly, on day I S
or 16 of estrus the female pigs) is injected with about 1000 units of pregnant
mare's serum
(PMS; available from Sigma and Calbiochem). Approximately 48 hours later, the
pigs) is
injected with about 1000 units of human chorionic gonadotropin) (hCG; Sigma)
and 24-48
hours later matured oocytes are collected from oviduct. These in vivo matured
pre-
fertilization oocytes are then injected with the desired retroviral
preparation as described
herein. Methods for the superowlation and collection of in vivo matured (i.e.,
oocytes at the
metaphase 2 stage) oocytes are known for a variety of mammals (e.g., for
superowlation of
mice, see Hogan et al., supra at pp. 130-133 (1994]; for superowlation of pigs
and in vitro
fertilization of pig oocytes see Cheng, Doctoral Dissertation, Cambridge
University,
Cambridge, United Kingdom [1995]).
Retroviral vectors capable of infecting the desired species of non-human
animal which
can be grown and concentrated to very high titers (e.g., >_ 1 x 10' cfu/ml)
are preferentially
employed. The use of high titer virus stocks allows the introduction of a
defined number of
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viral particles into the perivitelline space of each injected oocyte. The
perivitelline space of
most mammalian oocytes can accommodate about 10 picoliters of injected fluid
(those in the
art know that the volume that can be injected into the perivitelline space of
a mammalian
oocyte or zygote varies somewhat between species as the volume of an ooeyte is
smaller than
that of a zygote and thus, oocytes can accommodate somewhat less than can
zygotes).
The vector used may contain one or more genes encoding a protein of interest;
alternatively, the vector may contain sequences which produce anti-sense RNA
sequences or
ribozymes. The infectious virus is microinjected into the perivitelline space
of oocytes
(including pre-maturation oocytes) or one cell stage zygotes. Microinjection
into the
perivitelline space is much less invasive than the microinjection of nucleic
acid into the
pronucleus of an embryo. Pronuclear injection requires the mechanical puncture
of the
plasma membrane of the embryo and results in Lower embryo viability. In
addition, a higher
Level of operator skill is required to perform pronuclear injection as
compared to perivitelline
injection. Visualization of the pronucleus is not required when the virus is
injected into the
perivitelline space (in contrast to mlection into the pronucleus); therefore
injection into the
perivitelliae space obviates the di~culties associated with visualization of
pronuclei in species
such~..as cattle, sheep aud-.pigs:. .
'The virus stock may be titered and diluted prior to microinjection into the
perivitelline
space so that the number of proviruses integrated in the resulting transgenic
animal is
controlled. The use of a viral stock (or dilution thereof) having a titer of 1
x 10= cfu/m1
allows the delivery of a single viral particle per oocyte. The use of pre-
maturation oocytes or
mature fertilized oocytes as the recipient of the virus minimizes the
production of animals
which are mosaic for the provirus as the virus integrates into the genome of
the oocyte prior
to the occurrence of cell cleavage.
In order to deliver, on average, a single infectious particle per oocyte, the
micropipets
used for the injection are calibrated as follows. Small volumes (e.g., about 5-
10 p1) of the
undiluted high titer viral stock (e.g., a titer of about 1 x 10a cfu/ml) are
delivered to the wells
of a microtiter plate by pulsing the micromanipulator. The titer of virus
delivered per a given
number of pulses is determined by diluting the viral stock in each well and
determining the
titer using a suitable cell line (e.g., the 208F cell line) as described in
Ex. 2. The number of
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pulses which deliver, on average, a volume of virus stock containing one
infectious viral
particle (i.e., gives a MOI of 1 when titered on 208F cells) are used for
injection of the viral
stock into the oocytes.
Prior to microinjection of the titered and diluted (if required) virus stock,
the cumulus
cell layer is opened to provide access to the perivitelline space. The cumulus
cell layer need
not be completely removed from the oocyte and indeed for certain species of
animals (e.g.,
cows, sheep, pigs, mice) a portion of the cumulus cell layer must remain in
contact with the
oocyte to permit proper development and fertilization post-injection.
Injection of viral
particles into the perivitelline space allows the vector RNA (i.e., the viral
genome) to enter
the cell through the plasma membrane thereby allowing proper reverse
transcription of the
viral RNA.
IV. Detection of the Retrovirus Following Injection Into Oocytes or Embryos
The presence of the retroviral genotna in cells (e.g., oocytes or embryos)
infected with
IS pseudotyped retrovirus may be detected using a 'variety of means. The
expression of the gene
products) encoded by the retrovirus may be detected by detection of mRNA
corresponding to
the vector-encoded gene products using techniQues well known to the art (e.g.,
Northern blot,
dot blot, in situ hybridization and RT PCR analysis). Direct detection of the
vector-encoded
gene products) is employed when the gene product is a protein which either has
an enzymatic
activity (e.g., j3-galactosidase) or when an antibody capable of reacting with
the vector-
encoded protein is available.
Alternatively, the presence of the integrated viral genome may be detected
using
Southern blot or PCR analysis. For example, the presence of the LZRNL or LSRNL
genomes
may be detected following infection of oocytes or embryos using PCR as
follows. Genomic
.:25 DNA is extracted from the infected oocytes or embryos (the DNA may be
extracted from the
whole embryo or alternatively various tissues of the embryo may be examined)
using
techniques well known to the art. The LZRNL and LSRNL viruses contain the neo
gene and
the following primer pair can be used to amplify a 349-by segment of the neo
gene: upstream
primer: 5'-GCATTGCATCAGCCATGATG-3' (SEQ ID NO:1) and downstream primer: 5'-
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GATGGATTGCACGCAGGTTC-3' (SEQ ID N0:2). The PCR is carried out using well
known techniques (e.g., using a GeneArap kit according to the manufacturer's
instructions
[Perkin-Elmer]). The DNA present in the reaction is denatured by incubation at
94°C for 3
min followed by 40 cycles of 94°C for 1 min, 60°C for 40 sec and
72°C for 40 sec followed
S by a final extension at 72°C for 5 min. The PCR products may be
analyzed by
electrophoresis of 10 to 20% of the total reaction on a 2% agarose gel; the
349-by product
may be visualized by staining of the gel with ethidium bromide and exposure of
the stained
gel to IJV light. If the expected PCR product cannot be detected visually, the
DNA can be
transfsrted to a solid support (e.g., a nylon membrane) and hybridized with
a'iP-labeled neo
10. probe.
Southern blot analysis of genomic DNA extracted from infoctod oocytes and/or
the
resulting embryos, offspring and tissues derived therefrom is employed when
information
concerning the integration of the viral DNA into the host genome is desired.
To examine the
number of integration sites present in the host genome, the extract genomic
DNA is
15 typically digested with a restriction enzyme which ct~.at least once within
the vector
sequences. If the enzyme chosen cuts twice within the va~r sequences, a band
of lmovvn
(ie.,.prodictable) size is generated iti addition to .two figments of novel
length which can be
det~e~ed using appmopiia~e pmbes.
20 V. Detection of Foreign Protein Ezpressioa is Transgenie Animals
The present invention also provides transgenic animals that are capable of
expressing
foreign proteins in their milk, urine and blood. As indicated in Examples 8-
I0, the transgene
is stable, as it is shown to be passed from a transgenic bull to his offspring
(See, Example 8).
In addition, as shown in Examples 9 and 10, transgenic animals produced
according to the
25 present invention express foreign proteins in their body fluids (e.g..
milk, blood, and urine).
Thus, these data further demonstrate the utility of using the MoMLV LTR as a
promoter for
driving the constitutive production of foreign proteins in transgenic cattle.
It is also
contemplated that such a promoter could be used to control expression of
proteins that would
prevent disease andlor infection in the transgenic animals and their
offspring, .or be of use in
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the production of a consistent level of protein expression in a number of
different tissues and
body fluids.
For example, it is contemplated that the MoMLV LTR of the present invention
will
fmd use in driving expression of antibody to pathogenic organisms, thereby
preventing
infection and/or disease in'transgenic animals created using the methods of
the present
invention. For example, it is contemplated that antibodies directed against
organisms such as
E. coli, Salmonella ssp., Streptococcus ssp., Staphylococcus spp.,
Mycobacterium spp.,
produced by transgenic animals will find use preventing mastitis, scours, and
other diseases
that are common problems in young animals. It is also contemplated that
proteins expressed
by transgenic animals produced according to the present invention will find
use as
bacteriostatic, bactericidal, fungistatic, fungicidal, viricidal, and/or anti-
parasitic compositions.
Thus, it is contemplated that transgenic animals produced according to the
present invention
will be resistant to various pathogenic organisms.. Furthermore, the milk
produced by female
transgenic animals would contain substantial antibody levels. It is
contemplated that these
antibodies will find use in the protection of other animals ~(e.g., through
passive immunization
methods).
EXPE~RnV~NTA1.
The following examples serve to illustrate certain preferred embodiments and
aspects
of the present invention and are not to be construed as limiting the scope
thereof.
In the experimental disclosure which follows, the following abbreviations
apply: M
(molar); mM .(millimolar); pM (micromolar); nM (nanomolar); mol (moles); mmol
(millimoles); pmol (micromoles); nmol (nanomoles); gm (grams); mg
(milligrams}; pg
(micrograms);pg (picograms); L (liters); ml' (milliliters); p! (microliters);
em. (centimeters);
mm (millimeters); ~cm (micrometers); nm (nanometers); °C (degrees
Centigrade); AMP
(adenosine 5'-monophosphate); BSA (bovine serum albumin); cDNA (copy or
complimentary
DNA); CS (calf serum); DNA (deoxyribonucleic acid); ssDNA (single stranded
DNA);
dsDNA (double stranded DNA); dNTP (deoxyribonucleotide triphosphate); LH
(luteinizing
hormone); NIH (National Institutes of Health, Besthesda, MD); RNA (ribonucleic
acid); PBS
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(phosphate buffered saline); g (gravity); OD (optical density); HEPES
(N-[2-Hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]); HBS (HEPES buffered
saline);
PBS (phosphate buffered saline); SDS (sodium dodecyl sulfate); Tris-HCl
(tris[Hydroxymethyl]aminomethane-hydrochloride); Klenow (DNA polymerise I
large
(Klenow) fragment); rpm (revolutions per minute); EGTA (ethylene glycol-bis(D-
aminoethyi
ether) N, N, N', N'-tetraacetic acid); EDTA (ethylenediaminetetracetic acid);
bla (!3-lactamase
or ampicillin-resistance gene); ORI (plasmid origin of replication); lacI (lie
repressor); X-gal
(S-bromo-4=chloro-3-indolyl-~i-D-galactoside); ATCC (American Type Culture
Collection,
Rockville, MD); GIBCOBRL (GIBCOBRL, Grand Island, N~; Perkin-Elmer (Perkin-
Elmer,
Norwalk, CT); Abbott (Abbott Laboratories, Diagnostics Division, Abbott Park,
IL 60064);
and Sigma (Sigma Chemical Company, St. Louis, MO).
EXAMPLE 1
Generation of Cell Lines Stably Expressing the MoMLV Gag and Pol Proteins
The expression of the fusogenic VSV G protein on the surface of.cells results
in
syncytium formation and cell death. Therefore, in order to produce retroviral
particles
containing the VSV G protein as the membrane-associated protein a three step
approach was
taken. First, stable cell lines expressing the Gag and Pol proteins from MoMLV
at high
levels were generated (e.g., 293GP cells; Example 1). These stable cell lines
were then
infected using the desired retroviral vector which is derived from an
amphotrophic packaging
cell (e.g., PA317 cells transfected with the desired retroviral vector;
Example 2a). The
infected stable cell line which expresses the Gag and Pol proteins produces
noninfectious viral
particles lacking a membrane-associated protein (e.g., a envelope protein).
Third, these
infected cell lines are then transiently transfected with a plasmid capable of
directing the high
level expression of the VSV G protein (Example 2b). The transiently
transfected cells
produce VSV G-pseudotyped retroviral vectors which can be collected from the
cells over a
period of 3 to 4 days before the producing cells die as a result of syncytium
formation.
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The first step in the production of VSV G-pseudotyped retroviral vectors, the
generation of stable cell lines expressing the MoMLV Gag and Pol proteins is
described
below.
The human adenovirus 5-transformed embryonal kidney cell line 293 (ATCC GItL
1573) was cotransfected with the pCMVgag-pol and pFR400 plasmids using a ratio
of 10:1
(pCMVgag-pol and pFR400). pCMV gag-pol contains the MoMLV gag and pol genes
under
the control of the CMV promoter (pCMV gag-pol is available from the ATCC).
pFR400
encodes a mutant dihydrofolate reductase which has a reduced affinity for
methotrexate
(Simonsen et al., Proc. Natl. Acad. Sci. 80:2495 [1983]).
The plasmid DNA was introduced into the 293 cells using calcium phosphate co-
precipitation (Graham and Van der Eb, Virol., 52:456 [1973]). Approximately 5
x 10s 293
cells were plated into a 100 mm tissue culture plate the day before the DNA co-
precipitate
was added. A total of 20 Ng of plasmid DNA (18 lrg pCMV gag pol and 2 ~g
pFR400) was
added as a calcium-DNA co-precipitate to each 100 mm plate. Stable
transformants were
selected by growth in DMEM high glucose medium containing 10% FCS, 0.5 pM
methotrexate and 5 ~M dipyridimole (~ e., selective medium). Colonies which
grew in the
selective modium were screeneii for cxtracellular reverse transcxiptase
activity (Guff et al., J. .
Virol:, 38:239 [1981]) and intracellular p30°~ expression. p30°~
expression was determined
by Western blotting using a goat-anti p30 antibody (NCI antiserum 775000087).
A clone
which exhibited stable expression of the retroviral genes in the absence of
continued
methotrexate selection was selected. This clone was named 293GP (293 gag-poly.
The
293GP cell line, a derivative of the human Ad-5-transformed embryonal kidney
cell line 293,
was grown in DMEM-high glucose medium containing 10% FCS. The 293GP cell line
is
commercially available from Viagen, Inc., San Diego, CA.
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EXAMPLE 2
Preparation of Pseudotyped Retroviral Vectors Bearing the G Glycoprotein of
VSV
In order to produce VSV G protein pseudotyped retrovirus the following steps
were
taken. First, the 293GP cell line was infected with virus derived from the
amphotrophic
packaging cell line PA317. The infected cells packaged the retroviral RNA into
viral particles
which lack a membrane-associated protein (because the 293GP cell line lacks an
e~rv gene or
other gene encoding a membrane-associated protein). The infected 293GP cells
were then
transiently transfected with a plasmid encoding the VSV G protein to produce
pseudotyped
viral particles beating the VSV G protein.
a) Cell Lines and Plasmids
The amphotropic packaging cell line, PA317 (ATCC CRL 9078) was grown in
DMEM-high glucose medium containing 10% FCS. The 293QP cell lip was grcswn in
DMEM-high glucose medium containing 10% FCS. The titer of the pseudo-typod
virus may
lie determined using either 208F cells (Quade, Virol., 98:461 [1979D, or
NIHl3T3 calls
{ATCC CRL 1658); 208F and NIH/3T3 cells are grown is DMEM high gle~se medium
containing 10% CS.
The plasmid pLZRNL (Xu et al., Virol., 171:331 [1989v contains the gene
cacoding
E. coli ~-galactosidase (Lac2) under the lranscriptional control of the LTR of
the Moloney
marine sarcoma vines (MSV) followed by the gene encoding neomycin
phosphotransferase
(Neo) under the transcriptional control of the Rous sarcoma virus (RSV)
promoter. The
plasmid pLSRNL contains the gene encoding the hepatitis B surface antigen gene
(HBsAg)
under the transcriptional control of the MSV LTR followed by the Neo gene
under the control
of the RSV promoter (U.S. Patent No. 5,512,421).
The plasmid pl-ICMV-G contains the VSV G gene under the
transcriptional control of the human cytomegalovirus intermediate-early
promoter (Yee et al.
Meth. Cell Biol., 43:99 (1994)).
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b) Production and Titering of Pseudotyped LZRNL Virus
pLZRNL DNA was transfected into the amphotropic packaging line PA317 to
produced LZ1ZNL virus. The resulting LZRNL virus was then used to infoct 293GP
cells to
produce pseudotyped LZItTTI, virus bearing the VSV G protein (following
transient
transfection of the infected 293GP cells with a plasmid encoding the VSV G
protein). The
procedure for producing pseudotyped LZRNL virus was caniod out as described
(Yee et al.
Meth. Cell Biol., 43:99 [1994n.
Briefly, on day 1, approximately 5 x lOs PA317 cells were placed in a l00 mm
tissue
culture plate. On the following day (day 2), the PA317 cells were transfectod
with 20 pg of
pLZRNL plasmid DNA (plasmid DNA was purified using CsCI gradients) using the
standard
calcium phosphate co-precipitation pmccdute (Graham and Van da Eb, Viml.,
52:456
[1973]). A range of 10 to 40 iCg of plasmid DNA may be usod. Hocanse 293GP
cells may
take more than 24 hours to attach firmly to tissue culture plates, the 293GP
cells may be
placed in 100 mm plates 48 hours priar to transfection. The transfeetod PA317
calls provide
amphotropic LZRNL virus.
On day 3, approximately 1 x 10' 293GP cells ware placed in a 100 mm tissue
cx~l~e
plate 24 hours prior to the hazvrst of the ~c virns.from the transfoctod PA317
cells.
On day 4, culttm medium was harvcstod from the transfected PA317 cells 48
honrs.after the
application of the pLZRNL DNA. The culture medium was filtered through a 0.45
pnn filter
and polybrene was added to a final concentration of 8 pgfml. A stock sohrtion
of polybrene
was prepared by dissolving 0.4 gm hexadimethrine bromide (polybrene; Sigma) in
100 ml
sterile water; the stock solution was stored at 4°C. The culture medium
containing LZRNL
virus (containing polybren~*) was used to infect the 293GP cells as follows.
The culture
medium was removed from the 293GP cells and was replaced with the LZNRL virus
containing culture medium. The virus containing medium was allowed to remain
on the
293GP cells for 16 hours. Following the 16 hour infection period (on day 5),
the medium
was removed from the 293GP cells and was replaced with fresh medium containing
400
pglml 6418 (G1BCOBRL). The medium was changed every 3 days until 6418-
resistant
colonies appeared two weeks later. Care was taken not to disturb the 6418-
resistant colonies
when the medium was changed as 293GP cells attach rather loosely to tissue
culture plates. ~ .
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The 6418-resistant 293 colonies were picked using an automatic pipettor and
transferred directly into 24-well plates (i.e., the colonies were not removed
from the plates
using trypsin). The 6418-resistant 293 colonies (as termed "293GP/LZRNL"
cells) were
screened for the expression of the LacZ gene in order to identify clones which
produce high
titers of pseudotyped LZItNL virus. Clones in 24-well plates were transferred
to 100 mm
tissue culture plates and allowed to grow to confluency. Protein extracts are
prepared from
the confluent plates by washing the cells once with 10 ml PBS (137 mM NaCI,
2.6 mM KCI,
8.1 mM Na2HP0,, 1.5 mM KH~PO,). Two ml of 250 mM Tris-HCI, pH 7.8 was added
and
the cells were scrapped off the plate using a rubber policeman. The cells were
then collected
by centrifugation at room temperature and resuspended in 100 p1 250 mM Tris-
HCI, pH 7.8.
The cells were subjected to four rapid freeze/thaw cycles followed by
centrifugation at room
temperature to remove cell debris. The ~i-galactosidase activity present in
the resulting
protein extracts was determined as follows. Five microliters of protein
extract was mixed
with 500 E.tl ~i-gal buffer (50 mM Tris-HCI, pH 7.5, 100 mM NaCI, 10 mM MgCI~
IS containing 0.75 ONPG (Sigma). The mixhires were incubated at 37°C
until a yellow color
appeared. The reactions were stopped by the addition of 500 ~tl 10 mM FrDTA
and the
optical density of the reactions was. determined at 420 nm.
The 293GP/i.ZRNI. clone which generated the highest amount of [3-galactosidase
activity was then expanded and used subsequently for the production of
pseudotyped LZNRL
virus as follows. Approximately 1 x 106 293GP/LZItNL cells were placed into a
100 mm
tissue culture plate. Twenty-four hours later, the cells were transfected with
20 ~g of
pHCMV-G plasmid DNA using calcium phosphate co-precipitation. Six to eight
hours after
the calcium-DNA precipitate was applied to the cells, the DNA solution was
replaced with
fresh culture medium (lacking G418). Longer transfection times (overnight)
have been found
to result in the detachment of the majority of the 293GP/LZRNL cells from the
plate and are
therefore avoided. The transfected 293GP/L,ZRNL cells produce pseudotyped
LZRNL virus.
The pseudotyped LZRNL virus generated from the transfected 293GPlLZRNL cells
can be collected at least once a day between 24 and 96 hr after transfection.
The highest
virus titer was generated approximately 48 to 72 hr after initial pHCMV-G
transfection.
While syncytium formation became visible about 48 hr after transfection in the
majority of
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the transfected cells, the cells continued to generate pseudotyped virus for
at least an
additional 48 hr as long as the cells remained attached to the tissue culture
plate. The
collected culture medium containing the VSV G-pseudotyped LZRNL virus was
pooled,
filtered through a 0.45 pm filter and stored at -70°C.
The titer of the VSV G-pseudotyped LZRNL virus was then determined as follows.
5
x IOs rat 208F fibroblasts or NIH 3T3 cells were plated in a 100 mm culture
plate. Twenty-
fours hours after plating, the cells were infected with serial dilutions of
the LZRNL virus-
containing culture medium in the presence of 8 pg/ml polybrene. Sixteen hours
after
infection with virus, the medium was replaced with fresh medium containing 400
~g/ml 6418
and selection was continued for 14 days until 6418-resistant colonies became
visible. Viral
titers were typically about 0.5 to 5.0 x 106 colony forming units (cfu~ml. The
titer of the
vines stock could be concentrated to a titer of greater than 10' cfu/ml as
described below.
FILE 3
Concentration of Pseudotyped Retroviral Vectors
The VSV G-pseudotyped LZRhIL virus was concentrated to a high titer by two
cycles
of ultracentrifugation. The frozen culture medium collected as described in
Example 2 which
contained.pseudotyped LZRNL virus was thawed in a 37°C water bath and
was then
transferred to ultraclear centrifuge tubes (14 x 89 mm; Beckman, Palo Alto,
CA) which had
been previously sterilized by exposing the tubes to UV light in a laminar flow
hood overnight.
The virus was sedimented in a SW41 rotor (Beckman) at 50,000 x g (25,000 rpm)
at 4°C for
90 min. The culture medium was then removed from the tubes in a laminar flow
hood and
the tubes were well drained. The virus pellet was resuspended to 0.5 to 1% of
the original
volume of culture medium in either THE (50 mM Tris-HCI, pH 7.8; 130 mM NaCI; 1
mM
EDTA) or 0.1 X Hank's balanced salt solution ( 1 X Hank's balanced salt
solution contains 1.3
mM CaClz, 5 mM KCI, 0.3 mM KH~PO,, 0.5 mM MgCh6Hi), 0.4 mM MgSO,~7Hz0, 138
mM NaCI, 4 mM NaHCO~, 0.3 mM NaHZPO,~HzO; O.1X Hank's is made by mixing 1
parts
1X Hank's with 9 parts PBS]. The resuspended virus pellet was incubated
overnight at 4°C
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without swirling. The virus pellet could be dispersed with gentle pipetting
after the overnight
incubation without significant loss of infectious virus. The titer of the
virus stock was
routinely increase 100- to 300-fold after one round of ultracentrifugation.
The efficiency of
recovery of infectious virus varied between 30 and 100%.
The virus stock was then subjected to low speed centrifugation in a microfuge
for 5
min at 4°C to remove any visible cell debris or aggregated virions that
were not resuspended
under the above conditions (if the virus stock is not to be used for injection
into oocytes or
embryos, this centrifugation step may be omitted).
The virus stock was then subjected to another round of ultracentrifugation to
concentrate the virus stock further. The resuspended virus from the first
round of
centrifugation was pooled and pelleted by a second round of
ultraccntrifugation which was
performed as described above. Viral titers were increased approximately 2000-
fold after the
second round of ultracentrifugation (titers of the pseudotyped LZRNL virus
were typically
greater than or equal to I x I09 cfu/m1 after the second round of
ultracentrifugation).
The titers of the pre- and post-centrifugation fluids were determined by
infection of
208F (1~1IH 3T3 or Mao-T cells can also be employed) followed by selection of
6418-resistant
colonies as described above in Example 2: The concentrated viral stoclc.was
stable.(~e., did
not Lose infectivity) when stored at 4°C for several weeks.
EXAMPLE 4
Preparation of Pseudotyped Retrovirus For Infection of Oocytes and Embryos
The concentrated pseudotyped retrovirus were resuspended in O.1X HBS (2.5 mM
HEPES; pH 7.12, 14 mM NaCI, 75 pM NaZHPO,~Hz0) and 18 p1 aliquots were placed
in 0.5
ml vials (Eppendorf) and stored at -80°C until used. The titer of the
concentrated vector was
determined by diluting lpl of the concentrated virus 10''- or 10'a-fold with
O.1X HBS. The
diluted virus solution was then used to infect 208F and Mac-T cells and viral
titers were
determined as described in Example 2.
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Prior to infection of oocytes or embryos (by microinjection), 1 ~1 of
polybrene (25
ng/~1; the working solution of polybrene was generated by diluting a stock
solution having a
concentration of 1 mg/ml [in sterile H~OJ, in 0.1 HBS, pH 7.12) was mixed with
4 ~l of
concentrated virus to yield a solution containing 10'-10~ cfu/~l and 8 pglml
polybrene. This
solution was loaded into the injection needle (tip having an internal diameter
of approximately
2-4 pm) for injection into the perivitelline space of gametes (pre-maturation
oocytes, matured
oocytes) or one cell stage zygotes (early stage embryo). An Eppendorf
Transjector 5246 was
used for all microinjections.
EXAMPLE 5
Preparation and Microinjection of Gametes and Zygotes
Gametes (pre-maturation and pre-fertilization oocytes) and zygotes (fertilized
oocytes)
were prepared and microinjoctcd with retroviral stocks as described below.
a) Solndons
Tyrodes-Lactate with HEPES (TL-HEPES): 114 mM NaCI, 3.2 mM KCI, 2.0 mM
NaHC03, 0.4 mM NazIiiPO,,~HzO, 10 mM Na-lactate, 2 mM CaCh~2Hz0, 0.5 mM
MgCli 6Hz0, 10 mM HEPES, 100 IU/ml penicillin, 50 pg/ml phenol red, 1 mg/ml
BSA
fraction V, 0.2 mM pyruvate and 25 pg/ml gentamycin.
Maturation Medium: TC-199 medium (GIBCO) containing 10% FCS, 0.2 mM
pyruvate, 5 ug/ml NIH o-LH (NII-~, 25 ~g/ml gentamycin and l pg/ml estradiol-
17(3.
Sperm-Tyrodes-Lactate (Sperm-TL): 100 mM NaCI, 3.2 mM KCI, 25 mM NaHC03,
0.29 mM NazHZP04 HiO, 21.6 mM Na-lactate, 2.1 mM CaCIZ~2H20, 0.4 mM MgCh6H20,
10 mM HEPES, 50 pg/ml phenol red, 6 mg/ml BSA fraction V, 1.0 mM pyruvate and
25
~g/ml gentamycin.
Fertilization Medium: 114 mM NaCI, 3.2 mM KCI, 25 mM NaHC03, 0.4 mM
NazHzP04 HzO, 10 mM Na-lactate, 2 mM CaCIZ~2Hz0, 0.5 mM MgCli 6HZ0, 100 IU/ml
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penicillin, SO ltg/m! phenol red, 6 mg/m) BSA fatty acid free, 0.2 mM pyruvate
and 2S ltg/ml
gentamycin.
PHE: 1 mM hypotaurine, 2 mM penicillamine and 250 l~M epinephrine.
Embryo Incubation + Amino Acids (EIAA): 114 uM NaCI, 32 E,~M KCI, 25 pM
S NaHCO,, 1.6 pglml L(+)-lactate, 10.7 pg/ml L-glutamine, 300 itg/ml BSA fatty
acid free,
0.275 pg/ ml pyruvate, 25 lrg/ml gentaraycin, 10 u1 of 100X MEM amino acids
stock
(M7145, Sigma) per ml and 20 u1 of SOX BME amino acids stock (B6766, Sigma)
per ml.
0.1X HBS: 2.5 mM HEPES (pH 7.12), 14 mM NaCI and 75 1.~M NajHPO,~HiO.
b) Preparation, Injection, Matnration and
Fertilization of Pro-Maturation Oocytes
Oocytes were aspirated from small aatcal follicles on ovaries from dairy
cattle obtained
from a slaughterhouse. Freshly aspirated oocyta at the germinal vesicle (G~
stage, meiosis
arrested, with the cumulus mass attachod were selected (~e., pro-maturaBon
ootrytes). The
oocytes were then washed twice is freshly prepared TL-I~PES and traasfecmd
iato a I00 ~tl
drop of TIrHF.PES for miaroinjectio~a.
Concet~ated retroviral particles (p~r~ as descxi'bed in F~mple 3) were
resuspended in O.1X HBS, mixed with polybnne and loaded i>no t1~ injection
noed)e as
described in Example 4. Approximately 10 p1 of the virus solution was then
injected into the
perivitelline space of pre-maturation oocyies.
Following injection, the pre-maturation oocytes were washed twice in fresh TL-
HEPES
and transferred into maturation medium (10 oocytes in SO p1). The pre-
maturation oocytes
were then incubated in Maturation Medium for 24 hours at 37°C which
permits the oocytes to
mature to the metaphase II stage. The matured oocytes were then washed twice
in Sperm-TL
and 10 oocytes were then transferred into 44 p1 of Fertilization Medium. The
mature oocytes
(10 oocytes/44 p1 Fertilization Medium) were then fertilized by the addition
of 2 p1 of sperm
at a concentration of Z.5 x 10'/m1, 2 p1 of PHE and 2 p1 of heparin
(fertilization mixture).
Sperm was prepared by discontinuous percoll gradient separation of frozen-
thawed semen as
described (Kim et al., Mol. Reprod. Develop., 35:105 [1993]). Briefly, percoll
gradients
were formed by placing 2 ml of each of 90% and 4S% percoll~in a 1 S m1 conical
tube.
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Frozen-thawed semen was layered on top of the gradient and the tubes were
centrifuged for
minutes at 700xg. Motile sperm were collected from the bottom of the tube.
The oocytes were incubated for 16 to 24 hours at 37°C in the
fertilization mixture.
Following fertilization, the cumulus cells were removed by vortexing the cells
(one cell stage
5 zygotes, Pronucleus Stage) for 3 minutes to produce "nude" oocytes. The nude
oocytes were
then washed twice in embryo culture medium (EIAA) and 20 to 25 zygotes were
then
cultured in 50 p1 drop of EIAA (without serum until Day 4 at which time the
zygotes were
placed in EIAA containing 10% serum) until the desired developmental stage was
reached:
approximately 48 hours or Day 2 (Day 0 is the day when the matured oocytes are
co-cultured
10 with sperm) for morula stage (8 cell stage) or Day 6-7 for blastocyst
stage. Embryos at the
morula stage were analyzed for expression of (i-galactosidase as described in
Example 6.
Embryos derived from injected pre-maturation oocytes were also analyzed for ~i-
galactosidase
expression at the 2 cell, 4 cell, and blastocyst stage and all developmental
stages examined
were positive.
c) Preparation, Injection and Fert'dization of
pro-Fert~7ization Oocytes
Pre-maturation oocytes were harvested, washed twice with TL-HEPES as described
above. The oocytes were then cultured in Maturation Medium (10 oocytes per 50
p1
medium) for 16 to 20 hours to produce pre-fertilization oocytes (Metaphase II
Stage). The
pre-fertilization or matured oocytes were then vortexed for 3 minutes to
remove the cumulus
cells to produce nude oocytes. The nude oocytes were washed twice in TL-HEPES
and then
transferred into a 100 ltl drop of TL-HEPES for microinjection. Microinjection
was
conducted as described above.
Following microinjection, the pre-fertilization oocytes were washed twice with
TL-
HEPES and then placed in Maturation Medium until fertilization. Fertilization
was conducted
as described above. Following fertilization, the zygotes were then washed
twice in EIAA and
20 to 25 zygotes were then cultured per 50 ~1 drop of EIAA until the desired
developmental
stage was reached. The embryos were then examined for (3-galactosidase
expression (Ex. 6)
or transferred to recipient cows (Ex. 7).
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d) Preparation and Injection of One-Cell Stage Zygotes
Matured oocytes (Metaphase II stage) were generated as described above. The
matured oocytes were then co-cultured in the presence of sperm for 16 to 20
hours as
described above to generate zygotes at the pronucleus stage. Zygotes at the
pronucleus stage
were vortexed for 3 minutes to remove the cumulus cell layer prior to
microinjection.
Microinjection of retrovirus was conducted as described above. Following
microinjection, the
zygotes were washed four times in EIAA and then placed in an EIAA culture drop
(25
zygotes per 50 p1 drop of EIAA). The zygotes were cultured in EIAA (20 to 25
zygote per
50 p1 drop of EIAA) until the desired developmental stage was reached. The
embryos were
then examined for p-galactosidase expression (Ex. 6) or transferred to
recipient cows (Ex. 7).
F;~XAMPLE 6
Injection of Pseudotyped Retrovirus Into the Perivitelline Space of Maturing
Bovine Oocytes
Results in the Efficient Transfer of Vector Sequences
pocytes and one-cell zygotes which had been micminjected with pseudotyped
LZRNL
virus and cultured ~n vitro were eacamincd for expression of vector sequences
by staining for
~i-galactosidase activity when the embryos had reached the morula stage. (3-
galactosidase
activity was examined as follows. Embryos were washed twice in PBS then fixed
in 0.5%
glutaraldehyde in PBS containing 2mM MgCh for 40 min. at 4°C . The
fixed embryos were
then washed three times with PBS containing 2mM MgCh and then incubated at
37°C
overnight in X-gal solution (20mM K;Fe(CN)b, 20mM K,Fe(CI~6~HZO, 2 mM MgCh and
1
mg/ml X-gal). The presence of a blue precipitate indicates expression of ~3-
galactosidase
activity. The results are shown in Table 1 below.
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TABLE 1
at lnlectlon . ~t~&e at Analysis '/o Pos~tme )?or
yt~ge
; ~i gala~to~~~i~se
: ........ . . ..... . . . .. . .~~xe$~~~~
.. . . . ' . . ,.
.:
. Morula 47 (80/172)'
... . : :: . . .. .
Pre-Fertilization Oocyte
(injected 20-24 hrs after
exposure to Maturation
Medium)
Pronuclei Stage (injected Morula 25 (20/80)
18-20 hrs after exposure
to sperm)
One-Cell Zygote I Morula I 25 (20/80)
' Number positive/number injected.
From the results shown in Table 1, it is clear that infection of pre-
fertilization oocytes
IS .and zygotes using the methods of the present invention results in the
transfer and expression
of retrovirally encoded nucleic acid. While not limiting the present invention
to any
particular theory, it is currently believed that only half of the daughter
cells from an initial
founder cell infected with a retrovirus will contain the provirus because the
retroviral provirus
integrates into post-replication host DNA (Hajihosseini et al., EMBO J.,
12:4969 [1993]).
Therefore, the finding that 47% of the injected pre-fertilization oocytes are
positive for ~-
galactosidase expression suggests that 100% of these injected oocytes were
infected with the
recombinant retrovirus. Therefore, the methods of the present invention
provide an efficiency
of generating transgenic embryos which is superior to existing methods.
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EXAMPLE 7
Generation of Transgenic Cows Containing Integrated
Retroviral Nucleic Acid Sequences
Embryos derived from infected pre-fertilization oocytes and early zygotes were
transferred into recipient cows which were allowed to progress to term as
described below.
a) Treatment of Embryos Derived From Infected Oocytes and Zygotes
Pre-fertilization oocytes (infected about 17 hours after exposure to Matwation
Medium) and early stage zygotes (5 8 cell stage) were prepared and infected as
described in
Example 5 with the exceptions that 1) the VSV-G-pseudotyped virus used was the
LSRNL
virus which was prepared as described for the LZRNL virus in Ex. 2, and 2) at
day 4 post-
fertilization, embryos derived from injected pre-fertilization oocytes and
zygotes were placed
in freshly prepared EIAA medium containing 10% FCS and allowed to develop in
vitro until
transfer into recipient cows. Embryos at Day 7 were transferred into recipient
females which
were prepared as described below.
b) Preparation of Recipient Cows and Embryo Transfer
Recipient cows were synchronized by injecting 100 ~g of gonadotropin-releasing
hormone (GnRH; Sanofi Winthrop Pharmaceutical Inc., New York, NY) (Day 0).
Seven days
later, the recipients were injected with 25 mg of PGF2a (Upjohn Co.,
Kalamazoo, MI).
Thirty to 48 hours after injection of PGF2a, a second injection of 100 ~g of
GnRH was
given. Ovulation occurs about 24-32 hours post injection. Seven days after
ovulation
occurred, embryos derived from infected oocytes and zygotes (Day 7 embryos)
were then
transferred nonsurgically to the uteri the recipient cows. Two embryos were
transferred into
each recipient (it is expected that only one calf will be born from the
transfer of two embryos
into a single recipient).
A total of 20 embryos were transferred into recipients on three separate days.
In the
first transfer 8 embryos derived from infected pre-fertilization oocytes were
transferred into 4
recipients; four calves were born to these recipients and al! four were found
to be positive for
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the presence of vector proviral DNA (i.e., 100% were transgenic). In the
second transfer, 8
embryos derived from post-fertilization zygotes were transferred into 4
recipients; 2 calves
were born to these recipients and one of these animals was found to be
transgenic (in the
second transfer, one pregnancy was lost in the first month and another
pregnancy comprising
twins was lost in the eighth month; neither embryo from the 8 month pregnancy
was
transgenic). In the third transfer 4 embryos derived from infected zygotes
(infected at the 4-8
cell stage) were transferred into 2 recipients; 3 calves were born to these
recipients and none
were transgenic.
The nine calves appeared healthy at birth and continue to appear healthy at
the age of
6 months. Following the birth of offspring derived from the injected oocytes
and zygotes, the
offspring were examined by Southern blot and PCR analyses to determine whether
they
contained the retroviral transgenes and whether they exhibited somatic cell
mosaicism. Skin
tissue and white blood cells (huffy coat) was collected from the calves.
Genomic DNA was
extracted using standard techniques. Briefly, the tissue samples were digested
with 50 pg/ml
1 S proteinase K (GIBCO) at 55°C. The samples were then extracted
sequentially twice with an
equal volume of phenol, once with phenol:chloroform (1:1) and once with
chloroform. The
DNA present W the ~ ~1~~ ~Yer was then precipitated by the addition of 2
volumes of
isopropanol. The DNA was collected by centrifugation and the DNA pellet was
resuspended
in TE buffer (IOmM Tris-Cl, 1 mM EDTA, pH 8.0) and the concentration was
determined
spectrophotometrically. The DNA was then analyzed by Southern blotting and PCR
analysis.
The results are shown in Figures 2 and 3.
Figure 2 shows an autoradiography of a Southern blot of genomic DNA isolated
from
the skin (Fig. 2A) and blood (Fig. 2B) of the six calves derived from either
pre-fertilization
oocytes infected with VSV G-pseudotyped LSItNL virus at about 17 hours after
exposure to
Maturation Medium (calves numbered 17, 18, 20 and 21) or one cell zygotes
infected at about
12 hrs post-fertilization (calves numbered 15 and 16). The calf DNA was
digested with
HindIII which cuts the pLSRNL vector twice to generate a 1.6 kb fragment (Fig.
2C).
HindIII-digested DNA from the blood (lane labelled * 12 derived from a random,
nontransgenic calf), ovary and semen of nontransgenic cows (derived random
adult females
and males) were also included. Lanes labeled "3989 M and F" represent DNA
derived from
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two late term embryos that were born one month prematurely (these calves were
generated
from injected fertilized eggs and both are nontransgenic). Lanes labelled
"LSRNL pDNA"
contain HindIIl-digested pLSRNL plasmid DNA and provide controls for the
quantitation of
the copy number of the integrated proviruses in the offspring (DNA equivalent
to 5, 10 or 25
copies of LSRNL were applied in these lanes).
Approximately 10 pg of the HindIII-digested DNAs were electrophoresed on 0.8%
agarose gels, and blotted onto a nylon membrane. The membrane was hybridized
with a'~P-
labelled probe which hybridizes to the HBsAg gene present in the pLZRNL vector
(Fig. 2C).
The HBsAG probe was generated by PCR amplification of pLSRNL plasmid DNA using
the
upstream primer S-1 (5'-GGCTATCGCTGGATGTGTCT-3'; [SEQ ID N0:3]) and the
downstream primer S-3 (5'-ACTGAACAAATGGCACTAGT-3'; (SEQ ID N0:4]). The PCR-
generated probe (334 bp) was labeled using a Rediprime kit (Amersham,
Arlington Heights,
IL) according to the manufacturer's instructions. The autoradiographs shown in
Fig. 2 were
generated by exposure of the blots to X-ray film for 3 weeks at -80°C.
The results shown in Figure 2 demonstrates that calves 16, 17, 18, 20 and 21
contained retroviral vector DNA in~both the skin (Fig. 2A) and blood (Fig.
2B). As blood
cells (buS'y coat) are derived: from the mesoderm and skin cells are derived
from the
ectoderm, these results show that the transgenic animals do not display
somatic cell
mosaicism. Southern blotting analysis has shown that the majority (i.e.; 7/9)
of the
transgenic calves contain a single copy of the proviral sequence; a few (i.
e., 2/9) animals
appear to contain two copies of the integrated proviral sequence. These
results further
demonstrate that retroviral infection of both pre-fertilization oocytes and
early stage rygotes
was successful in integrating the viral sequences into the genome of the
resulting transgenic
animals.
In order to confirm the presence of integrated retroviral sequences in the
genome of
the transgenic animals' somatic cells, PCR analysis (Fig. 3) was performed
using genomic
DNA isolated from the five transgenic calves which were determined by Southern
blot
analysis to be transgenic for the retroviral sequences. Figure 3 shows the
results of the PCR
analysis following amplification of two different regions (i.e., the neo gene
and the HBSAg
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gene) of the LZRNL retroviral genome which was injected into the oocytes.
Genomic DNA
from the skin and blood of each of the five transgenic calves was amplified
using the
upstream and downstream primers (SEQ ID NOS:1 and 2 and NOS:3 and 4; described
supra)
for the neo (Fig. 3A) and HBsAg (Fig. 3B) genes, respectively. The PCRs were
conducted
using the following thermocycling conditions: 94°C ( 4 min);
(94°C [2 min]; 50°C [2 min];
72°C (2 min]) ~ ~,; 72°C (10 min). Amplification yielded the
expected size of amplified
sequence with the neo (349 bp) and HBsAg (334 bp) primers in both the blood
and skin of
each of the five transgenic calves. Genomic DNA isolated from the blood of non-
transgenic
calves as well as from semen and ovary of non-transgenic cattle were used as
negative
controls in the PCRs. pLSRNL DNA was used as the positive control.
These data demonstratc that the infection of pre-fertilization oocytes results
in the
efficient transfer of retroviral vector DNA (100% or 4 transgenic calves/4
calves born from
embryos derived from infected pre-fertilization oocytes). In addition to
providing a means for
effciently generating transgenic animals. The methods of the present invention
providc a
IS means for generating transgenic animals which do not display somatic cell
mosaicism.
Further, these methods permit the production of transgenic animals which
contain a single
copy of the transgcne:
In order to confirm germ line transmission of the integrated viral sequences,
the
transgenic offspring are bred with non-transgenic cattle and the presence of
the viral
sequences (i. e., the transgene) determined using Southern blot analysis or
PCR amplification
as described above. Animals which are heterozygous or homozygous for the
transgene are
produced using methods well known to the art (e.g., interbreeding of animals
heterozygous for
the transgene).
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EXAMPLE 8
Detection of the HBsAg Transgene in the Sperm of Transgenic Bulls
Semen was collected from two transgenic bulls, # 16 and #21. DNA was isolated
from
the semen samples using methods known in the art. PCR was then conducted on
the sample
DNA, using the Primers S 1 and S3, as described below. The PCR results
indicated that both
bulls had the transgene in their sperm.
These results demonstrated that transgenic bulls produced either by
perivitelline space
injection of an unfertilized oocyte (#21) or by perivitelline space injection
of a fertilized
zygote (#16) have the transgene present in their sperm, and are thus capable
of passing the
transgene on to their offspring. Indeed, as described in Example 9 below, bull
# 16 has
produced two live transgenic offspring.
Confirmation of Transgene Stability
To confirm the transgene staMlity of a transgenic bull produced as described
in the
previous Examples, and to determine whether the gene was behaving in a normal
Mendelian
fashion, a transgenic bull (designated as #16) produced through one-cell
zygotic injection, was
naturally mated with a non-transgenic cow. This mating resulted in the
production of twin
calves, one female (designates( as #42) and one male (designated as #43).
Blood and skin
samples were taken from each of the calves, and their DNA was isolated using
methods
known in the art (See e.g., Hogan et al." Manipulating the Mouse Embryo: A
Laboratory
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY [1986]). PCR was
performed on these DNA samples, using the methods described in Example 7,
above. Two
sets of primers were used to analyze both the blood and skin samples. One set
of primers
("Neol" and "Neo2") was used to detect the neomycin resistance gene in the
LSRNL vector.
The location and description of these primers is shown in Figure 3A. The
second set of
primers (S1 and S3) were used to detect a portion of the Hepatitis B surface
antigen (HBsAg)
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gene in the LSRNL vector. The location and description of these primers is
shown in Figure
3B. Both the skin and blood samples from these calves were positive for the
LSRNL
transgene, indicating that the gene can be transmitted from an original
transgenic bull created
by one-cell zygotic injection, to his offspring. Figure 4 shows the results of
PCR screening of
skin samples from these calves. In this gel, the control animal is indicated
as #12, while the
offspring are indicated as #42 and #43, as described above. Lane one contains
DNA size
standards, and lanes 2-4 contain the DNA samples analyzed using the neo PCR
primers, while
lanes 5-7 contain the same DNA samples analyzed using the HBsAg PCR primers.
The
correct size for the neo band is 349 base pairs, while the correct size for
the HBsAg band is
l0 slightly smaller, at 334 base pairs.
These data demonstrate that transgenic animals can be successfully created by
perivitelline space injection of a one-cell zygote with a pseudotyped
replication-defective
retrovirus. In addition, these data also demonstrate that the incorporated
transgene is passed
on the offspring of the transgenic animal.
EXAMPLE IO
Production of .HBsAg in Nfillc of Transgenic Cows
In this experiment, female founder transgenic heifers (designated as #17 and
#18),
were artificially induced to lactate at 22 months of age, using a protocol
described by
Dommer (Dommer, "Artificial Induction of Lactation in Nulligravida Heifers,'
MS Thesis,
University of Wisconsin, Madison, 1996; and Dommer and Bremel, J. Dairy Sci.,
79 (Suppl.
1):146 [1996]). After induction of lactation and the subsequent secretion of
milk, the milk
was assayed for the presence of HBsAg.
Milk samples were collected from # 17 and # 18, and five control heifers that
had also
bean artificially induced to lactate using the same protocol and at the same
time as #17 and
#18. Whole milk samples were analyzed using the AUSZYME~ Monoclonal Antibody
Assay
(Abbott), for the detection of HBsAg.
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The milk samples collected from #I? and #18 tested positive for HBsAg. The
milk
samples from the five control heifers were all negative for the antigen. The
estimated level of
Hgspg production, based on the AUSZYME~ kit and its positive control, as well
as a
dilution series of the milk samples, was found to be 200 ng HBsAg/ml milk, for
#17, and 700
ng HBsAg/ml milk, for #18.
'these data clearly demonstrate that transgenic animals produced by
perivitelline space
injection of an unfertilized oocyte are capable of producing substantial
levels of foreign
proteins in their milk. In addition, these experiments also demonstrate the
utility of using the
MoMLV LTR as a promoter for driving the production of foreign proteins in the
milk of
transgenic cattle, as this promoter was shown to be capable of causing the
production of
HBsAg in the milk of these transgenic animals. In addition, the expression of
an exonless
~~~ (i_ e., with the LTR of the present invention) indicates that the LTR is
also
functioning as an enhancer. Furthermore, these data clearly show that the
expression system
of the present invention is capable of preferential mannmary exPr~on, even
though the
MoMLV LTR is not a "mannmary-spe~o~ Promoter.
EXAN~LE 11
Presence HBsAg in the Serum and Urine
of Transgenic Cattle
In addition to milk samples, blood and urine samples were also collected from
the two
female founder transgenic heifers #17 and #18. The serum was separated from
the whole
blood using methods known in the art (i. e., centrifugation). The urine and
serum samples
were assayed for the presence of HBsAg using the AUSZYME~ system, as per the
kit
manufacturer's instructions. The urine and serum of #17 and #18 all tested
positive for the
presence of HBsAg, while the urine and serum samples from the control animals
all tested
negative. Based on this test system, the estimated level of HBsAg production
for #17 was
2.58 ng HBsAglml of serum, and 0.64 ng HBsAg/ml of urine. For #18, the values
were 0.64
ng HBsAg/ml of 'serum, and 0.97 ng HBsAg/ml of urine.
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These data demonstrate that transgenic animals produced by perivitelline space
injection of an unfertilized oocyte are capable of producing substantial
levels of foreign
proteins in their serum and urine. These data further demonstrate the utility
of using the
MoMLV LTR as a promoter for driving the constitutive production of foreign
proteins in
transgenic cattle, as this promoter was shown in these experiments to cause
the production of
HBsAg in milk, serum, and urine of transgenic cattle. As used herein, the term
"constitutive"
refers to a relatively low level of expression throughout the animal's body.
In contrast, the
term "preferentially expressed" indicates that a relatively high level of
expression is achieved
in certain tissues or body fluids, as compared to other tissues and fluids.
For example, in
preferred embodiments of the present invention, foreign proteins of interest
are preferentially
expressed is such fluids as mills.
It is contemplated that such a promoter could be used to control expression of
proteins
that would prevent disease and/or infection in the transgenic animals and
their ofl'spring,~ or be
of use in the production of a consistent level of protein expression in a
number of different
I S tissues ~ and body fluids.
From.the above it is clear that the invention provides improved methods and
compositions for the production of transgenic non-human animals. The methods
of the
present invention provide for the production of transgenic non-human animals
with improved
efficiency and a reduced incidence of generating animals which are mosaic for
the presence of
the transgene.
Various modifications and variations of the described method and
system of the invention will be apparent to those skilled in the art without
departing from the
scope and spirit of the invention. Although the invention has been described
in connection
with specific preferred embodiments, it should be understood that the
invention as claimed
should not be unduly limited to such specific embodiments. Indeed, various
modifications of
the described modes for carrying out the invention which are obvious to those
skilled in
molecular biology, transgenic animals, or related fields are intended to be
within the scope of
the following claims.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Bremel, Robert D.
Char, Anthony W.S.
Burns , Jane C .
Bleck, Gregory T.
(ii) TITLE OF INVENTION: Methods For Creating Transgenic Animals
(iii) NUMBER OF SEQUENCES: 10
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Medlen & Carroll, LLP
(B) STREET: 220 Montgomery Street, Suite 2200
(C) CITY: San Francisco
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(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) CpMpUTBR: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Pateritla Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUN~ER: US
(8) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGBNT I~R~TION:
(A) N~; Irigolia, Diane 8.
(B) xEaISTRATION NoMSER: 40,0~~
(c) ~8/r rroMSER: wARF-o21e4
TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (415) 705-8410
(B) TBLEFAX: (415) 397-8338
(2) INFORMATION FOR SEQ ID NO: l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: l:
GCATTGCATC AGCCATGATG
-62-
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(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
GATGGATTGC ACGCAGGTTC
(2) INFORMATION,FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDfiDNESS: single
(D) TOPOhOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA"
(xi) 88QUSNCE DESCRIPTION: SEQ ID N0:3:
GGCTATCGCT GGATGTGTCT 20
(2) INFORMATION FOR 88Q ID N0:4:
(i) sEQvENCS csARAC~rERISTICS:
(A) LENGTH: 20 base pairs
(8) TYPE: nucleic~acid
(C) STRANDBDNESS: single
(D) TOPOhOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
- (A) DESCRIPTION: /dese = "DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
ACTGAACAAA TGGCACTACiT 2 0
(2) INFORMATION FOR SEQ ID NO: S:
(i) SEQUENCE CHARACTERISTICS:
(AI LENGTH: 1590 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA"
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(ix) FEATURE:
(A) NAME/KEY: CDS
(8) LOCATION: 1..1587
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: S:
ATG GAT CTC TTT CCC ATT TTG GTC GTG GTG CTC ATG ACA GAT 48
ACT GTC
Met Asp Leu Phe Pro Ile Leu Val Val Val Leu Met Thr Asp
Thr Val
1 5 10 15
~A ~ pp,G TTT CAA ATT GTC TTC CCG GAT CAG AAT GAA CTG 96
GAG TGG
Leu Gly Lys Phe Gln Ile Val Phe Pro Asp Gln Asn Glu Leu
Glu Trp
20 25 30
AGA CCA GTT GTG GGT GAC TCT CGG CAT TGC CCA CAG TCA TCA 144
GAA ATG
Arg Pro Val Val Gly Asp Ser Arg His Cys Pro Gln Ser Ser
Glu Met
35 40 45
CAA TTC GAT GGA AGC AGA TCC CAG ACC ATA CTG ACT GGG AAA 192
GCT CCC
Gln Phe Asp Gly Ser Arg Ser Gln Thr Ile Leu Thr Gly Lys
Ala Pra
50 55 60
GTG GGG ATC ACG CCC TCT.~ ~ GAT GGA TTT ATC TGC CAT GCC 240
GCA
Val Gly Ile Thr Pro Ser Lys Ser Asp Gly Phe Ile Cys His
Ala Ala
65 70 75 80
AAA TGG GTG ACA ACA TGT GAT TTC AGG TGG TAT GGG CCG AAA 288
TAC ATC
Lys Trp Val Thr Thr Cys Asp Phe Arg Trp Tyr Gly Pro Lys
Tyr Ile
85 90 95
ACT CAT TCA ATA CAT CAT CTG AGA CCG ACA ACA TCA GAC TGT 336
GAG ACA
Thr His Ser Ile His His Leu Arg Pro Thr Thr Ser Asp ~
Glu Thr
100 105
GCT CTC CAA AGG~TAT AAA GAT GGG AGC TTA ATC AAT CTT GGA 384
TTC CCC
Ala.Leu Gln Arg Tyr LYH ~P iaYO Ser Leu Ile Asn Leu Gly
Phe Pro
115 125
CCA GAA TCC TGC GGT TAT GCA ACA GTC ACA GAT TCT GAG GCA 432
ATG TTG
Pro Glu Ser Cys Gly Tyr Als Thr Val Thr Asp Ser'Glu Ala
Met Leu
130 140
GTC CAA GTG ACT CCC CAC CAC GTT GGG GTG GAT GAT TAT AGA 480
GGT CAC
Val Gln Val Thr Pro His His Val Gly Val Asp Asp TYr Arg
Gly 160
145 150 155
TGG ATC GAC CCA CTA TTT CCA GGA GGA GAA TGC TCC ACC AAT 528
TTT TGT
Trp Ile Asp Pro Leu Phe Pro Gly Gly Glu Cys Ser Thr Asn
Phe Cys
165 170 175
GAT ACA GTC CAC AAT TCA TCG GTG TGG ATC CCC AAG AGT CAA 576
AAG ACT
Asp Thr Val His Asn Ser Ser Val Trp Ile Pro Lys Ser Gln
Lys Thr
180 185 190
GAC ATC TGT GCC CAG TCT TTC AAA AAT ATC AAG ATG ACC GCA 624
TCT TAC
Asp Ile Cys Ala Gln Ser Phe Lys Asn Ile Lys Met Thr Ala
Ser Tyr
195 200 205
CCC TCA GAA GGA GCA TTG GTG AGT GAC AGA TTT GCC TTC CAC 672
AGT GCA
Pro Ser Glu Gly Ala Leu Val Ser Asp Arg Phe Ala Phe His
Ser Ala
210 215 220
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TAT CAT CCA AAT ATG CCG GGG TCA ACT GTT TGC ATA ATG GAC 720
TTT TGC
Tyr His Pro Asn Met Pro Gly Sex Thr Val Cys Ile Met Asp
Phe Cys
225 230 235 240
GAA CAA AAG GGG TTG AGA TTC ACA AAT GGA GAG TGG ATG GGT 768
CTC AAT
Glu Gln Lys Gly Leu Arg Phe Thr Asn Gly Glu Trp Met Gly
Leu Asn
245 250 255
GTG GAG CAA TCC ATC CGA GAG AAG AAG ATA AGT GCC ATC TTC 816
CCA AAT
Val Glu Gln Ser Ile Arg Glu Lys Lys Ile Ser Ala Ile Phe
Pro Asn
260 265 270
TGT GTT GCA GGG ACT GAA ATC CGA GCC ACA CTA GAA TCA GAA 864
GGG GCA
Cys Val Ala Gly Thr Glu Ile Arg Ala Thr Leu Glu Ser Glu
Gly Ala
275 280 285
AGA ACT TTG ACG TGG GAG ACT CAA AGA ATG CTA GAT TAC TCT 912
TTG TGT
Arg Thr Leu Thr Trp Glu Thr Gln Arg Met Leu Asp Tyr Ser
Leu Cys
290 295 300
CAG AAC ACC TGG GAC AAA GTT TCC AGG AAA GAA CCT CTC AGT 960
CCG CTT
Gln Asn Thr Trp Asp Lys Val Ser Arg Lys Glu Pro Leu Ser
Pro Leu
310 315 320
305
GAC TTG AGC TAT CTG TCA CCA AGG GCT CCA GGG AAA GGC ATG 1008
GCC TAT
Asp Leu Ser Tyr Leu Ser Pro Arg Ala Pro Gly Lys Gly Met
Ala Tyr
3Z5 330 335
C GTC ATA AAC GGA ACC CTG CAT TCG GCT CAT GCT AAA TAC 1056
ATT AGA
AC
Thr Val Ile Asn Gly Thr Leu His Ser Ala His Ala Lys Tyr
Ile Arg
340 345 350
ACC TGG ATT GAT TAT GGA GAA ATG AAG GAA ATT AAA GGT GGA ll04
CGT GGA
Thr Trp Ile Asp Tyr Gly Glu Met Lys Glu Ile Lys Gly Gly
Arg Gly
355 ~ . .. 360 . . X65
GAA TAT TCC AAG GCT CCT GAG CTC CTC TGG TCC CAG TGG TTC 1152
GAT TTT
Glu Tyr Ser Lys Ala Pro Glu Leu Leu Trp Ser Gln Trp Phe
Asp Phe
370 375 380
CCG TTC AAA ATT GGA CCG AAT GGA CTC CTG CAC ACA GGG AAA 1200
ACC
GGA
Gly Pro Phe Lys Ile Gly Pro Asn Gly Leu Leu His Thr Gly
Lys Thr
385 390 395 400
TTT AAA TTC CCT CTT TAT TTG ATC GGA GCA GGC ATA ATT GAC 1248
GAA GAT
Phe~Lys Phe Pro Leu Tyr Leu Ile Gly Ala Gly Ile Ile Asp
Glu Asp
405 410 415
CTG CAT GAA CTA GAT GAG GCT GCT CCC ATT GAT CAC CCA CAA ATG CCT 1296
Leu His Glu Leu Asp Glu Ala Ala Pro Ile Asp His Pro Gln Met Pro
420 425 . 430
GAC GCG AAA AGC GTT CTT CCA GAA GAT GAA GAG ATA TTC TTC GGA GAC 1344
Asp Ala Lys Ser Val Leu Pro Glu Asp Glu Glu Ile Phe Phe Gly Asp
435 440 445
ACA GGT GTA TCC AAA AAC CCT ATC GAG TTG ATT CAA GGA TGG TTC TCA 1392
Thr Gly Val Ser Lys Asn Pro Ile Glu Leu Ile Gln Gly Trp Phe Ser
450 455 460
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AAT TGG AGA GAG AGT GTA ATG GCA ATA GTT CTA CTC ATC 1440
GTC GGA ATT
Asn Trp Arg Glu Ser Val Met Ala Ile Val Leu Leu Ile
Val Gly Ile
465 470 475. 480
GTT GTG ACA TTT CTG GCG ATC AAG ACG CTT AAT TGT CTC 1488
GTC CGG GTG
Val Val Thr Phe Leu Ala Ile Lys Thr Leu Asn Cys Leu
Val Arg Val
485 490 495
TGG AGA CCC AGA AAG AAA AGA ATC GTC GTA GAT GTT GAA 1536
AGA CAA GAA
Trp Arg Pro Arg Lys Lys Arg Ile Val Val Asp Val Glu
Arg Gln Glu
500 505 510
TCC CGA CTA AAC CAT TTT GAG ATG AGA GAA TAT GTT AAG 1584
GGC TTT CCT
Ser Arg Leu Asn His Phe Glu Met Arg Glu Tyr Val Lys
Gly Phe Pro
515 520 525
AGA TAA 1590
~'9
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 529 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECOLB TYPE: protein
(xi) SEQUENCE DESCRIPTION: S8Q ID N0:6:
Met Asp Leu Pro Ile Leu Thr Asp Thr
Phe Leu Val Met Val
Val Val
1 5 10. ~ 15
Leu Gly Lys Gln Ile Phe Pro aln Glu Leu Glu
~Phe Val Asp Asn Trp
a0 25 30
Arg Pro Val Gly Asp Arg His Pro Ser ser Glu
Val~ Ser Cys Gln Met
35 40 45
Gln Phe Asp Ser Arg Gln Thr Leu Gly Lys Ala
Gly Ser Ile Thr Pro
50 55 60
Val Gly Ile Pro Ser Ser Asp Phe Cys His Ala
Thr Lys Gly Ile Ala
65 70 . 75 80
Lys Trp Val Thr Cys _Phe Arg Tyr Pro Lys Tyr
Thr Asp Trp Gly Ile
85 90 95
Thr His Ser His His Arg Pra Thr Asp Cys Glu
Ile Leu Thr Ser Thr
100 105 110
Ala Leu Gln Tyr Lys Gly Ser Ile Leu Gly Phe
Arg Asp Leu Asn Pro
115 120 125
Pro Glu Ser Gly Tyr Thr Val Asp Glu Ala Met
Cys Ala Thr Ser Leu
130 135 140
Val Gln Val Pro His Val Gly Asp Tyr Arg Gly
Thr His Val Asp His
145 150 155 160
Trp Ile Asp Leu Phe Gly Gly Cys Thr Asn Phe
Pro Pro Glu Ser Cys
165 170 175
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Asp Thr Val His Asn Ser Ser Val Trp Ile Pro Lys Ser Gln Lys Thr
180 185 190
Asp Ile Cys Ala Gln Ser Phe Lys Asn Ile Lys Met Thr Ala Ser Tyr
195 200 205
Pro Ser Glu Gly Ala Leu Val Ser Asp Arg Phe Ala Phe His Ser Ala
210 215 220
Tyr His Pro Asn Met Pro Gly Ser Thr Val Cys Ile Met Asp Phe Cys
230 235 240
225
Glu Gln Lys Gly Leu Arg Phe Thr Asn Gly Glu Trp Met Gly Leu Asn
245 250 255
Val Glu Gln Ser Ile Arg Glu.Lys Lys Ile Ser Ala Ile_Phe Pro Asn .
260 265 270
Cys Val Ala Gly Thr Glu Ile Arg Ala Thr Leu Glu Ser Glu Gly Ala
275 280 285
Arg Thr Leu Thr Trp Glu Thr Gln Arg Met Leu Asp Tyr Ser Leu Cys
290 295 300
Gln Asn Thr Trp Asp Lys Val Ser Arg'Lys Glu Pro Leu Ser Pro Leu
305 310 315 320
Asp Leu Ser Tyr Leu Ser Pro Arg Ala Pro Gly Lys Gly Met Ala Tyr
325 330 335
Thr Val Ile Asn Gly Thr Leu His Ser Ala His Ala Lys Tyr Ile Arg
340 345 350
Thr Trp Ile Asp~Tyr Gly Glu Met Lys Glu~Ile LYs 365 Gly Arg Gly
355 360
Glu Tyr Ser Lys Ala Pro Glu Leu Leu Trp Ser Gln Trp Phe Asp Phe
370 375 380
Gly Pro Phe Lys Ile Gly Pro Asn Gly Leu Leu His Thr Gly Lys Thr
385 390 395 400
Phe Lys Phe Pro Leu Tyr Leu Ile Gly Ala Gly Ile Ile Asp Glu Asp
405 . 410 415
Leu His Glu Leu Asp Glu Ala Ala Pro Ile Asp His Pro Gln Met Pro
420 425 430
Asp Ala Lys Ser Val Leu Pro Glu Asp Glu Glu Ile Phe Phe Gly Asp
435 440 445
Thr Gly Val Ser Lys Asn Pro Ile Glu Leu Ile Gln Gly Trp Phe Ser
450 455 460
Asn Trp Arg Glu Ser Val Met Ala Ile Val Gly Ile Val Leu Leu Ile
465 470 475 480
Val Val Thr Phe Leu Ala Ile Lys Thr Val Arg Val Leu Asn Cys Leu
485 490 495
Trp Arg Pro Arg Lys Lys Arg Ile Val Arg Gln Glu Val Asp Val Glu
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500 505 510
Ser Arg Leu Asn His Phe Glu Met Arg Gly Phe Pro Glu Tyr Val Lys
515 520 525
Arg
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1590 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA"
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1587
(xi) SEQDENCB DESCRIPTION: SEQ ID N0:7:
ATG GAT CTC TTT CCC ATT TTG GTC GTG GTG CTC ATG ACA 48
GAT ACT GTC
u Phe Pro Ile Leu Val Val Val Leu Met Thr Asp Thr Val
L
e
Met Asp
15
1 5
TTA GGG AAG TTT CAA ATT GTC TTC CCG GAT CAG AAT GAA 96
CTG GAG TGG
Leu Gly Lys Phe Gln Ile Val Phe Pro Asp Gln Asn Glu
Leu Glu Trp
25 30
AGA CCA GTT GTG GGT GAC TCT CGG CAT TGC CCA CAG TCA 144
TCA GAA ATG
Pro Val Val Gly Asp 8er Arg His Cys Pro Gln Ser Ser
Glu Met
Arg
40 ~ 45
35
CAA TTC GAT GGA AGC AGA TCC CAG ACC ATA CTG ACT GGG I92
AAA GCT CGC
Gln Phe Asp Gly Ser Arg Ser Gln Thr Ile Leu Thr Gly
Lys Ala Pro
50 55 60
G ATC ACG CCC TCT AAA TCA GAT GGA TTT ATC TGC CAT GCC 240
GCA
~
GG
GTG
Val Gly Ile Thr Pro Ser Lys Ser Asp Gly Phe Ile Cys
His Ala Ala
65 70 75 80
AAA TGG GTG ACA ACA TGT GAT TTC AGG TGG TAT GGG CCG 288
AAA TAC ATC
Lys Trp Val Thr Thr Cys Asp Phe Arg Trp Tyr Gly Pro
Lys 95 Ile
85 90
ACT CAT TCA ATA CAT CAT CTG AGA CCG ACA ACA TCA GAC 336
TGT GAG ACA
Thr His Ser Ile His His Leu Arg Pro Thr Thr Ser Asp
Cys Glu Thr
100 105 110
GCT CTC CAA AGG TAT AAA GAT GGG AGC TTA ATC AAT CTT 384
GGA TTC CCC
Ala Leu Gln Arg Tyr Lys Asp Gly Ser Leu Ile Asn Leu
Gly Phe Pro
115 120 125
CCA GAA TCC TGC GGT TAT GCA ACA GTC ACA GAT TCT GAG 432
GCA ATG TTG
Pro Glu Ser Cys Gly Tyr Ala Thr Val Thr Asp Ser G1u
Ala Met Leu
130 135 140
GTC CAA GTG ACT CCC CAC CAC GTT GGG GTG GAT GAT TAT 480
AGA GGT CAC
Val Gln Val Thr Pro His His Val Gly Val Asp Asp Tyr
Arg Gly His
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145 150 155 160
TGG ATC GAC CCA CTA.TTT CCA GGA GGA GAA TGC TCC ACC 528
AAT TTT TGT
Trp Ile Asp Pro Leu Phe Pro Gly Gly Glu Cys Ser Thr
Asn Phe Cys
165 170 175
GAT ACA GTC CAC AAT TCA TCG GTG TGG ATC CCC AAG AGT 576
CAA AAG ACT
Asp Thr Val His Asn Ser Ser Val Trp Ile Pro Lys Ser
Gln Lys Thr
180 185 190
GAC ATC TGT GCC CAG TCT TTC AAA AAT ATC AAG ATG ACC 624
GCA TCT TAC
Asp Ile Cys Ala Gln Ser Phe Lys Asn Ile Lys Met Thr
Ala Ser Tyr
195 200 205
CCC TCA GAA GGA GCA TTG GTG AGT GAC AGA TTT GCC TTC 672
CAC AGT GCA
Pro Ser Glu Gly Ala Leu Val Ser Asp Arg Phe Ala Phe
His Ser Ala
210 215 220
TAT CAT CCA AAT ATG CCG GGG TCA ACT GTT TGC ATA ATG 720
GAC TTT TGC
Tyr His Pro Asn Met Pro Gly Ser Thr Val Cys Ile Met
Asp Phe Cys
225 230 235 240
~ CpA AAG GGG TTG AGA TTC ACA AAT GGA GAG TGG ATG GGT 768
CTC AAT
Glu Gln Lys Gly Leu Arg Phe Thr Asn Gly Glu Trp Met
Gly Leu Asn
245 250 255
GTG GAG CAA TCC ATC CGA GAG AAG AAG ATA AGT GCC ATC 816
TTC CCA AAT
Val Glu Gln Ber Ile Arg Glu Lys Lys Ile Ser Ala Ile
Phe Pro Asn
260 265 270
TGT GTT GCA GGG ACT GAA ATC CGA GCC ACA CTA GAA TCA 864
GAA GGG GCA
Cys Val Ala Gly Thr Glu Ile Arg Ala Thr Leu Glu Ser
Glu Gly Ala
a~5 aao ass
AGA ACT TTG ACG TGG GAG ACT CAA AGA ATG CTA GAT TAC 912
TCT TTG TGT
Arg Thr Leu Thr Trp Glu Thr Gln Arg Met Leu Asp Tyr
Ser Leu Cys
290 295 300
CAG AAC ACC TGG GAC AAA GTT TCC AGG AAA GAA CCT CTC 960
AGT CCG CTT
Gln Asa Thr Trp Asp Lys Val Ser Arg Lys Glu Pro Leu
Ser Pro Leu
305 310 315 320
GAC TTG AGC TAT CTG TCA CCA AGG GCT CCA GGG AAA GGC 1008
ATG GCC TAT
Asp Leu Ser Tyr Leu Ser Ero Arg Ala Pro Gly Lys Gly
Met Ala Tyr
325 330 335
ACC GTC ATA AAC GGA ACC CTG CAT TCG GCT CAT GCT AAA 1056
TAC ATT AGA
Thr Val Ile Asn Gly Thr Leu His Ser Ala His Ala Lys
Tyr Ile Arg
340 345 350
ACC TGG ATT GAT TAT GGA GAA ATG AAG GAA ATT AAA GGT ll04
GGA CGT GGA
Thr Trp Ile Asp Tyr Gly Glu Met Lys Glu Ile Lys Gly
Gly Arg Gly
355 360 365
GAA TAT TCC AAG GCT CCT GAG CTC CTC TGG TCC CAG TGG 1152
TTC GAT TTT
~Glu Tyr Ser Lys Ala Pro Glu Leu Leu Trp Ser Gln Trp
Phe Asp Phe
370 375 380
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GGA CCG AAA GGA CCG GGA CTC CAC ACA GGG AAA ACC
TTC ATT AAT CTG 1200
Gly Pro Lys Gly Pro Gly Leu His Thr Gly Lys Thr
Phe Ile Asn Leu
385
390 395 400
TTT AAA CCT TAT TTG GGA GCA ATA ATT GAC GAA GAT
TTC CTT ATC GGC 1248
Phe Lys Pro Tyr Leu Gly Ala Ile Ile Asp Glu Asp
Phe Leu Ile Gly
405 410 415
CTG CAT GAA CTA GAT GAG GCT GCT CCC ATT GAT CAC CCA 1296
CAA ATG CCT
Leu His Glu Leu Asp Glu Ala Ala Pro Ile Asp His Pro
Gln Met Pro
420 425 430
GAC GCG AAA AGC GTT CTT CCA GAA GAT GAA GAG ATA TTC 1344
TTC GGA GAC
Asp Ala Lys Ser Val Leu Pro Glu Asp Glu Glu Ile Phe
Phe Gly Asp
435 440 445
ACA GGT GTA TCC AAA AAC CCT ATC GAG TTG ATT CAA GGA 1392
TGG TTC TCA
Thr Gljr Val Ser Lys Asn Pro Ile Glu Leu Ile Gln Gly
Trp Phe Ser
450 455 460
G AGA GAG AGT GTA ATG GCA ATA GTC GGA ATT GTT CTA CTC 1440
ATC
AAT TG
Asn Trp Arg Glu Ser Val Met Ala Ile Val Gly Ile Val
Leu Leu Ile
470 475 480
465
TT GTG ACA TTT CTG GCG ATC AAG ACG GTC CGG GTG CTT 1488
AAT TGT CTC
G
Val Val Thr Phe Leu Ala Ile Lys Thr Val Arg Val Leu
Asn Cys Leu
485 490 495
G AGA CCC AGA AAG AAA AGA ATC GTC-AGA CAA GAA GTA GAT 1536
GTT GAA
TG
Trp Arg Pro Arg Lys Lys Arg Ile Val Arg Gln Glu Val
Asp Val Glu
500 505 510
TCC CGA CTA AAC CAT TTT GAG ATG _AGA.~ ~ ~T ~ TAT'GTT 1584
AAG
Ser Arg Leu Asn His Phe Glu Met Arg Gly Phe Pro Glu
Tyr Val Lys
' 515 520 525.
1590
~'g
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 529 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
Met Asp Leu Phe Pro Ile Leu Val Val Val Leu Met Thr Asp Thr Val
1 5 10 15
Leu Gly Lys Phe Gln Ile Val Phe Pro Asp Gln Asn Glu Leu Glu Trp
20 25 30
Arg Pro Val Val Gly Asp Ser Arg His Cys Pro Gln Ser Ser Glu Met
35 40 45
Gln Phe Asp Gly Ser Arg Ser~Gln Thr Ile Leu Thr Gly Lys Ala Pro
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SO 55 60
Val Gly Ile Thr Pro Ser Lys Ser Asp Gly Phe Ile Cys His Ala Ala
65 70 75 80
Lys Trp Val Thr Thr Cys Asp Phe Arg Trp Tyr Gly Pro Lys Tyr Ile
g5 90 95
Thr His Ser Ile His His Leu Arg Pro Thr Thr Ser Asp Cys Glu Thr
100 105 110
Ala Leu Gln Arg Tyr Lys Asp Gly Ser Leu Ile Asn Leu Gly Phe Pro
115 120 125
Pro Glu Ser Cys Gly Tyr Ala Thr Val Thr Asp Ser Glu Ala Met Leu
130 135 140
Val Gln Val Thr Pro His His Val Gly Val Asp Asp Tyr Arg Gly His
145 150 155 160
Trp Ile Asp Pro Leu Phe Pro Gly Gly Glu Cys Ser Thr Asn Phe Cys
165 170 175
Asp Thr Val His Asn Ser Ser Val Trp Ile.Pro Lys Ser Gln Lys Thr
180 185 190
Asp Ile Cys Ala Gln Ser Phe Lys Asn Ile Lys Met Thr Ala Ser Tyr
195 200 205
Pro Ser Glu Gly Ala Leu Val Ser Asp Arg Phe Ala Phe His Ser Ala
210 215 220
Tyr His Pro Asn Met Pro Gly Ser Thr Val Cys Ile Met A_sp Phe Cys
225 230 235 240
Glu Gln Lys Gly Leu Arg Phe Thr Asn Gly Glu Trp.Met Gly Leu Asn
245 ~ 250 255
Val Glu Gln Ser Ile Arg Glu Lys Lys Ile Ser Ala Ile Phe Pro Asn
260 265 270
Cys Val Ala Gly Thr Glu Ile Arg Ala Thr Leu Glu Ser Glu Gly Ala
275 . 280 285
Arg Thr Leu Thr Trp Glu Thr Gln Arg Met Leu Asp Tyr Ser Leu Cys
290 295 300
Gln Asn Thr Trp Asp Lys Val Ser Arg Lys Glu Pro Leu Ser Pro Leu
305 310 315 320
Asp Leu Ser Tyr Leu Ser Pro Arg Ala Pro Gly Lys Gly Met Ala Tyr
325 330 335
Thr Val Ile Asn Gly Thr Leu His Ser Ala His Ala Lys Tyr Ile Arg
340 345 350
Thr Trp ile Asp Tyr Gly Glu Met Lys Glu Ile Lys Gly Gly Arg Gly
355 360 365
Glu Tyr Ser Lys Ala Pro Glu Leu Leu Trp Ser Gln Trp Phe Asp Phe
370 375 380
_'
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Gly Pro Phe Lys Ile Gly Pro Asn Gly Leu Leu His Thr Gly Lys Thr
385 390 395 400
Phe Lys Phe Pro Leu Tyr Leu Ile Gly Ala Gly Ile Ile Asp Glu Asp
405 410 415
Leu His Glu Leu Asp Glu Ala Ala Pro Ile Asp His Pro Gln Met Pro
420 425 430
Asp Ala Lys Ser Val Leu Pro Glu Asp Glu Glu Ile Phe Phe Gly Asp
435 440 945
Thr Gly Val Ser Lys Asn Pro Ile Glu Leu Ile Gln Gly Trp Phe Ser
450 455 460
Asn Trp Arg Glu Ser Val Met Ala Ile Val Gly Ile Val Leu Leu Ile
465 470 475 480
Val Val Thr Phe Leu Ala Ile Lys Thr Val Arg Val Leu Asn Cys Leu
485 490 495
Trp Arg pro Arg Lys Lys Arg Ile Val Arg Gln GIu Val Asp Val Glu
500 505 510
Ser Arg Leu Asn His Phe Glu Met Arg Pro Glu Tyr Val Lys
Gly Phe
515 520 525
Arg
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQiJE~TCB CHARACTERISTICS:
(A) LBrIGTB .' 1569 base pairs
(B) TYPE: nucleic acid
~C) STRANDEDNBSS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic
acid
(A) DESCRIPTION: /desc = "DNA"
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) IACATION: 1..1566
(xi) SEQUENCE DESCRIPTION: SEQ ID
N0:9:
ATG AAT ATA CCT TGC TTT GCT GTG ATC TTA GCT ACT ACA CAT 48
CTC AGC
Met Asn Ile Pro Cys Phe Ala Val Ile Leu Ala Thr Thr His
Leu Ser
1 5 10 15
TCT CTG GGA GAA TTC CCC TTG TAT ACG GAG AAA ATA GAG AAA 96
ATT CCC
Ser Leu Gly Glu Phe Pro Leu Tyr Thr Glu Lys Ile Glu Lys
Ile Pro
20 25 30
TGG ACC CCC ATA GAC ATG ATC CAT CTT CCT AAT AAC ATG CTG 144
AGT TGC
Trp Thr Pro Ile Asp Met Ile His Leu Pro Asn Asn Met Leu
Ser Cys
35 40 45
TCT GAG GAA GAA GGT TGC AAT ACA GAG TTC ACC TAC TTC GAG 192
TCT CCT
Ser Glu Glu Glu Gly Cys Asn Thr Glu Phe Thr Tyr Phe Glu
Ser Pro
50 55 60
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CTC AAG AGT GGT TAC CTA GCC CAT CAG AAG GTC CCA GGA TTT 240
ACA TGC
Leu Lys Ser Gly Tyr Leu Ala His Gln Lys Val Pro Gly Phe
Thr Cys
65 70 75 80
ACT GGG GTT GTG AAT GAG GCA GAG ACA TAC ACA AAC TTT GTC 288
GGA TAT
Thr Gly Val Val Asn Glu Ala Glu Thr Tyr Thr Asn Phe Val
Gly Tyr
85 90 95
GTC ACC ACC ACC TTC AAA AGG AAG CAC TTT AAA CCT ACA GTG 336
GCT GCT
Val Thr Thr Thr Phe Lys Arg Lys His Phe Lys Pro Thr Val
Ala Ala
100 105 110
TGT CGT GAT GCC TAC AAC TGG AAA GTA TCA GGG GAC CCC CGA 384
TAT GAA
Cys Arg Asp Ala Tyr Asn Trp Lys Val Ser Gly Asp Pro Arg
Tyr Glu
115 120 125
GAA TCT CTA CAC ACC CCG TAT CCC GAC AGC AGC TGG TTA AGG 432
ACT GTG
Glu Ser Leu His Thr Pro Tyr Pro Asp Ser Ser Trp Leu Arg
Thr Val
130 135 140
ACC ACA ACC AAA GAA GCC CTT CTT ATA ATA TCG CCA AGC ATT 480
GTA GAG
Thr Thr Thr Lys Glu Ala Leu Leu Ile Ile Ser Pro Ser Ile
Val Glu
150 155 160
1~5
ATG GAC ATA TAT GGC AGG ACC CTT CAC TCT CCC ATG TTC CCT 528
TCG GGG
Met Asp Ile Tyr Gly Arg Thr,Leu His Ser Pro Met Phe Pro
Ser Gly
165 ~ 170 ' i75
.
GTC CCC TCT TGT ACA ACC AAC CAT 576
AAA'TGT TCC AAG CTC TAT CCT TCT
Ly8 Cys Ser Lye Leu Tyr Pro Ser Val Pro Ser Cys Thr Thr
Asn His
180 185 190
GAT TAC ACA TTG TGG TTG CCA GAA GAT TCT AGT CTG AGT TTG 624
ATT TGC
Asp,Tyr Thr Leu Trp Leu Pro Glu Asp Ser Ser Leu Ser Leu
Ile Cys
195 200 205
GAC ATC TTC ACT TCC AGC AGT GGA CAG AAG GCC ATG.AAT GGG.TCT672
CGC
Asp Ile Phe Thr ser Ser Ser Gly Gln Lys Ala Met Asn Gly
Ser Arg
210 215 220
ATC TGC GGA TTC SAG GAT GAA AGG GGA TTT TAC AGA TCC TTG 720
AAG GGA
Ile Cys Gly Phe Lys Asp Glu Arg Gly Phe Tyr Arg Ser Leu
Lys Gly
230 235 240
225
TCC TGT AAG CTG ACA TTG TGC GGG AAA CCT GGA ATT AGG CTG 768
TTC GAC
Ser Cys Lys Leu Thr Leu Cys Gly Lys Pro Gly Ile Arg Leu
Phe Asp
245 250 255
GGA ACT TGG GTC TCT TTT ACA AAG CCG GAC GTT CAT GTG TGG 816
TGC ACT
Gly Thr Trp Val Ser Phe Thr Lys Pro Asp Val His Val Trp
Cys Thr
26'0 265 270
CCC AAC CAG TTA GTC AAT ATA CAT AAC GAC AGA CTA GAT GAG 864
GTT GAA
Pro Asn Gln Leu Val Asn Ile His Asn Asp Arg Leu Asp Glu
Val Glu
275 280 285
CAT CTG ATC GTG GAC GAT ATC ATC AAG AAG AGA GAG GAG TGT 912
TTA GAC
His Leu Ile Val Asp Asp Ile Ile Lys Lys Arg Glu Glu Cys
Leu Asp
290 295 300
ACG CTG GAA ACT ATA CTT ATG TCT CAA TCA GTT AGT TTT AGA 960
CGG TTG
Thr Leu Glu Thr Ile Leu Met Ser Gln Ser Val Ser Phe Arg
Arg Leu
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310 315 320
305
AGC CAT TTC AGA AAG TTA GTT CCA GGA TAT GGA AAA GCT 1008
TAC ACT ATT
Ser His Phe Arg Lys Leu Val Pro Gly Tyr Gly Lys Ala
Tyr Thr Ile
325 330 335
TTG AAC GGC AGC TTA ATG GAA ACA AAT GTC TAC TAC AAA 1056
AGA GTT GAC
Leu Asn Gly Ser Leu Met Glu Thr Asn Val Tyr Tyr Lys
Arg Val Asp
340 345 350
AGG TGG GCG GAC ATT TTG CCT TCT AGG GGA TGT CTG AAA ll04
GTC GGA CAA
Arg Trp Ala Asp Ile Leu Pro Ser Arg Gly Cys Leu 365
Val Gly Gln
355 360
CAG TGC ATG GAC CCT GTC AAA GGG GTC CTC TTC AAC GGA 1152
ATT ATC AAG
Gln Cys Met Asp Pro Val Lys Gly Val Leu Phe Asn Gly
Ile Ile Lys
370 375 380
GGT CCG GAT GGA CAA ATA TTG ATT CCA GAG ATG CAG TCA 1200
GAG CAG CTC
Gly Pro Asp Gly Gln Ile Leu Ile Pro Glu Met Gln Ser
Glu Gln Leu
390 395 400
385
AAA CAG CAT ATG GAT CTG TTG AAA GCA GCT ATG TTT CCT 1248
CTC CGT CAT
Lys Gln His Met Asp Leu Leu Lys Ala Ala Met Phe Pro
Leu Arg His
405 410 415
CCT TTA ATC AAC AGA GAG GCA GTC TTC AAG AAG GAT GGA 1296
AAT GCC GAT
Pro ~Leu Ile Asn Arg Glu Ala Val Phe Lys Lys Asp Gly
Asa Ala Asp
420 425 430
GAT TTT GTT GAT CTC CAT ATCi CCT GAT GTT CAA AAA TCT 1344
GTG TGG GAT
Asp Phe Val Asp Leu His Met Pro Asp Val Gla Lys Ser
Val ser Asp
435 440 445
GTC GAC CTG QGC CTG.CCT CAT TGG GGG TTC TGG TTG TTA 1392
GTC GQG GCA
Val Asp Leu Gly Leu Pro His Trp Gly Phe Trp Leu Leu
Vah Gly Ala
450 455 460
ACA GTA GTA GCC TTT GTG GTC TTG GCG TGC TTG CTC CGT GTA TGT TGT 1440
Thr Val Val Ala Phe Val Val Leu Ala Cys Leu Leu Arg Val Cys Cys
470 475 480
465
AGG AGA ATG AGA AGG AGA AGG TCA CTG CGT GCC ACT CAG GAT ATC CCC 1488
Arg Arg Met Arg Arg Arg Arg Ser Leu Arg Ala Thr Gln Asp Ile Pro
485 490 495
CTC AGC GTT GCC CCT GCC CCT GTC CCT CGT GCC AAA GTG GTG TCA TCA 1536
Leu.Ser Val Ala Pro Ala Pro Val Pro Arg Ala Lys Val Val Ser Ser
500 505 510
TGG GAG TCT TCT AAA GGG CTC CCA GGT ACT TGA 1569
Trp Glu Ser Ser Lys Gly Leu Pro Gly Thr
515 520
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 522 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Met Asn Ile Pro Cys Phe Ala Val Ile Leu Ser Leu Ala Thr Thr His
1 5 10 15
Ser Leu Gly Glu Phe Pro Leu Tyr Thr Ile Pro Glu Lys Ile Glu Lys
20 25 30
Trp Thr Pro Ile Asp Met Ile His Leu Ser Cys Pro Asn Asn Met Leu
35 40 45
Ser Glu Glu Glu Gly Cys Asn Thr Glu Ser Pro Phe Thr Tyr Phe Glu
50 55 60
Leu Lys Ser Gly Tyr Leu Ala His Gln Lys Val Pro Gly Phe Thr Cys
65 70 75 80
Thr Gly Val Val Asn Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr
85 90 95
Val Thr Thr Thr Phe Lys Arg Lys His Phe Lys Pro Thr Val Ala Ala
100 105 110
Cys Arg Asp Ala Tyr Asn Trp Lys Val Ser Gly Asp Pro Arg Tyr Glu
115 120 1Z5
~Glu Ser Leu His Thr Pro Tyr Pro Asp Ser Ser Trp Leu Arg Thr Val
130 , 135 140
Thr Thr Thr Lys Glu Ala Leu Leu Ile Ile ser Pro Ser Ile Val Glu
145 150 155 160
Met Asp Ile Tyr Gly Arg Thr Leu His Ser Pro Met Phe Pro Ser Gly
165 170 175
Lys Cys Ser Lys Leu Tyr Pro Ser Val Pro Ser Cys Thr Thr Asn His
180 185 190
Asp Tyr Thr Leu Trp Leu Pro Glu Asp Ser.Ser Leu Ser Leu Ile Cys
195 200 205
Asp Ile Phe Thr Ser Ser Ser Gly Gln Lys Ala Met Asn Gly Ser Arg
210 215 220
Ile Cys Gly Phe Lys Asp Glu Arg Gly Phe Tyr Arg Ser Leu Lys Gly
225 230 235 240
Ser Cys Lys Leu Thr Leu Cys Gly Lys Pro Gly Ile Arg Leu Phe Asp
245 250 255
Gly Thr Trp Val Ser Phe Thr Lys Pro Asp Val His Val Trp Cys Thr
260 265 270
Pro Asn Gln Leu Val Asn Ile His Asn Asp Arg Leu Asp Glu Val Glu
275 280 285
His Leu Ile Val Asp Asp Ile Ile Lys Lys Arg Glu Glu Cys Leu Asp
290 295 300
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Thr Leu Glu Thr Ile Leu Met Ser Gln Ser Val Ser Phe Arg Arg Leu
305 310 315 320
Ser His Phe Arg Lys Leu Val Pro Gly Tyr Gly Lys Ala Tyr Thr Ile
325 330 335
Leu Asn Gly Ser Leu Met Glu Thr Asn Val Tyr Tyr Lys Arg Val Asp
340 345 350
Arg Trp Ala Asp Ile Leu Pro Ser Arg Gly Cys Leu Lys Val Gly Gln
355 360 365
Gln Cys Met Asp Pro Val Lys Gly Val Leu Phe Asn Gly Ile Ile Lys
370 375 380
Gly Pro Asp Gly Gln Ile Leu Ile Pro Glu Met Gln Ser Glu Gln Leu
390 395 400
385
Lys Gln His Met Asp Leu Leu Lys Ala Ala Met Phe Pro Leu Arg His
405 410 415
Pro Leu Ile Asn Arg Glu Ala Val Phe Lys Lys Asp Gly Asn Ala Asp
420 425 430
Asp Phe Val Aap Leu His Met Pro Asp Val Gln Lys Ser Val Ser Asp
435 440 445
Val Asp Leu Gly Leu Pro His Trp Gly Phe Trp Leu Leu Val Gly Ala
450 455 460
Thr Val Val Ala Phe Val Val Leu Ala Cya Leu Leu Arg Val Cys Cys
470 475 480
465
Arg.Arg Met Arg Arg Arg Arg Ser Leu Arg Ala Thr Gln Asp Ile Pro
485 490 495
Leu Ser Val Ala Pro Ala Pro Val Pro Arg Ala Lys Val Val.Ser Ser
500 505 510
Trp GIu Ser Ser Lys Gly Leu Pro Gly Thr
515 520
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