Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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The present invention relates to vectors, in particular .vectors that are
suitable for use as
s or with or in the preparation of a yeast artificial chromosome.
A yeast artificial chromosome - otherwise known as a YAC - comprises the
structural
components of a yeast chromosome into which it is possible to clone very large
pieces of
DNA. By way of example, it is generally possible to clone into a YAC stretches
of
DNA that are up to about 1000kb long - which are much larger than when
compared
with the stretches of DNA that can be cloned into other cloning vectors such
as plasmids
(typically up to 20kb stretches of DNA), bacteriophage ~, (typically up to
25kb stretches
of DNA), cosmids (typically up to 45kb stretches of DNA) and the P1 vector
(typically
up to 100kb stretches of DNA) (see Lodish et al 1995 Molecular Cell Biology
3rd
15 Edition, Pub. Scientific American Books, page 233).
YACs were initially proposed by Burke et al (Burke, D.T., G.F. Carte and M.V.
Olson
(1987) Cloning of large segments of exogenous DNA into yeast by means of
artificial
chromosome vectors. Science 236: 806-812). General introductory teachings on
YACs
2o have been presented by T.A. Brown 1995 (Gene Cloning, An Introduction, 3rd
Edition,
page 325, Pub. Chapman & Hall, pages 139-142).
Typically, a YAC contains the following essential functional elements: a
centromere,
two telomeres and one or more origins of replication. The centromere is
required to
2s correctly distribute the chromosome to daughter cells during cell division;
the telomeres
are required to ensure correct replication; and the replication origins) is
(are) present to
ensure initiation of DNA replication. The origins of replication are sometimes
referred
to as ARS elements.
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YACs are typically prepared from YAC vectors. These vectors are typically
circular.
When they are needed to be used to prepare the YAC they are then linearised -
such as
by use of specific restriction enzymes.
s To date, a number of YAC vectors have been proposed in the literature.
Examples of
such vectors are pYAC2 and pYAC4 which are discussed in US-A-4889806. Other
YAC vectors are disclosed in WO-A-95/03400. Other YAC vectors include pYAC3
and
pYACS.
o YAC vectors - such as pYAC3, pYAC4 and pYACS - are essentially a pBR322
plasmid
into which a number of yeast genes have been inserted. These genes include a
yeast
centromere region (called CENT, two telomere regions (called TEL), and two
selectable
marker genes (called URA3 and TRPI). The TEL sequences do not correspond to
the
full genomic telomere sequences. Nevertheless these partial sequences still
function as
~s telomeric sequences. One replication origin (called ori, such as ARSI) is
positioned
intermediate CEN4 and TRPl. When used for cloning, the YAC vectors are cut
with
BarnHI and a second restriction enzyme (SmaI for pYAC3, EcoRI for pYAC4 and
NotI
for pYACS) to produce two vector arms. The fragments are then ligated with a
nucleotide sequence of interest (which for ease of reference shall be called
"NOI") which
2o has been digested with corresponding restriction enzymes.
The resultant linear structure (which is not drawn to scale) is presented as
Figure 17.
This resultant linear structure - which is a YAC - comprises in the correct
orientation the
essential functional features of a chromosome.
YACs have been - and are still being - used to map the human genome. In this
case, the
NOI is a gene or fragment thereof whose full sequence (or even function) may
not have
been determined. In these studies, YACs are used to create genomic libraries
which are
then screened. By way of example, the physical map of the human Y chromosome
and
3o the long arm of chromosome 21 have been determined through analysis of long
segments
of human DNA cloned into YACs by inter alia sequence tagged sites. This work
is
summarised in Lodish et al 1995 (ibid, page 285).
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However, the use of YACs is by no means limited to mapping of the human
genome.
For example, the use of YACs has led to the preparation of physical maps of
the
Drosophila X chromosome containing the shibire (ship locus (Bliek and
Meyerowitz
1991 Nature 351 441). YACs have also been proposed to map plant genomes such
as
s the Arabidopsis genome.
In addition to their usage in genome mapping, YACs have now another important
use.
In this regard, it has been recently found that under certain conditions YACs
can be
introduced into mammalian cells (such as murine cells), whereupon they can
behave
1o functionally the same as (or very similar to) endogenous chromosomes. In
this regard, it
is possible to deliver - such as by use of cell fusion techniques,
microinjection techniques
or transfection techniques - YACs containing large fragments of human DNA
(i.e. the
NOI) into the mouse germline. This was initially achieved with a 670kb YAC
containing a human X-chromosome fragment (Jacobovitis et al 1993 Nature 362
pages
~s 255-258). By way of further example, WO-A-94/23049 reports on a YAC
containing
the gene coding for ~i-amyloid precursor protein.
Hence, YACs now enable workers to analyse an or the in vivo functional
behaviour of a
NOI, such as a human NOI. For these studies, prior knowledge of the sequence
and/or
2o function of the NOI need not be necessary.
A review of these in vivo functional studies has been presented by Jacobovitis
(Current
Biology 1994 vol 4 No 8 pages 761-763), who states:
25 "The ability to replace mouse genes with their human equivalents using
yeast artifccial chromosome technology provides a powerful new technique
for studying the regulation and function of human genes" .
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Other reviews and details on these studies and techniques have been presented
by Larin
and Lehrach (Genet. Res. Camb 1990 56 pp 203-208), Schedl et al (Nucleic Acids
Research vol 20 No. 20 pages 3073-3077), and Montoliu et al (Reprod. Fert.
Dev. 1994
6 577-584).
YACs may even be used to study the functional behaviour of mutant genes. In
this
regard, WO-A-95/14769 reports on a method of producing a mouse that expresses
human mutant protein sequences that utilises the "pop-in/pop-out" method in
combination with YAC technology to insert mutations into YACs and thereby
derive
io stem cells capable of being used in the development of transgenic mice.
This particular
method comprises obtaining a gene contained within a YAC, introducing a
predetermined mutant human DNA sequence (which is the NOI) into the YAC by
homologous recombination, utilising transgenics to insert the mutant gene into
embryonic stem cells, and injecting the stem cells into blastocysts to derive
a transgenic
i5 mouse that expresses the mutant protein sequences. The "pop-in/pop-out"
method is
described by Rothstein ( 1991 Methods In Enzymology vol 194, Guide To Yeast
Genetics
and also in Molecular Biology. Eds. Gutherie at al, San Diego: Academic Press.
Pages
281-301) and McCormick et al (TCM Vol 6 No. 1 1996 pages 16-24).
20 According to Schedl et al (reference 28): the transfer of YAC DNA into
mammalian
cells came into focus of interest soon after the first report of yeast
artificial
chromosomes. In a successfully transgenic cell the genes contained on the YAC
are
embedded in an almost natural chromosomal context, which should ensure
regulation of
expression comparable to their endogenous counterparts. Therefore, such a
method
25 should allow a more rapid identification of genes by complementation
analysis as well as
detailed studies of function of genes and their regulation in vivo. Schedl et
al (ibic~
describe a method for the isolation of purified and concentrated YAC DNA as
well as
protocols for microinjection into somatic cells in culture and fertilized
mouse oocytes.
3o Despite the fact that YACs have many important applications, there are
nevertheless
problems associated with their production and their usage.
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For example, the low efficiency with which transgenic animals are produced
using YAC
DNA ( 1-5 Rb ) compared to DNA from conventional vectors (approximately 10
°b ) is
probably caused by the low concentration of YAC DNA available for injection.
Assuming that 2 pl of 540kb YAC at a concentration of 1 ng/~1 is injected into
a
5 pronucleus, a fertilised egg receives only 1 molecule of YAC DNA.
Amplification of
YACs in yeast should provide a possible method for the isolation of more
concentrated
YAC DNA which should lead to more successful generation of YAC transgenic
animals
(i.e. a transgenic mammal that comprises a YAC). However, to date, there has
not been
a totally acceptable solution to this problem.
to
For example, Smith et al proposed use of the YAC vector pCGS990 (Smith et al
Mammalian Genome 1993 4 pages 141-147). Even though that YAC vector goes some
way to overcoming this problem, it nevertheless comprises the TK gene (i.e.
the herpes
simplex virus thymidine kinase gene) as a selectable marker. This is
problematic as
~5 expression of this gene can cause male sterility in transgenic animals.
Alternatively, or in addition, with current techniques it is not currently
possible to
readily monitor the in vivo expression pattern of a NOI that has been
introduced into an
organism - such as a mouse. Current techniques - such as in situ
hydridisation,
2o Polymerase Chain Reaction (PCR) and Northern Blotting - are laborious to
carry out.
In addition, and by way of further example, .none of the earlier reported
studies has
reported on the co-expression of a NOI and a reporter gene in a YAC.
z5 Thus there are problems associated with the known vectors for preparing
YACs.
The present invention seeks to improve upon the existing techniques associated
with the
preparation of and usage of YACs.
3o In this regard, the present invention seeks to provide two types of vectors
that can be
used on their own or in combination with each other and thereby overcome at
least one
of the above-mentioned problems.
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According to a first aspect of the present invention there is provided one or
more of the
following embodiments, which for ease have been presented as numbered
paragraphs:
1. A YAC vector comprising an IRES.
2. A YAC vector comprising a reporter gene wherein the expression product of
the
reporter gene is capable of producing a visually detectable signal.
3. A YAC vector comprising a reporter gene wherein the expression product of
the
to reporter gene is capable of producing an immunologically detectable signal.
4. A YAC vector comprising an IRES and a reporter gene wherein the expression
product of the reporter gene is capable of producing a visually detectable
signal.
5. A YAC vector comprising an IRES and a reporter gene wherein the expression
product of the reporter gene is capable of producing an immunologically
detectable
signal.
6. pYNl.
7. pYN2.
8. pYIV3.
9. pYIV4.
10. A YAC prepared by the vector (such as the insertion thereof) according to
any
one of the above-mentioned embodiments.
11. Use of an IRES to modify a YAC vector or a YAC.
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According to a second aspect of the present invention there is provided one or
more of
the following embodiments, which for ease have been presented as numbered
paragraphs:
s 1. A YAC vector comprising a nucleotide sequence wherein the nucleotide
sequence
comprises the sequence presented as SEQ ID No.l or SEQ ID No. 4 a variant,
homologue or derivative thereof.
2. A vector capable of modifying a YAC or a YAC vector wherein the vector
to comprises a nucleotide sequence wherein the nucleotide sequence comprises
the sequence
presented as SEQ ID No.l or SEQ ID No. 4 or a variant, homologue or derivative
thereof.
3. pYAM4.
is
4. A YAC prepared by the vector according to any one of the above-mentioned
embodiments.
5. Use of a nucleotide sequence comprising the sequence presented SEQ ID No.l
or
2o SEQ ID No. 4 or a variant, homologue or derivative thereof in a vector to
prepare a
YAC vector or a YAC.
6. Use of a nucleotide sequence comprising the sequence presented SEQ ID No.l
or
SEQ ID No. 4 or a variant, homologue or derivative thereof in a vector to
increase the
2s expression efficiency of one or more NOIs within a YAC vector or a YAC.
For convenience, the vector of the first aspect of the present invention is
sometimes
referred to herein as being an insertion vector; whereas the vector of the
second aspect
of the present invention is sometimes referred to herein as being an
amplification vector.
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However, the term "vector" as used herein in the general sense means the
vector of the
first aspect of the present invention andlor the vector of the second aspect
of the present
invention.
s According to a third aspect of the present invention there is provided the
combination of
at least any one of the embodiments of the first aspect of the present
invention and at
least any one of the embodiments of the second aspect of the present
invention.
With the third aspect of the present invention - namely the combination of the
vector of
to the first aspect of the present invention and the vector of the second
aspect of the present
invention - the term "combination" means that the resultant YAC or
transgenic/transformed cell, organ or organism is prepared by use of both the
vector of
the first aspect of the present invention and the vector of the second aspect
of the present
invention.
is
The third aspect of the present invention is not limited to preparative
techniques wherein
both the vector of the first aspect of the present invention and the vector of
the second
aspect of the present invention have to be used at the same time when
preparing the
YAC, let alone the transformed/transgenic cell, organ or organism. In this
regard, it is
2o sometimes advantageous for the vector of the first aspect of the present
invention to be
used at a different stage than the vector of the second aspect of the present
invention
during the preparation of the YAC, or even the transformed/transgenic cell,
organ or
organism.
2s According to a further aspect of the present invention there is provided a
YAC vector or
a YAC comprising a selection gene, wherein that selection gene is specifically
removable
from the YAC vector or the YAC.
Other aspects of the present invention include:
A YAC transgenic mammal co-expressing an NOI and a reporter gene wherein
the expression pattern of the NOI can be determined by measuring a detectable
signal
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(such as a visually or an immunologically detectable signal) produced by the
expression
product of the reporter gene.
A YAC transgenic mammal expressing a reporter gene under the control of a
s regulatory sequence from a human NOI.
Use of a YAC transgenic mammal to test for potential pharmaceutical and/or
veterinary agents.
1o An assay method for identifying an agent that can affect the expression
pattern of
an NOI or the EP ("expression product") activity thereof,
the assay method comprising
~5 administering an agent to a YAC transgenic mammal according to the present
invention;
determining whether the agent modulates (such as affects the expression
pattern
or activity) the NOI or the EP by means of the detectable signal.
An assay method according to the present invention wherein the assay is to
screen
for agents useful in the treatment of disturbances in any one of: circadian
function, sleep
disorders, eating disorders, pre-menstural syndrome, autoimmune disorders,
birth
defects in women and/or sexual dysfunction.
An agent identified by the method according to the present invention.
A process comprising the steps of:
(a) performing the assay according to the present invention;
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(b) identifying one or more agents that affect the expression pattern of the
NOI or the
EP activity thereof;
(c) preparing a quantity of those one or more identified agents.
5 A process comprising the steps of:
(a) performing the assay according to the present invention;
(b) identifying one or more agents that affect the expression pattern of the
NOI or the
to EP activity thereof;
(c) preparing a pharmaceutical composition comprising one or more identified
agents.
A process comprising the steps of:
(a) performing the assay according to the present invention;
(b) identifying one or more agents that affect the expression pattern of the
NOI or the
2o EP activity thereof;
(c) modifiying one or more identified agents to cause a different effect on
the
expression pattern of the NOI or the EP activity thereof.
2s Use of a YAC according to the present invention or any one of the vectors
according to the present invention to screen for agents capable of affecting
the expression
pattern of an NOI or the EP activity thereof in a transgenic mammal.
Use of an agent in the preparation of a pharmaceutical composition for the
3o treatment of a disorder or condition associated with the expression pattern
of an NOI or
the EP activity thereof, the agent having an effect on the expression pattern
of the NOI
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or the EP activity thereof when assayed in vitro by the assay according to the
present
invention.
Use of an agent identified by an assay according to the present invention in
the
s manufacture of a medicament which affects the expression pattern of an NOI
or the EP
activity thereof.
Use of an agent identified by an assay according to the present invention in
the
manufacture of a medicament which affects the expression pattern of an NOI or
the EP
1o activity thereof.
In accordance with the present invention, at least part of the assay can be
carried out in
living tissue.
15 A preferred - but non-limiting - example of an NOI is the human. serotonin
transporter
(SERT), preferably the the human serotonin transporter (SERT) presented as SEQ
ID
No. 2 or a variant, homologue or derivative thereof.
The terms "variant", "homologue", or "derivative" in relation to this aspect
of the present
2o invention include any substitution of, variation of, modification of,
replacement of,
deletion of or addition of one (or more) nucleic acid from or to the sequence
providing the
resultant expression product of the nucleotide sequence has the same activity
as the
expression product of SEQ ID No. 2, preferably having at least the same level
of activity
of the expression product of SEQ LD. No. 2. In particular, the term
"homologue" covers
2s identity with respect to structure and/or function providing the resultant
nucleotide
sequence has promoter activity. With respect to sequence identity (i.e.
similarity),
preferably there is at least 75 % , more preferably at least 85 % , more
preferably at least
90% sequence identity. More preferably there is at least 95%, more preferably
at least
98%, sequence identity. These terms also encompass allelic variations of the
sequences.
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Another preferred - but non-limiting - example of an NOI is the VIP2 receptor
(VIPR2).
The VIP2 receptor is referred to interchangeably throughout the text as the
VIP2
receptor, VIPR2 or VPAC2R (Harmar et al 1998 Pharmacological Reviews 50: 265-
270). Preferably, the VIPR2 is the VIP2 receptor (VIPR2) presented as SEQ ID
No. 3
s or a variant, homologue or derivative thereof.
The terms "variant", "homologue", or "derivative" in relation to this as~ct of
the present
invention include any substitution of, variation of, modification of,
replacement of,
deletion of or addition of one (or more) nucleic acid from or to the sequence
providing the
to resultant expression product of the nucleotide sequence has the same
activity as the
expression product of SEQ ID No. 3, preferably having at least the same level
of activity
of the expression product of SEQ LD. No. 3. In particular, the term
"homologue" covers
identity with respect to structure and/or function providing the resultant
nucleotide
sequence has promoter activity. With respect to sequence identity (i.e.,
similarity),
15 preferably there is at least 75 l , more preferably at least 85 ~ , more
preferably at least
90 l sequence identity. More preferably there is at least 95 ~ , more
preferably at least
98°6, sequence identity. These terms also encompass allelic variations
of the sequences.
The present invention also encompasses modified YACs comprising these aspects
of the
2o present invention, transformed cells comprising these aspects of the
present invention,
transgenic organisms comprising these aspects of the present invention,
processes for
making all of these aspects, and methods of expressing all of these aspects.
The term "affects" includes any one or more of: treats, prevents, suppresses,
alleviates,
25 restores, modulates, influences or to otherwise alter an existing state.
The term "agent" includes any entity (such as one or more chemical compounds,
including peptide sequunces and variants/homologues/derivatives/fragments
thereof)
which is capable of affecting the expression pattern of the NOI or the EP
activity
3o thereof. It also includes mimics and equivalents and mutants thereof. It
also includes
agonists and antagonists and antibodies. Non-limiting antibodies include:
polyclonal,
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monoclonal, chimeric, single chain, Fab fragments, fragments produced by a Fab
expression library and humanised monoclonal antibodies.
The term "expression product" or "EP" means the expressed protein per se but
also
includes fusion proteins comprising all or part of same. The EP may be the
same as the
naturally occuring form or is a variant, homologue, fragment or derivative
thereof.
The term "disturbances in circadian function" means disorders which may lead
to
impaird physical and mental well-being that can occur through extremes in work
patterns
1o such as shift work, in normal ageing, when travelling through time zones
(jet lag), and
in dementia.
There are a number of advantages associated with the present invention.
is For example, with the use of the amplification vector it is possible to
obtain high copy
numbers of YACs.
For example, with the use of the insertion vector it is possible to readily
monitor the in
vivo expression pattern of a NOI.
Also, with the use of the insertion vector, it is possible to express - such
as over-express
- a NOI and a reporter gene contained within or as a YAC.
It provides a means for producing YAC transgenic animals and the analysis of
these
animals in terms of expression, regulation and function of NOIs present in YAC
DNA.
It facilitates the determination of sites/regions where an NOI is expressed
and the
identification of agents which may affect the expression pattern of the NOI or
the EP
activity thereof.
Other advantages associated with the present invention will be apparent from
the
following text.
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With the present invention, the vector may comprise at least one NOI.
With the present invention, the term NOI (i.e. nucleotide sequence of
interest) includes
any suitable nucleotide sequence, which need not necessarily be a complete
naturally
s occuring DNA sequence. Thus, the DNA sequence can be, for example, a
synthetic
DNA sequence, a recombinant DNA sequence (i.e. prepared by use of recombinant
DNA techniques), a cDNA sequence or a partial genomic DNA sequence, including
combinations thereof. The DNA sequence need not be a coding region. If it is a
coding
region, it need not be an entire coding region. In addition, the DNA sequence
can be in
to a sense orientation or in an anti-sense orientation. Preferably, it is in a
sense
orientation. Preferably, the DNA is or comprises cDNA.
The vectors of the present invention may be used to prepare a modified YAC or
a
modified YAC vector. The modified YAC or the modified YAC vector can be used,
for
1 s example, for expression and/or regulation and/or functional studies of the
NOI. In
addition, the vectors of the present invention can be used to prepare modified
YACs or
modified YAC vectors that can be used for expression and/or regulation and/or
functional studies of the NOI in combination with other entities, such as
other NOIs,
compounds or compositions. In addition, the modified YAC or modified YAC
vector
2o can be used to test potential pharmaceutical agents (including veterinary
agents).
Here the term "modified YAC or modified YAC vector" means a modified YAC or
modified YAC vector having a modified genetic structure. With the present
invention,
the YAC or YAC vector has a modified genetic structure since part or all of
the vector
2s according to the present invention has been incorporated into the YAC or
YAC vector.
The vectors of the present invention may be used to prepare transformed cells
that can be
used, for example, for functional studies of the NOI. In addition, the vectors
of the
present invention can be used to prepare transformed cells that can be used
for functional
3o studies of the NOI in combination with other entities, such as other NOIs,
compounds or
compositions. In addition, the transformed cells can be used to test potential
pharmaceutical agents (including veterinary agents).
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The vectors of the present invention may be used to prepare transformed cells
that
comprise mutated genes - such as by use of the pop-in/pop-out technique
(mentioned
above).
s Here the term "transformed cell" means a cell having a modified genetic
structure. With
the present invention, the cell has a modified genetic structure since a
vector according
to the present invention has been introduced into the cell.
The term "cell" includes any suitable organism. In a preferred embodiment, the
cell is a
to mammalian cell. In a highly preferred embodiment, the cell is a murine
cell.
The cell can be an isolated cell or a collection of cells. The cell or cells
may even be
part of a tissue or organ or an organism (including an animal).
15 The cell can be transformed in vivo or in vitro, or combinations thereof.
Typically, the cell will be transformed by any one of the following methods:
transfection, microinjection, electroporation or microprojectile bombardment,
including
combinations thereof.
Preferably, the cell will be transformed by, or by at least, transfection.
For some applications, the transformed cells may be prepared by use of the
modified
YAC according to the present invention.
The vectors of the present invention can also be used to prepare transgenic
organisms
that can be used for functional studies of the NOI. In addition, the vectors
of the present
invention can be used to prepare transgenic organisms that can be used, for
example, for
functional studies of the NOI in combination with other entities, such as
other NOIs,
3o compounds or compositions. In addition, the transgenic organisms can be
used to test
potential pharmaceutical agents (including veterinary agents).
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Here the term "transgenic organism" means an organism comprising a modified
genetic
structure. With the present invention, the organism has a modified genetic
structure
since a vector according to the present invention has been introduced into the
organism.
The term "organism" includes any suitable organism. In a preferred embodiment,
the
organism is a mammal. In a highly preferred embodiment, the organism is a
mouse.
For some applications, the transgenic organisms may be prepared by use of the
transformed cells of the present invention.
to
In a preferred embodiment, the insertion vector of the present invention is
itself a YAC
vector.
For some applications, it may be advantageous that the amplification vector of
the
Is present invention is itself a YAC vector.
With the present invention, the vector of the present invention may
additionally comprise
one or more selection genes to enable the vector and any resultant entity
comprising the
same or made from the same (such as a modified YAC vector, a YAC or a specific
yeast
2o strain comprising any one of the same) to be selectively grown and/or
screened. These
selection genes can be chosen from suitable selection genes that are
available. Examples
of suitable selection genes include LYS2 (see Barnes and Thorner 1986, Mol and
Cell
Biol 6: pp 2828-2838), LEU2 (see Beach and Nurse 1981 Nature vol 290 pp 140-
142),
and ADE2 (see Stotz and Linder 1990 Gene 95 pp 91-98).
In a preferred aspect any one or more of the selection gene is specifically
removable
from the vector, the modified YAC vector and the modified YAC according to the
present invention. In this regard, the term "specifically removable" means
being able to
remove the one or more selection gene without disrupting any other region in
the vector,
3o the modified YAC vector and the modified YAC according to the present
invention. For
example, the selection gene may be flanked by unique restriction sites.
Alternatively,
the selection gene may be flanked by a LoxP element which is removable by use
of Cre
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recombinase. Teachings on the use of the LoxP element and Cre recombinase have
been
published by Deursen et al (1995 PNAS Vol 93 pages 7376-7380), Kuhn et al
(1995
Science Vol 269 pages 1427-1429) and Araki et al (1995 PNAS Vol 92 pages 160-
164).
By way of example, the selection gene flanked by the LoxP element may
therefore be
removed prior to or after formation of the transgenic animal stem cell.
Removal of the
selection gene is highly desirable as it means that the transgenic organism is
not
expressing the selection gene and so there can be no affect of that gene on
the organism
or even on the expression of the NOI being studied. In addition, removal of
the
selection gene means that the NOI is nearer to any 3' regulatory regions that
may be
io present on the YAC.
With the present invention, the YAC vector may additionally comprise one or
more
NOIs. The NOI need not be of known function and/or structure. Preferably, the
NOI is
of human origin.
is
In accordance with one aspect of the present invention, the YAC vector
comprises an
internal ribosomal entry site (i.e. an IRES).
A review on IRES is presented by Mountford and Smith (TIG May 1995 vol 11 No.
5
2o pages 179 - 184). A suitable IRES has also been disclosed by Mountford et
al
(Mountford et al 1994 PNAS 91 pages 4303-4307).
IRES sequences are also mentioned in WO-A-93/03143, WO-A-97/14809, WO-A-
94/24301, WO-A-95/32298, and WO-A-96/27676. These references do not disclose
or
25 suggest the use of an IRES unit in preparing or being a part of a YAC.
According to WO-A-97/14809, IRES sequences act on improving translation
efficiency
of RNAs in contrast to a promoter's effect on the transcription of DNAs. A
number of
different IRES sequences are known including those from encephalomyocarditis
virus
30 (EMCV) (Ghattas, LR., et al., Mol. Cell. Biol., 11:5848-5859 (1991); BiP
protein
[Macejak and Sarnow, Nature 353:91 (1991)]; the Antennapedia gene of
drosphilia
(exons d and e) [Oh, et al., Genes & Development, 6:1643-1653 (1992)] as well
as those
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18
in polio virus [Pelletier and Sonenberg, Nature 334: 320-325 ( 1988); see also
Mountford
and Smith, TIG 11, 179-184 (1985)].
According to WO-A-97/ 14809, IRES sequences are typically found in the 5' non-
coding
region of genes. In addition to those in the literature they can be found
empirically by
looking for genetic sequences that affect expression and then determining
whether that
sequence affects the DNA (i.e. acts as a promoter or enhancer) or only the RNA
(acts as
an IRES sequence).
to Thus the present invention is not intended to be limited to a specific IRES
sequence.
Instead, the sequence to be used can be any sequence that is capable of acting
as an IRES
sequence - i.e. it is capable of improving translation efficiency of an RNA.
A preferred IRES sequence is that presented as SEQ ID No. 1 or a variant,
homologue,
1 s derivative or fragment thereof.
The terms "variant", "homologue", "derivative" or "fragment" in relation to
this aspect of
the present invention include any substitution of, variation of, modification
of, replacement
of, deletion of or addition of one (or more) nucleic acid from or to the
sequence providing
2o the resultant nucleotide sequence has IRES activity, preferably having at
least the same
activity of the IRES shown as SEQ LD. No. 1. In particular, the term
"homologue"
covers identity with respect to structure and/or function providing the
resultant nucleotide
sequence has promoter activity. With respect to sequence identity (i.e.
similarity),
preferably there is at least 75 ~ , more preferably at least 85 ~ , more
preferably at least
2s 90 ~ sequence identity. More preferably there is at least 95 ~ , more
preferably at least
98°x, sequence identity. These terms also encompass allelic variations
of the sequences.
Another preferred IRES sequence is that presented as SEQ ID No. 4 or a
variant,
homologue, derivative or fragment thereof.
The terms "variant", "homologue", "derivative" or "fragment" in relation to
this aspect of
the present invention include any substitution of, variation of, modification
of, replacement
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19
of, deletion of or addition of one (or more) nucleic acid from or to the
sequence providing
the resultant nucleotide sequence has IRFF,S activity, preferably having at
least the same
activity of the IRES shown as SEQ LD. No. 4. In particular, the term
"homologue"
covers identity with respect to structure and/or function providing the
resultant nucleotide
s sequence has promoter activity. With respect to sequence identity (i.e.
similarity),
preferably there is at least 75 % , more preferably at least 85 k , more
preferably at least
90 ~O sequence identity. More preferably there is at least 95 % , more
preferably at least
98 % , sequence identity. These terms also encompass allelic variations of the
sequences.
to Sequence identity with respect to any of SEQ ID 1-4 can be determined by a
simple
"eyeball" comparison (i.e. a strict comparison) of any one or more of the
sequences with
another sequence to see if that other sequence has, for example, at least 75 %
sequence
identity to the sequence(s).
15 Relative sequence identity can also be determined by commercially available
computer
programs that can calculate ~ identity between two or more sequences using any
suitable
algorithm for determining identity, using for example default parameters. A
typical
example of such a computer program is CLUSTAL. Advantageously, the BLAST
algorithm is employed, with parameters set to default values. The BLAST
algorithm is
2o described in detail at http://www.ncbi.nih.gov/BLAST/blast help.html, which
is
incorporated herein by reference. The search parameters are defined as
follows, can be
advantageously set to the defined default parameters.
Advantageously, "substantial identity" when assessed by BLAST equates to
sequences
25 which match with an EXPECT value of at least about 7, preferably at least
about 9 and
most preferably 10 or more. The default threshold for EXPECT in BLAST
searching is
usually 10.
BLAST (Basic Local Alignment Search Tool) is the heuristic search algorithm
employed
3o by the programs blastp, blastn, blastx, tblastn, and tblastx; these
programs ascribe
significance to their findings using the statistical methods of Karlin and
Altschul (see
http://www.ncbi.nih.gov/BLAST/blast help.html) with a few enhancements. The
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BLAST programs were tailored for sequence similarity searching, for example to
identify homologues to a query sequence. For a discussion of basic issues in
similarity
searching of sequence databases, see Altschul et al (1994) Nature Genetics
6:119-129.
5 The five BLAST programs available at http://www.ncbi.nlm.nih.gov perform the
following tasks:
blastp - compares an amino acid query sequence against a protein sequence
database.
o blasts - compares a nucleotide query sequence against a nucleotide sequence
database
blastx - compares the six-frame conceptual translation products of a
nucleotide query
sequence (both strands) against a protein sequence database.
~5 tblastn - compares a protein query sequence against a nucleotide sequence
database
dynamically translated in all six reading frames (both strands).
tblastx - compares the six-frame translations of a nucleotide query sequence
against the
six-frame translations of a nucleotide sequence database.
BLAST uses the following search parameters:
HISTOGRAM - Display a histogram of scores for each search; default is yes.
(See
parameter H in the BLAST Manual).
DESCRIPTIONS - Restricts the number of short descriptions of matching
sequences
reported to the number specified; default limit is 100 descriptions. (See
parameter V in
the manual page).
3o EXPECT - The statistical significance threshold for reporting matches
against database
sequences; the default value is 10, such that 10 matches are expected to be
found merely
by chance, according to the stochastic model of Karlin and Altschul (1990). If
the
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21
statistical significance ascribed to a match is greater than the EXPECT
threshold, the
match will not be reported. Lower EXPECT thresholds are more stringent,
leading to
fewer chance matches being reported. Fractional values are acceptable. (See
parameter E
in the BLAST Manual).
CUTOFF - Cutoff score for reporting high-scoring segment pairs. The default
value is
calculated from the EXPECT value (see above). HSPs are reported for a database
sequence only if the statistical significance ascribed to them is at least as
high as would
be ascribed to a lone HSP having a score equal to the CUTOFF value. Higher
CUTOFF
to values are more stringent, leading to fewer chance matches being reported.
(See
parameter S in the BLAST Manual). Typically, significance thresholds can be
more
intuitively managed using EXPECT.
ALIGNMENTS - Restricts database sequences to the number specified for which
high-
scoring segment pairs (HSPs) are reported; the default limit is 50. If more
database
sequences than this happen to satisfy the statistical significance threshold
for reporting
(see EXPECT and CUTOFF below), only the matches ascribed the greatest
statistical
significance are reported. (See parameter B in the BLAST Manual).
2o MATRIX - Specify an alternate scoring matrix for BLASTP, BLASTX, TBLASTN
and
TBLASTX. The default matrix is BLOSUM62 (Henikoff & Henikoff, 1992). The valid
alternative choices include: PAM40, PAM120, PAM250 and IDENTITY. No alternate
scoring matrices are available for BLASTN; specifying the MATRIX directive in
BLASTN requests returns an error response.
STRAND - Restrict a TBLASTN search to just the top or bottom strand of the
database
sequences; or restrict a BLASTN, BLASTX or TBLASTX search to just reading
frames
on the top or bottom strand of the query sequence.
3o FILTER - Mask off segments of the query sequence that have low
compositional
complexity, as determined by the SEG program of Wootton & Federhen (1993)
Computers and Chemistry 17:149-163, or segments consisting of short-
periodicity
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internal repeats, as determined by the XNU program of Claverie & States (1993)
Computers and Chemistry 17:191-201, or, for BLASTN, by the DUST program of
Tatusov and Lipman (see http://www.ncbi.nlm.nih.gov). Filtering can eliminate
statistically significant but biologically uninteresting reports from the
blast output (e.g.,
s hits against common acidic-, basic- or proline-rich regions), leaving the
more
biologically interesting regions of the query sequence available for specific
matching
against database sequences.
Low complexity sequence found by a filter program is substituted using the
letter "N" in
o nucleotide sequence (e.g., "NNNNNNNNNNNNN") and the letter "X" in protein
sequences (e.g., "XXXXXXXXX").
Filtering is only applied to the query sequence (or its translation products),
not to
database sequences. Default filtering is DUST for BLASTN, SEG for other
programs.
It is not unusual for nothing at all to be masked by SEG, XNU, or both, when
applied to
sequences in SWISS-PROT, so filtering should not be expected to always yield
an effect.
Furthermore, in some cases, sequences are masked in their entirety, indicating
that the
statistical significance of any matches reported against the unfiltered query
sequence
2o should be suspect.
NCBI-gi - Causes NCBI gi identifiers to be shown in the output, in addition to
the
accession and/or locus name.
Most preferably, sequence comparisons are conducted using the simple BLAST
search
algorithm provided at http://www.ncbi.nlm.nih.gov/BLAST.
Other computer program methods to determine identify and similarity between
the two
sequences include but are not limited to the GCG program package (Devereux et
al
1984 Nucleic Acids Research 12: 387) and FASTA (Atschul et al 1990 J Molec
Biol
403-410).
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23
In some aspects of the present invention, no gap penalties are used when
determining
sequence identity.
The present invention also encompasses nucleotide sequences that are
complementary to
the sequences presented herein, or any fragment or derivative thereof. If the
sequence is
complementary to a fragment thereof then that sequence can be used as a probe
to identify
similar promoter sequences in other organisms etc.
The present invention also encompasses nucleotide sequences that are capable
of
o hybridising to the sequences presented herein, or any fragment or derivative
thereof.
Hybridization means a "process by which a strand of nucleic acid joins with a
complementary strand through base pairing" (Coombs J (1994) Dictionary of
Biotechnology, Stockton Press, New York NY) as well as the process of
amplification as
carried out in polymerase chain reaction technologies as described in
Dieffenbach CW
and GS Dveksler (1995, PCR Primer, a Laboratory Manual, Cold Spring Harbor
Press,
Plainview NY).
Also included within the scope of the present invention are nucleotide
sequences that are
2o capable of hybridizing to the nucleotide sequences presented herein under
conditions of
intermediate to maximal stringency. Hybridization conditions are based on the
melting
temperature (Tin) of the nucleic acid binding complex, as taught in Berger and
Kimmel
{1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152,
Academic Press, San Diego CA), and confer a defined "stringency" as explained
below.
Maximum stringency typically occurs at about Tm-5°C (5°C below
the Tm of the
probe); high stringency at about 5°C to 10°C below Tm;
intermediate stringency at about
10°C to 20°C below Tm; and low stringency at about 20°C
to 25°C below Tm. As will
be understood by those of skill in the art, a maximum stringency hybridization
can be
3o used to identify or detect identical nucleotide sequences while an
intermediate (or low)
stringency hybridization can be used to identify or detect similar or related
nucleotide
sequences.
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24
In a preferred aspect, the present invention covers nucleotide sequences that
can hybridise
to the nucleotide sequences of the present invention under stringent
conditions (e.g. 65 °C
and O.IxSSC).
The present invention also encompasses nucleotide sequences that are capable
of
hybridising to the sequences that are complementary to the sequences presented
herein, or
any fragment or derivative thereof. Likewise, the present invention
encompasses
nucleotide sequences that are complementary to sequences that are capable of
hybridising
to the sequence of the present invention. These types of nucleotide sequences
are examples
of variant nucleotide sequences. In this respect, the term "variant"
encompasses sequences
that are complementary to sequences that are capable of hydridising to the
nucleotide
sequences presented herein. Preferably, however, the term "variant"
encompasses
sequences that are complementary to sequences that are capable of hydridising
under
stringent conditions (eg. 65°C and 0. IxSSC { IxSSC = 0.15 M NaCI,
0.015 Na3 citrate pH
7.0}) to the nucleotide sequences presented herein.
Insertion of the IRES into a YAC by use of the insertion vector of the present
invention -
and thus forming a modified YAC - enables the modified YAC to express, in
particular
over-express, at least two nucleotide sequences. Of these two nucleotide
sequences one
2o may be a NOI, such as a NOI of human origin. The other of those nucleotide
sequences
may be another NOI. Alternatively, and in a preferred aspect, the other
nucleotide
sequence is a reporter gene according to the present invention.
The present invention also encompasses a vector or a YAC obtained therefrom
comprising more than one IRES. In this embodiment, the vector or the YAC
obtained
therefrom preferably comprises more than NOI.
In a preferred aspect of the first aspect of the present invention, the YAC
vector
comprises a reporter gene whose expression product is capable of producing a
visually
3o detectable signal. Examples of such reporter genes include: LacZ (see
Mansour et al
1990 PNAS vol 87 pp 7688-7692), green fluorescent protein (see Chiocchetti et
al 1997
Biochim Biophys Acta 1352: pp 193-202; and Chalfie et al 1994 Science vol 263
pp 802-
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805), chloroamphenicol acetyl transferase (see Gorman et al 1982 Mol Cell Biol
2(9) pp
1044-1051; and Frebourg and Brison 1988 Gene vol 65 pp 315-318), or luciferase
(see
de Wet et al 1987 Mol Cell Biol 7(2) pp 725-737; and Rodriguez et al 1988 PNAS
vol
85 pp 1667-1671).
5
In another preferred embodiment of the first aspect of the present invention,
the YAC
vector comprises a reporter gene whose expression product is capable of
producing, or
being detected by an agent capable of providing, an immunologically detectable
signal.
o In a preferred aspect, the reporter gene when fused to the NOI leads to the
production of
a fusion protein that can be detected by commercially available antibodies,
such as a
haemagglutinin tag (see Pati 1992 Gene 15; 114(2): 285-288), a c-myc tag (see
Emrich et
al 1993 Biocem Biophys Res Commun 197(1): 214-220), or the FLAG epitope (Ford
et
al 1991 Protein Expr Purif Apr; 2(2):95-107).
By using a reporter gene according to the present invention it is possible to
readily
observe the functionality of NOIs contained within YAC libraries, such as YAC
human
DNA libraries.
2o For example, if the NOI in the YAC has an expression regulatory role (such
as a
promoter) then expression of the reporter gene according to the present
invention by a
transgenic organism according to the present invention enables workers to
readily
determine in or at which sites or regions that expression regulatory element
is active. In
addition, workers will be able to readily test agents etc. that may affect the
expression
ability or pattern of that regulatory element.
By way of further example, if the NOI in the YAC has a functional role other
than an
expression regulatory role then by use of the insertion vector according to
the invention
workers can fuse (either directly or indirectly such as by means of one or
more spacing
3o nucleotide sequences) the NOI to the reporter gene according to the present
invention.
Thus, if the NOI is fused to the reporter gene according to the present
invention and is
present in a transgenic organism according to the present invention, then
workers can
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26
readily determine which sites or regions that NOI is expressed. In addition,
workers
will be able to readily test agents etc. that may affect the expression
pattern of that NOI.
A further advantage is that by being able to readily monitor the expression
pattern or
level of the NOI enables workers to determine the phenotype.
One aspect of the present invention concerns the use of SEQ ID No. l or SEQ ID
No. 4
or a variant, homologue or derivative thereof. Here, the term "variant,
homologue or
derivative thereof" includes any addition, substitution or deletion of one or
more nucleic
acids providing the resultant entity can still function as an IRES.
0
For a preferred aspect of the present invention it is envisaged that any
variant, homologue
or derivative of the IRES sequence comprises at least 100 by of SEQ ID No.l or
SEQ ID
No. 4. Preferably, any variant, homologue or derivative comprises at least 200
by of SEQ
ID No.l or SEQ ID No. 4. Preferably, any variant, homologue or derivative
comprises at
s least 300 by SEQ ID No.l or SEQ ID No. 4. Preferably, any variant, homologue
or
derivative comprises at least 400 by of SEQ ID No.l or SEQ ID No. 4.
Preferably, any
variant, homologue or derivative comprises at least S00 by of SEQ ID No.l or
SEQ ID
No. 4. Preferably there is at least 809 sequence identity, preferably at least
85°Xo
sequence identity, preferably at least 90 % sequence identity, preferably at
least 95 ~
2o sequence identity, more preferably there is at least 98l sequence identity
with the
sequence shown as SEQ ID No. l or SEQ ID No. 4.
Preferably, the nucleotide sequence is the sequence presented as SEQ ID No.l
or SEQ
ID No. 4.
As indicated, the YAC vector of the present invention comprises the nucleotide
sequence
presented as SEQ ID No.l or SEQ ID No. 4 or a variant, homologue or derivative
thereof. Here the nucleotide sequence increases the expression efficiency of
one or more
NOIs within a YAC vector or a YAC.
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27
The YAC vector may additionally comprise one or more marker genes. These genes
can
be chosen from suitable marker genes that are available. An example of a
suitable
marker gene is PGK Hyg (see Nara et al 1993 Curr Genet 23(2}: pp 134-140).
The nucleotide sequence of the present invention can be used to modify a YAC
or a
YAC vector, such as pYAC 1, pYAC2, pYAC3 or pYAC4 etc.
The present invention also encompasses combinations of the above-mentioned
aspects.
The following samples were deposited by the MRC Brain Metabolism Unit, Royal
Edinburgh Hospital, Morningside Park, Edinburgh, EH 10 5HF in accordance with
the
Budapest Treaty at the recognised depositary The National Collections of
Industrial and
Marine Bacteria Limited (NCIMB) at 23 St. Machar Drive, Aberdeen, Scotland,
United
Kingdom, AB2 1RY on 24 November 1997
JM 109 pYIV 1 - deposit number NCIMB 40907
JM109 pYIV2 - deposit number NCIMB 40908
JM109 pYIV3 - deposit number NCIMB 40909
JM109 pYIV4 - deposit number NCIMB 40910
2o JM109 pYAM4 - deposit number NCIMB 40906
The present invention will now be described only by way of example, in which
reference
shall be made to the following Figures:
2s Figure 1 which is a diagrammatic representation of pYIVl;
Figure 2 which is a diagrammatic representation of pYIV2;
Figure 3 which is a diagrammatic representation of pYIV3;
Figure 4 which is a diagrammatic representation of pYIV4;
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28
Figure 5 which is a photographic image;
Figure 6 which is a diagrammatic representation of pYAM4;
Figure 7 which is a picture of a gel;
Figure 8 which is a photographic image;
Figure 9 which is a photographic image;
Figure 10 which presents a nucleotide sequence;
Figure 11 which is a picture of a gel;
Figure 12 which is a PCR map of the integrated SERT 35D8/D6 YAC DNA;
Figure 13 which is a PCR map of the integrated VIPR2 HSC7E526/V 12 YAC DNA;
Figure 14 which is a photographic image;
Figure 15 which is a photographic image;
Figure 16 which is a graphical representation of ~i-Gal enzyme activity
determined using a
chemiluminescent reporter assay system; and
Figure 17 which is a schematic diagram.
In slightly more detail:
Figure 7 shows implication of YAC DNA by pYAM4. The endogenous chromosomal DNA
from S. cerevisiae is shown in lane 3 and sizes in kb at the left. All other
lanes were loaded with
DNA plugs form Lys+ YAC clones and cultured in medium with galactose but
lacking lysine
after retrofitting with pYAM4. The migration position of YAC DNA in each clone
is indicated
with an arrow. Levels of amplification of the YAC DNA are based on comparison
of ethidium
RECTIFIED SHEET (RULE 91)
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29
bromide staining of YAC DNA to that of endogeous chromosomes of similar size.
Lane 1, 350
kb, 8-fold, lane 2, 630 kb, 3-fold, lane 4, 615 kb, 3-fold, lane 5, 500 kb, 4-
fold, lane 6, 200 kb,
5-fold, lane 7, 200 kb, 6-fold; lane 8, 230 kb, 2-fold; lane 9, 150 kb, 3-
fold, lane 10/11/12, 230
kb, 5-fold.
Figure 10 shows the IRES sequence which is derived from Encephalomycocarditis
virus. The
sequence has been genetically modified at the 3' end to introduce a Hindlll
restriction site.
Figure 14 shows the immunohistochemical staining of the LacZ reporter gene in
the
t 0 suprachiasmatic nuclei of transgenic mice expressing a YAC containing the
human VPAC2R
gene. The single cell resolution obtainable with the immunohistochemical
approach is worthy of
note.
Figure 15 illustrates the histochemical staining of (3-galactosidase activity
in transgenic mice
containing the YAC HSC7E526/V 12.
Figures 15a and 15b show staining patterns for (3-galactosidase activity in a
coronal slice from
the brain of a transgenic mouse for whom mouse A108.2 was the father. In (a)
the stained
suprachiasmatic nuclei are indicated with arrowheads, in (b) an enlarged view
is shown. Figure
15c shows staining in the pancreas from the same transgenic mouse (tg) and in
a wild type (wt)
littermate.
30
Figure 16 illustrates the tissue distribution of (3-galactosidase activity in
transgenic mice
containing the YAC HSC7E526/V 12.
~galactosidase (LacZ) activity was determined in tissue extracts from control
mice and two
independent lines expressing the hVPAC2R-HA-IacZ transgene. Enzyme activity
was determined
using a chemiluminescent reporter assay system (Galacto-Light Plus, Tropix).
ND indicates
tissues in which no (3-galactosidase activity was detected.
For ease of reference, some parts of the following text has been split
according to the vector of
the first aspect of the present invention or according to the vector of the
second aspect of the
present invention.
RECTIFIED SHEET (RULE 9~
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INSERTION VECTOR
5 Construction of YAC Insertion vectors
For genetic manipulation of YAC DNA, we have generated a series of
modification
cassettes which can be inserted into any YAC DNA:
1o pYIVi
A cassette containing a lacZ reporter gene flanked by IRES and the LEU2
selective
marker. The vector can be used for YACs which have been introduced into a Leu
yeast
strain. The details of the construction procedure were as follows:
~s
A 3.5 kb cassette containing the lacZ gene and polyadenylation sequences was
isolated
from pMRl3-lac~PA (23) by SaII (complete) and EcoRI (partial) digestion and
inserted
into the EcoRI-SaII sites of pBluescript SK-, generating pSK-IacZ PA. The IRES
was
introduced into pSK-IacZ PA by replacing the 1. l kb XbaI (in the polylinker)-
EcoRV (in
2o the lacZ sequence) fragment with a 1.7 kb XbaI-EcoRV fragment (IRES-5'-
lacZ) from
pIRES-bgeo (24), resulting in pIRES-lacZ-PA. A 2.2 kb XhoI-SaII fragment
containing
the LEU2 gene was isolated from pDB248 (25) and inserted into the compatible
SaII site
in pIRES-IacZ PA in the same orientation as the IacZ gene, resulting a plasmid
(pYIVl)
containing the IRES-lac~PA-LEU2 cassette in pBluescript SK- (Fig. 1). Five
unique
25 restriction sites flank the cassette (SacII, NotI and XbaI between the T3
primer and the
IRES, SaII and XhoI sites between LEU2 gene and the T7 primer) which allow
insertion
of genomic DNA on both sides of the cassette.
3o pYIV2 is similar to pYIV 1 except that the LEU2 gene is replaced by the
ADE2 gene so
that the plasmid can be directly introduced into the conventional yeast strain
(AB 1380)
used for the construction of most YAC libraries. A 2.5 kb BglII fragment
containing the
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31
ADE2 gene was isolated from pASZ 11, filled in and inserted into the filled in
HindIII
site of pBluescript SK- in an orientation such that the T7 promoter is
adjacent to the 5'
end of the ADE2 gene (pSK-ADE2). The IRES-lacZ-PA cassette was released from
pIRES-IacZ PA by SaII digestion, filled in and then cut with NotI. The
fragment was
inserted into the EcoRI (filled in) and Notl sites of pSK-ADE2, resulting a
plasmid
(pYIV2) containing and IRES-lacZ ADE2 cassette (Fig.4). Four unique sites
(SacII,
NotI, SaII and XhoI) are available for cloning.
pYN3
to
For many genes, there is no antibody available to detect the encoded protein.
We
introduced a haemaggluttinin (HA) epitope tag into pYIV2 so that a
commercially
available antibody can be used to localise the distribution of the protein
product of a
YAC transgene within cells. The HA tag was introduced as follows:
The translation stop colon of the human VIP2 receptor cloned in the vector
pcDNA3
was converted into an XhoI site by PCR-based mutagenesis. A linker encoding
the HA
epitope tag flanked by Xhol-XbaI sites
5'-TC GAG TAC CCA TAC GAT GTT CCA GAT TAC GCC TCC CTC TAG-3'
3'-ATG GGT ATG CTA CAA GGT CTA ATG CGG AGG GAG ATC AGA TC-5'
was cloned into the XhoI-XbaI sites at the end of the VIPZ receptor. The VIP2
receptor-
HA fragment was released from the pcDNA3 vector by BamHI and XbaI (filled-in)
digestion and cloned into pBluescript SK- (in which the XhoI site was removed
by filling
in) at the BamHI-EcoRV sites generating pSK-VIP2R-HA.
The IRES-lacZ ADE2 cassette was isolated from pYIV2 with NotI (filled in) and
Salt
3o restriction enzymes and inserted into the HindIII (filled in) and SaII
sites of pSK-VIP2R-
HA, yielding a plasmid containing VIP2R-HA-IRES-IacZ ADE2. The HA-IRES-lacZ-
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32
ADE2 cassette was isolated by XhoI-SaII digestion and inserted into pGEMIIZ at
the
XhoI-SaII sites, resulting in pYIV3 (Fig.3). In pYIV3, the HA-IRES-lacZ-ADE2
cassette is flanked by NotI and XhoI restriction sites at the 5' side and by
SaII and SfiI
sites at the 3' side, facilitating the cloning of genomic fragments of
interest for YAC
manipulation.
pYIV4
pYIV4 is similar to pYIV3 except that the orientation of the ADE2 gene is
opposite to
1o that in pYIV3 and two loxP elements, in same orientation, were introduced,
one at the
BgIII site between the IacZ and PA, and another following the ADE2 gene.
The loxP sequence from pBG was cloned into pBluescript SK- at EcoRI and SaII
sites
(pSK-IoxP). The ADE2 gene was excised from pSK ADE2 by EcoRI and CIaI (filled
in)
digestion and cloned into pSK-loxP at the EcoRI and SmaI sites (pSK-IoxP
ADE2). The
EcoRI-SaII loxP fragment from pSK-IoxP was isolated, blunt ended with Klenow
and
inserted into the BgIII (filled in) site between the lacZ and poly A sequences
of pSK-
IRES-lacZ PA generating pSK-IRES-lacZ IoxP-PA. The IRES-lacZ loxP-PA cassette
was isolated by NotI and SaII (filled in) and cloned into pSK-loxP-ADE2 at the
NotI and
2o BamHl (filled in) sites, yielding a construct pYIV4 (Fig.4). The pYIV4
vector permits
deletion of the SV40 PA and ADE2 gene in the YAC transgenic animals using Cre
recombinase so that a transgene and IacZ reporter gene can be followed by its
own 3'-
untranslated region.
Yeast DNA preparation
Yeast DNA was isolated with the combined methods of Schedl et al. (26) and
Bellis et
al. (27). Clones were inoculated into 15 ml of medium (Ura-/Lys-) with 2 % of
galactose
instead of glucose as the carbon source. When cells had grown to late log
phase after 2-
4 days, plugs were taken and subjected to novozyme digestion for 4-6 hours as
described
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33
by Schedl et al. (28). Then, plugs were washed in 50 mM EDTA (2 x 30 min) and
digested with proteinase K (2mg/ml) in a buffer containing 0.5 M NaCI, 0.125 M
Tris
pH 8.0, 0.25 M Na2EDTA, 1 ~O Lithium sulphate, and 0.5 M ~-mercaptoethanol at
55°C overnight. Plugs then were washed with TE and stored at 4°C
in 0.5 M EDTA.
Pulsed field gel electrophoresis
DNA plugs were washed in TE (3 x 30min), loaded on a 1 °6 agarose gel
and sealed with
1 % agarose in 0.5 x TBE buffer. Gels were run in 0.5 x TBE buffer at 6V/cm
for 24
1o hours at 14°C with 60 sec. switch time. After running, gels were
stained with ethidium
bromide and photographed.
AMPLIFICATION VECTOR
Construction of pYAM4
pYAM4 was constructed using pYAC4, pBluescript SK- and pBG. pBG is a
modification of pCGS990 in which the SaII site has been converted to a NotI
site and a
PGK-Hyg-loxP cassette has been introduced between the LYS2 and TK genes in
2o pCGS990.
pBG was constructed as follows: The unique SaII site in pCGS990 was converted
into
NotI with the XhoI-NotI-XhoI linker
5'-TCGAGCGGCCGC-3'
3'-CGCCGGCGAGCT-5'
3o resulting in pCGS990N. A 2044 by PstI fragment containing the
chloramphenicol
resistance gene (cm) flanked by two loxP sites was excised from pUC91ox2cm,
blunt
ended with T4 DNA poiymerase and ligated to the filled-in SphI site of pHA58
which
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34
contains a Hygromycin B resistance gene flanked by the mouse Pgkl
(phosphoglycerate
kinase 1) promoter and polyadenylation signal. The resulting plasmid
(pHA581ox2cm.1)
was digested with BgIII, a 3.5 kb BgIII fragment harbouring a IoxP-cm-loxP-Hyg
cassette was isolated, filled in with Klenow and inserted into the EcoRI site
(filled in)
between the TK and LYS2 genes in pCGS990N, obtaining pCGS990N-Hyglox2cm. The
chloramphenicol resistance gene was removed from pCGS990N-Hyglox2cm with
purified Cre recombinase. DNA was purified, transformed into E. Coli XL-1 Blue
and
chloramphenicol sensitive colonies were selected, leading to the production of
pBG.
to A 572 by (SmaI-CIaI) fragment between the cloning site and CEN4 in pYAC4
was
cloned into SmaI-CIaI sites of pBluescript SK- vector. The fragment was
excised with
CIaI and NotI and inserted into the CIaI-NotI sites of pBG, resulting in
pYAM3.
A 705 by XhoI-BamHI fragment of the telomere (TEL) from pYAC4 was blunt ended
~5 with Klenow and inserted into the filled in SacI-SacII sites of pBluescript
SK- vector.
The orientation of the TEL in the resulting plasmid (pSK-TEL) was confirmed by
sequencing with T7 and reverse primers. pSK-TEL was digested with NotI-SaII
restriction enzymes and replaced the corresponding region (pBR322-TEL-TK LoxP)
in
pYAM3, leading to the generation of pYAM4 (Fig. 6 ).
Transformation
pYAM4 was linearised with NotI. Yeast were inoculated into 10 ml SD medium
lacking
uracil and tryptophan. When yeast had grown to 2 x 107 cells/ml, they were
harvested
and washed with 5 ml of LTE (O.1M LiOAc, lOmM Tris pH7.5, and 1 mMNa2EDTA).
After resuspension in 100 ~l of LTE, cells were incubated at 30°C for
one hour with
regular inversion. One ~.g of linearised pYAM4 and 5 ~,1 of carrier DNA
(salmon sperm
DNA,10 mg/ml) were added to the cells and mixed. After incubation at
30°C for 30
minutes, 0.7 ml of PEG/LTE (40 k PEG 3,300 in LTE) was mixed with the cells
and
3o incubated at 30°C for 30 minutes. Then cells were heat-shocked at
42°C for 5 minutes.
Cells were spun down for 2 min at 500 x g and washed twice with TE. After
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resuspension in 400 ~cl TE, cells were plated out on SD medium lacking uracil
and
lysine. Plates were kept at 30°C for 3-5 days. Single colonies were
picked and plated
onto dishes lacking lysine or tryptophan. Clones which could not grow on the
medium
lacking tryptophan were selected for further analysis.
s
Yeast DNA preparation
Yeast DNA was isolated with the combined methods of Schedl et al. (26) and
Bellis et
al. (27). Clones were inoculated into 15 ml of medium (Ura-/Lys-) with 29b of
o galactose instead of glucose as the carbon source. When cells had grown to
late log
phase after 2-4 days, plugs were taken and subjected to novozyme digestion for
4-6
hours as described by Schedl et al. (28). Then, plugs were washed in 50 mM
EDTA (2
x 30 min) and digested with proteinase K (2mg/ml) in a buffer containing 0.5 M
NaCI,
0.125 M Tris pH 8.0, 0.25 M Na2EDTA, 1 °b Lithium sulphate, and 0.5 M ~-
~5 mercaptoethanol at 55°C overnight. Plugs then were washed with TE
and stored at 4°C
in 0.5 M EDTA.
Pulsed field gel electrophoresis
2o DNA plugs were washed in TE (3 x 30min), loaded on a 1 % agarose gel and
sealed with
1 °~fo agarose in 0.5 x TBE buffer. Gels were run in 0.5 x TBE buffer
at 6V/cm for 24
hours at 14°C with 60 sec. switch time. After running, gels were
stained with ethidium
bromide and photographed.
25 COMBINATION OF INSERTION VECTOR AND AMPLIFICATION VECTOR
Combination of the pYlV3 and pYAM4 for YAC transgenic study
The vector pYIV3 was used to introduce the haemagglutinin (HA) tag and IacZ
reporter
3o gene into two YAC clones,35D8 (SOOkb) and HSC7E526 (630kb), which contain
the
human serotonin transporter (SERT) and VIP2 receptor (VIPR2) genes
respectively. In
order to integrate the lacZ reporter gene into each of the YAC clones by
homologous
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recombination, genomic DNA sequences (at least a few hundred bp) flanking the
stop
colon of each gene were introduced on either side of the HA-IRES-IacZ-Ade2
sequences
of the pYIV3 vector.
Construction of the pLacZVIPR2+ vector
The XhoI site in the polylinker of pBluescript SK- was removed by digesting
the vector
with XhoI and filling in the recessed 3' termini with Klenow fragment of E.
coli DNA
polymerise I, generating pSKX. A BamHI-XbaI fragment containing the human
VIPR2
to cDNA with the HA tag at the C-terminus of the coding sequence (see McDonald
et al
1997 Biochem Soc 25:442S) was subcloned from the pcDNA3 vector into pSKX at
the
EcoRV site in an orientation such that the 5' end of the cDNA was adjacent to
the T3
primer in the pSKX vector, generating pSK-VIPR2-HA. A NotI-PstI fragment of
pSK-
VIPR2-HA containing VIPR2 cDNA sequences was then replaced with a 1.2 kb NotI-
t5 PstI fragment of VIPR2 genomic DNA (PstI cuts in the last coding exon of
the human
VIPR2 gene), generating pVHA. The IRES-IacZ PA-Ade2 cassette was excised from
pYIV2 as a SaII - NotI (blunt ended) fragment and inserted into the SaII-
HindIII (blunt
ended) sites of pVHA, resulting in pVHAIZA.
2o By PCR using primers
32366 (5'-CAA ACG GAG ACC TCG GTC CTC GAG CCC CAC-3')
and
32496 (5'-CGG GTA CCA AAA TGG TGG GTT GTT CTG TAA-3')
XhoI and KpnI restriction sites were introduced at ends of a 1.6 kb fragment
of genomic
DNA 3' of the stop colon of the human VIPR2 gene. The fragment was subcloned
into
3o the XhoI and KpnI sites of the pSK- vector, generating p3'VIPR2.
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37
The NotI-SaII fragment of pVHAIZA which contains VIPR2-HA-IRES-lacZ-PA-Ade2
was ligated into NotI-XhoI digested p3'VIPR2, generating a final construct,
pLacZVIPR2+. For efficient homologous recombination in yeast, genomic DNA
sequences at least a few hundred by in length must flank the stop codon of the
target
s gene either side of the HA-IRES-IacZ-Ade2 sequences of pYIV3. In pLacZVIPR2+
there is 1.3 kb of VIPR2 genomic sequence upstream and 1.6 kb of VIPR2 genomic
sequence downstream of the HA-IRES-lacZ-Ade2 cassette.
Construction of the pLacZSERT'' vector
1o
The XhoI site in the polylinker of pBluescriptSK' was removed by digesting the
vector
with XhoI and filling in the recessed 3' termini with Klenow fragment of E.
coli DNA
polymerase, generating pSKX. A BamHI-XbaI fragment containing the human VIPR2
cDNA with the HA tag at the C-terminus of the coding sequence was subcloned
from the
1 s pcDNA3 vector into pSKX at the EcoRV site, in an orientation that the 5'
end of the
cDNA is adjacent to the T3 primer in the pSKX vector, generating pSK-VIPR2-HA.
A
kb human genomic DNA fragment contaning intron 13 and exon 14 of the SERT gene
was cloned into NotI-XhoI sites of pBluescriptSK- using PCR primers
20 32365 (5'ACT GCA TAG CGG CCG SAT CTT TCA TTT GCA TCC CC 3')
and
32853 (5' TGT GCT CGA SAG CAT TCA AGC GGA TGT 3')
generating pInl3. To introduce the HA tag into the C-terminus of the SERT gene
product, the 5 kb SERT intron 13 sequence (NotI-XhoI fragment) was used to
replace the
NotI-XhoI fragment in pSK-VIPR2-HA, generating pInl3-HA. The intron 13
sequence
and the HA tag were isolated as a SacII - CIaI (blunt ended) fragment and
inserted into
3o SacII and (blunt ended) NotI sites of pYIV2, generating pInl3-HA-IZA. The
sequences
downstream of the stop codon in exon 14 of the SERT gene were isolated by PCR
of
human genomic DNA using primers
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38
32358 (5' CTC sZ~ AGG AAA AAG GCT TCT 3' )
and
32359 (5' TAB ~T,A, ~C TGT TCT CTC CTA CGC AGT TT 3' )
and cloned into the XhoI-KpnI sites of pBluescriptSK- generating p3'SERT.
Finally, the
intronl3-HA-IRES-IacZ Ade2 fragment was isolated by NotI and SaII digestion of
pInl3-HA-IZA and inserted into the NotI and XhoI sites of p3'SERT, resulting
in
to pLacZSERT+
Introduction of the Insertion Constructs into the YAC DNA
The pLacZVIPR2+ and pLacZSERT+ constructs were linearised with NotI and
introduced into the YAC clones HSC7E526 and 35D8 respectively: The
transformants
which incorporated HA-lacZ Ade2 sequences into YAC DNA by homolgous
recombination were selected by growing on plates lacking uracil, tryptophan
and
adenine. The integration of the HA-IRES-lacZ Ade2 sequence into the YAC DNA
was
confirmed by Southern hybridization with an Ade2 probe.
Amplification of Modified YAC DNA
YAC subclones which incorporated the HA-IacZ Ade2 cassette were transformed
with
NotI linearised pYAM4. Recombinants were isolated on selective medium lacking
uracil,
adenine and lysine and replica plated on plates lacking uracil, adenine and
tryptophan.
Successful replacement of the YAC left arm (containing TRPl gene) by pYAM4
would
result in yeast capable of growth on medium lacking uracil, adenine and lysine
but not
on the counter selection medium lacking tryptophan.
3o Tryptophan sensitive clones were cultured in selective medium (Ura /Ade
/Lys~ with
galactose as carbon source instead of glucose. In such medium, the GAL1
promoter
adjacent to the CEN4 in the pYAM4 vector will be induced. Activation of
transcription
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39
from the GAL1 promoter interferes with the function of the CEN4 leading to
non-segregation of the YAC DNA and a consequent increase the YAC DNA copy
number per cell.
TR_ANSFOR1~FD CELLS/TR_ANSOENIC ORGANISMS
A YAC according to the present invention may be transfered into mammalian
cells by
appropriately adapting the teachings of Schedl et al (reference 28). By the
term
"adapting" we mean following the teachings but using the vectors of the
present
1o invention where appropriate. It is to be understood that other techniques
may be used to
transfer a YAC according to the present invention in mammalian cells and these
other
techniques are well documented in the art (e.g. for example see WO-A-95/14769
and/or
Gietz et al 1995 Yeast vol 11 No. 4, pp 355-360).
According to Schedl et al (ibis, possibly the most straight forward approach
to generate
transgenic cell lines is the transfer of YACs by sphaeroblast fusion (this is
a technique
disclosed in the previous chapter of Reference 28). This method, however,
normally
leads to integration of the entire yeast genome in addition to the YAC, which
might
obscure the results of some experiments. In contrast, direct microinjection of
DNA into
2o the nucleus of a recipient cell allows purification of a YAC prior to the
transfer.
Some teachings of Schedl et al (ibic~ are as follows:
Materials
1. SE: 1M Sorbitol, 20mM EDTA (pH8.0).
2. TENPA: IOmM Tn's-HCI (pH 7.5), ImM EDTA (pH8.0), 100mM NaCI, 30pM
spermine, 70~M spermidine.
3. IB: IOmM Tris-HCI (pH7.5), O.ImM EDTA (pH8.0), 100mM NaCI, 30~M
spermine, 70~M spermidine.
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WO 99/28449 PCT/GB98/03558-
4. LIDS: 1 l lithium-dodecyl-sulphate, 100mM EDTA (pH8.0).
5. Zymolyase, ICN Biomedicals Inc., Costa Mesa, CA, USA.
s 6. Nusieve low melting point (LMP) agarose, FMC, Rockland, ME, USA.
7. Seaplaque low melting point (LMP) agarose, FMC, Rockland, ME, USA.
8. Automatic Injection System, Zeiss, Germany.
to
9. Femptotips, Eppendorf,
10. Insert moulds (plug formers), Pharmacia, Uppsala, Sweden.
1s 11. CHEF-DR 11, pulsed-field-gel-electrophoresis (PFGE) system, BIO-RAD
Laboratories, Richmond, CA, USA.
12. ~3-mercaptoethanol (14M stock).
20 {Note: Heavy metal ions present in buffers even in traces will lead to
degradation of the
YAC DNA during the agarase treatment. Make sure to use water of highest
quality for
the preparation of buffers.}
Methods
Preparation of high density agarose plugs for preparative Pulsed Field Gel
Electrophoresis (PFGE)
1. Inoculate 500 ml of of SD medium (-URA) with your yeast clone and grow the
3o culture to late log phase.
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41
2. Prepare a solution of 1 °~ Seaplaque LMP agarose in SE buffer
containing l4mM
(3-mercaptoethanol and keep at 45°C until use.
3. Spin down cells at 4000 rpm for Smin (Sorvall RT6000). and resuspend the
pellet
in SOmI SE. Transfer the cell suspension into a SOmI Falcon tube.
4. Seal the bottom of Pharmacia plug formers (insert moulds) with strips of
tape and
place them on ice.
l0 5. Wash cells twice with SE (4000 rpm, Smin.).
6. After the last washing step, discard the supernatant, and carefully remove
all
liquid by cleaning the inside of the tube with a paper towel. The cell pellet
should be
about 1 to 1. Sml.
7. Add 200 pl of SE buffer. With a cut off tip try to resuspend the pellet.
The
suspension will be very thick and difficult to pipet.
8. Transfer O.SmI aliquots of the cell suspension into 2m1 Eppendorf tubes and
keep
2o at 37°C.
9. Just before use dissolve lOmg Zymolyase in 2.Sml of the LMP agarose
solution.
{Note: Zymolyase does not completely dissolve at this concentration. Weigh in
the
required amount and work with a protein suspension. }
10. Transfer O.SmI of this solution to the yeast cell suspension and mix
thoroughly
the agarose with the cells by pipetting up and down using a blue cut off tip.
{Note: Only a completely homogenous mixture will yield in high quality plugs
with even
distribution of DNA. }
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42
Keep the solution at 45°C at all times to avoid setting of the
agarose.
11. Using a cut off yellow tip pipet 80p1 aliquots of the mixture into plug
formers
kept on ice. Leave for 10 min to allow the agarose to set.
12. Transfer the plugs into SE buffer containing l4mM ~i-mercaptoethanol and
lmg/ml Zymolyase. Incubate at 37°C for 4 to 6h.
13. Replace the buffer with LIDS buffer using at least O.Smllplug and incubate
at
to 37°C with gentle rocking. After 1 h refresh the LIDS buffer and
continue incubation
overnight.
14. Wash plugs extensively in TE pH8.0 until no more bubbles can be seen.
Store
plugs in O.SM EDTA at 4°C until use.
{Note: DNA plugs prepared this way can be stored without degradation for at
least one
year.
Isolation of intact YAC DNA for microinjection
1. Cast a gel using 0.25xTAE, 1 % agarose using a comb of which you have
sealed
several teeth of the comb with tape to obtain a preparative lane of
approximately Scm.
{Note: To ensure straight bands it is recommended to run the preparative lane
in the
center of the gel. Bands of preparative lanes bigger than 5cm, may show
"smiling"
effects, which leads to imprecise excision of the DNA and, hence, a lower
yield/ final
concentration. }
If the DNA will be concentrated by a second gel run standard agarose can be
used.
3o Otherwise use an LMP agarose.
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43
2. Wash the high density plugs for 4x15min in TE pH8 with gentle shaking on a
rocking platform.
3. Load the plugs next to one another into the preparative lane.
{Note: Best results are achieved using rectangular plugs (such as produced in
Pharmacia
plug formers), which can be loaded next to one another without intervening
spaces. Use
90m1 of the 1 % gel for a small BioRad casting chamber (l4cm x 12.7cm). The
plugs
should occupy the entire height of the gel. Therefore, when casting the gel,
make sure
to that the comb is touching the bottom of the casting chamber. Make sure that
the casting
chamber as well as the PFGE-chamber are absolutely leveled to avoid any loss
of DNA
during the gel run. }
Then seal the slot with 1 % LMP agarose (0.25xTAE).
4. Run the PFGE in a cooled buffer (O.ZSxTAE) using conditions optimized to
separate the YAC from the endogenous chromosomes.
{Note: Best separation results from endogenous chromosomes are achieved using
a
2o single pulse time instead of a time ramp for the entire run. It is worth
while to test out
several conditions before starting the isolation procedure}.
5. After the gel run cut off marker lanes on either side including about 0.5cm
of the
preparative lane and stain them in 0.25xTAE buffer containing O.S~g/ml
ethidium
bromide. Mark the position of the YAC band under UV light using a sterile
scalpel
blade.
6. Reassemble the gel and excise the part of the preparative lane coresponding
to the
YAC-DNA. Cut also slices above and below the YAC DNA to serve as marker lanes
3o for the second gel run.
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44
7. Position the gel slices on a minigel tray with the YAC slice in the middle
and cast
a 4 ~O LMP agarose (Nusieve, FMC) gel 0.25xTAE around them.
8. Run the gel at a 90° angle to the PFGE run for approximately 6-8h at
4V/cm in
0.25xTAE (circulating buffer). The running time depends on the size of the gel
slice as
well as on the agarose used for the PFGE run.
9. Cut off and stain the two marker lanes to localize the DNA.
o {Note: If the DNA has not yet completely run into the Nusieve LMP gel
continue the
electrophoresis. Since it is impossible to digest normal agarose with the
enzyme
agarase, it is important to excise only LMP material. }
10. Excise the concentrated DNA from the corresponding position of the YAC DNA
is lane.
11. Equilibrate the gel slice on a rocking platform in 20m1 of TENPA buffer
for at
least l.Sh.
20 12. Transfer the gel slice into a l.Sml Eppendorf tube and remove all
additional
buffer using a fine tipped pipette.
13. Melt the agarose for 3min at 68°C, centrifuge for lOs to bring down
all of the
molten agarose and incubate for an additional Smin at 68°C.
14. Transfer the tube to 42°C for Smin. Add 2U of agarase (New Englands
BioLabs)
per 0.1 ml of molten gel slice.
{Note: Do not add agarase directly from the -20°C freezer, which can
lead to setting of
3o part of the LMP agarose. Load the enzyme into the tip and allow to warm up
for a few
secoonds by placing into the molten agarose. Carefully release the enzyme
while stiring
slowly with the tip. Mixing can be achieved by releasing air bubbles into the
solution}.
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WO 99/28449 - PCT/GB98/03558-
Incubate for further 3h at 42°C.
15. Dialyse the resulting DNA solution for 2h on a floating dialysis membrane
(Millipore, pore size O.OSpm) against microinjection buffer (lOmM Tris:HCI,
pH7.5,
5 O.ImM EDTA, 100mM NaCI, 30pM spermine, 70p,M spermidine).
16. To determine the DNA concentration, check 2p1 on a thin 0. 8 Rb agarose
gel with
very small slots, using ~, DNA of known concentration as a standard.
to {Note: It is useful to prepare a 2ng/pl stock solution of DNA. Loading of
2, 5, 10 and
20ng of this standard should allow a relatively accurate determination of the
YAC DNA
concentration}
17. The integrity of the DNA can be checked running 20p.1 of the preparation
on a
15 PFGE gel (use a comb with small slots).
Iq~ection into cultured cells
The Zeiss Automatic Injection System (AIS) can be used for rapid injection of
large
2o numbers of cells growing on cell culture dishes. A digital camera attached
to a
microsope transmits an image to the computer screen. An interactive computer
program
is then used to position the pipette above a "reference cell" and to mark the
tip of the
needle on the screen. This position is stored by the computer and serves as a
reference
point for the rest of the injections. Nuclei of other cells visible on the
screen can now be
25 marked by clicking on them with a computer mouse and injections are
performed
automatically by the computer. The amount of DNA injected can be regulated by
the
injection time as well as the pressure set at the Eppendorf injection system.
High
pressures result in higher efflux of the DNA containing solution. The pressure
to be set
depends on the viscosity of the DNA solution and the size of the needle
opening and,
3o therefore, has to be adjusted individually in each experiment. The pressure
in a standard
experiment will vary between 20 and 150 hectopascal. Almost confluent dishes
are best
to inject. A too low cell density allows only a few cells to be injected per
frame,
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46
whereas cells on confluent plates do not grow in one plane making it
impossible to inject
all cells into the nuclei. The efficiency of microinjection will depend
greatly on the cell
type. Best results are achieved using cells with big and easily visible
nuclei.
1. Grow cells on a Scm dish to 80 % confluency in the medium required by the
cell
type.
2. Immediately before injection cover the cells with Sml of fresh medium and
top
layer the dish with Sml of liquid paraffin. Liquid paraffin is preferably used
to prevent
~o contamination of the cells as well as evaporation of the medium during
injections.
3: Switch on computer, microscope, monitor, Eppendorf microinjector and pump
and place the culture dish on the stage.
~5 4. Choose the command STAGE from the main menue to select a region of the
dish
which is almost confluent but in which the cells are still growing in one
plane. The
stage can be moved by clicking (always use the top/yellow button) onto the
crossed
double arrows. The direction of the arrow indicates the direction in which the
stage will
move. The distance from the center of the cross determines the speed with
which the
2o stage moves.
5. Return to the main menu and select MARK/INJECT. A new menu will appear
which allows to choose from the following options:
25 STORE DATA: Allows to generate a file in which the positions of the
injected cells will
be stored. To use this option the bottom of the dish has to be marked to give
the
machine left and right hand references (scratch crosses at either side). Find
the marks
after the plate has been place on the stage and click cursor on the
appropriate box to
record the references. If you generate a file you must enter an operator and a
sample
30 name.
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APPEND: Allows you to go back to a previous file to find the cells which have
been
microinjected.
NO FILE: This option does not record the cells that are injected and is
sufficient for
most applications.
6. Select the number of frames you want to inject by filling in numbers lower
than
for X and Y values. A frame is the window visible on the screen and,
therefore,
represents the field in which cells can be marked and injected at a time. Each
frame has
to specific X and Y coordinates. The computer moves along the x-axis first. An
array of 5
x 10 frames will allow you to inject more than 1000 cells depending on the
confluency of
the plate.
7. Click on DATA OK.
8. Load l.Sp,1 of DNA solution into an Eppendorf microloader and insert it
into an
Eppendorf Femptotip placing it at the very bottom of the tip. Slowly release
the DNA
solution trying to avoid the introduction of air bubbles, which can block the
needle.
9. Twist the tip carefully to remove it from its cover and load the needle by
screwing it into the injection needle holder at the microscope.
10. Choose the option adjust from the menu. Use the mouse to lower the needle
by
clicking onto the arrow in the center of the screen. The distance from the
center
determines the speed of the movement. Start with high speed and slow down when
you
approach the surface of the dish. Once the needle touches the medium find it
in low
power magnification and use the micrometer screws on the pipette holder to
center the
needle in the frame. Change the lens to higher magnification. Focus on a plane
intermediate between the cells and the needle and bring the needle down into
focus.
3o Repeat this procedure until the tip of the needle is pressing down on a
cell. This will
result in a small halo surrounding the needle tip as you press down on the
cell.
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11. The following options are available to adjust the position of the needle
STEP DOWN: Lowers the needle in the smallest possible increment.
s MARK TIP: Allows to set the reference point for the computer software. To
adjust
click on the very tip of the injection needle.
INJECTION TIME: Determines the time the needle remains within the cell and is,
therefore, one parameter for the volume delivered to the nucleus. This time
has to be
1o varied depending on the pressure, tip size etc. A time of 0.2s is a good
value to start
with.
MOVE STAGE: Allows to move the stage directed with the mouse.
15 RESTART: Takes you back to the main menu and you can reset any of the
parameters.
HOME: Takes the needle back to the original position.
POSITION OK: Click on this when you are ready to start injecting.
12. To perform the injections click on MARK NEXT. This will allow to direct
the
computer to the nuclei of cells to be injected. Click on MARK and subsequently
onto
the nuclei. To start the injections click on INJECT. The computer will perform
the
injections into the marked cells. Successfully injected cells can be
identified by a
temporary dramatic swelling of the nucleus. If no change of cells can be
observed after
a number of injections check the following possibilities:
The injection needle is blocked: Use the high pressure button (P3) at the
injection
machine to release DNA. If this does not help the needle has to be replaced.
~ The computer is injecting in the wrong plane: Stop the injections by
pressing the
yellow button and try lowering or lifting the needle in single step
increments. Be
careful not to break the needle on the surface of the dish by lowering it too
much.
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Too low pressure: Increase the pressure for P1. Be aware that too high
pressure will
result in bursting of the cells.
Press the mouse button at any time during irjections to adjust the needle
height or
remark the tip of the needle to inject the whole frame again press RESTART.
Alternatively you can carry on with CONTINUE.
13. To finish the injections press RESTART, MARKJINJECT, HOME, EXIT.
1o Pronuclear injections into fertilised mouse oocytes
The procedure of generating transgenic mice includes isolation of fertilized
oocytes from
superovulated females, microinjection of DNA into pronuclei and the transfer
of injected
oocytes into pseudopregnant foster mothers. A detailed description of these
steps can be
t5 found in for example Hogan, Murphy and Carter (1993 Transgenesis in the
mouse in
"Transgenesis Techniques", Methods in Molecular Biology vol. 18 Ed. Murphy and
Carter, pp 109-176. Humana Press, Totowa, New Jersey) and reference 28
(subsequent
chapter) - the contents of each of which are incorporated herein by
reference).
2o Preparation of DNA constructs for injection normally involves a filtration
step in which
the DNA is passed through a membrane with 0.2pm pore size. This step is
recommended to avoid blocking of the injection needle by ~ dust particles in
the DNA
solution. YAC DNA preparations should not be subjected to filtration, because
of
shearing forces occuring during this step. We have found that blockage of the
needle is
25 a relatively infrequent event if the agarose digestion was successful. In
some cases it
might be preferable to centrifuge the DNA for 5min (12000rpm Eppendorf
centrifuge) to
remove undigested gel pieces. However, since small particles of agarose can
trap DNA
we would strongly recommend to determine the DNA concentration after the
centrifugation step.
Some DNA preparations are very sticky, which is probably due to incomplete
agarose
digestion. In these cases a higher proportion of injected oocytes will be
found to lyse
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and the injection needle has to be exchanged more frequently. Prepare a new
batch of
DNA for the next injection day and take care to digest all agarose. Even more
than with
normal constructs, try to avoid touching the pronuclei during injections. Once
touched
they will stick to the needle and being pulled out. If that happens replace
the
5 microinjection pipet immediately. The percentage of lysed oocytes should not
be
markedly higher, when compared with normal constructs. Injected oocytes can be
either
transfered on the same day to the oviduct of pseudopregnant foster mothers or
incubated
overnight at 37°C in M16 buffer. Normal survival rates (20 to
30°x) of transfered
embryos even at DNA concentrations as high as lOng/p.l should be obtained.
Transgenic animals can be identified by PCR or Southern blot analysis with DNA
isolated from tail tips. With 250kb constructs about 10 to 20% of the
offspring should
have YAC DNA incorporated. Once a transgenic line has been established it is
important
to confirm the integrity of the integrated construct. This can be achieved by
15 conventional PFGE mapping with several probes scattered over the YAC,
which,
however, requires a detailed knowledge of the restriction map of the
construct.
Alternatively, the RecA approach can be used to release the entire YAC from
the mouse
genome.
20 ~$~~
INSERTION VECTOR
General
2s The analysis in transgenic animals of genes expressed in YACs can be
greatly facilitated
by the use of a reporter gene for the accurate and sensitive detection of
cellular sites of
transcription. We have constructed a series of YAC modification vectors
(pYIVi,
pYIV2, pYIV3 and pYIV4) which can be inserted into YACs after the translation
initiation or stop colon. The common feature of all of these vectors is that
they contain
3o a lacZ reporter gene downstream of a viral internal ribosome entry site
(IRES), together
with selective markers. In transgenic animals expressing these constructs, the
IacZ
reporter gene will be expressed in the same pattern as the transgene so that
the
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expression, regulation and function of the transgene can be analysed using
simple
histochemical staining procedures. This approach may provide a more complete
picture
of the pattern of expression of the transgene than standard procedures such as
in situ
hybridisation. The pYIV 1 vector can be used for YACs which have been
introduced
into a Leu- yeast strain, while pYIV2, pYIV3, and pYIV4 can be directly
introduced
into the yeast strain (AB1380) which was used for construction of most YAC
libraries.
One of the vectors (pYIV3) permits the HA epitope tag sequence (from influenza
hemagglutinin) to be fused to the carboxyl terminus of the expression product
of the gene
of interest, so that the protein product of the transgene can be detected
using the
to commercially available 12CA5 monoclonal antibody. pYIV4 contains loxP
elements
flanking the SV40 polyadenylation signal and the ADE2 gene. In transgenic
animals
generated using pYIV4, the polyA sequence and the ADE2 gene sequences can be
deleted using Cre recombinase so that the transgene and lacZ reporter gene are
flanked
by the authentic 3'-untranslated region of the transgene. A comparison between
animals
is containing the SV40 polyadenylation signal and the ADE2 gene with those in
which these
sequences have been removed will reveal the function of the 3' sequence of the
transgene.
Expression of the IRES-lacZ from pYlVl in YAC transgenic mice
We have examined the expression of the lacZ reporter gene in transgenic mice
expressing a YAC clone modified using the pYIV 1 insertion vector. Insulin-
like growth
factor II (IGF2) cDNA was introduced 3' of the Wilm's tumor (wtl) gene
promoter
(isolated from a 480 kb human YAC clone). Then the promoter and cDNA were
2s inserted at the NotI-XbaI sites 5' of the IRES-lacZ LEU2 cassette in the
pYIVl vector.
The first intron of the wtl gene was cloned 3' of the LEU2 gene. The construct
was
introduced to the 480 kb YAC by homologous recombination. After amplification,
the
modified YAC DNA was isolated and micro-injected into fertilised eggs. Eight
lines of
transgenic mice were produced, 5 of which expressed the lacZ reporter gene.
All
3o expressing lines produced an X-Gal (it is understood that the terms X-Gal,
~-Gal and
LacZ are synonymous) staining pattern (Fig.S) identical to that of the human
gene from
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52
which the promoter was derived. These data demonstrated that the IRES-IacZ
reporter
gene is functional in the YAC insertion vectors.
AMPLIFICATION VECTOR
The improved homologous recombination efjlciency of pYAM4
To assess the homologous recombination efficiency of pYAM4, the plasmid was
linearised with NotI and introduced into a variety of YAC clones from the ICI,
ICRF and
to chromosome-7-specific YAC libraries. Recombinants were isolated on
selective medium
lacking uracil and lysine and replica plated on plates lacking uracil and
tryptophan.
Successful replacement of the left arm (containing TRPl gene) by pYAM4 would
result
in yeast capable of growth on medium lacking uracil and lysine but not on the
counter
selection medium lacking tryptophan.
Of a total of 1266 Lys + clones analysed, 167 clones could not grow on medium
lacking
tryptophan (Table 1). That is, the homologous recombination leading to the
loss of the
TRP1 gene occurred in 167 clones. The retrofitting efficiency of pYAM4 overall
is
13.3% which is much higher than pCGS990 and pCGS966 (0.5-2.5°x) (11,
12).
Table I. Retrofitting efficiency of pYAM4 in a variety of YAC clones from
different
YAC libraries
YAC clone Insert Size Ura-/Lys- Ura-/Trp- (not growing)
ICI YAC Clones
1 (16FC9) 51 4
2. (27FE5) 320 55 3
3. (30FH70 350 105 33
4. (3HG4) 300 46 2
5. (36AH3) 300 77 16
6. ( 12GG6) 300 141 31
Subtotal 475 89 ( 18 .7 k )
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53
ICRF YAC clones
1. (49A9) 340 37 3
2. (35D8) 500 103 8
3. (132C6) 630 133 19
Subtotal 274 30 ( 10.9 ~ )
Chromosomal YAC clones
HSC7E526 550 107 8 (7.5 ~)
E145A7 200 129 11 (8.59b)
ywss922 350 57 8
ywss 1545 420 59 8
~ 5 ywss2056 400 15 2
ywss3844 300 151 11
Subtotal 282 29 ( 10. 3 rb )
2o Total 1266 167 ( 13 .3 9b )
The increased efficiency of retrofitting is probably due to the introduction
of a 572 by
CIaI-SmaI fragment from pYAC4 adjacent to the CEN4. When the SmaI-CIaI
fragment
of pYAM4 was deleted by NotI-CIaI digestion, or pYAM4 was linearised with
CIaI, the
25 frequency of tryptophan sensitive clones was not significantly different
from that
obtained with pCGS990.
Amplification of YAC DNA by pYAM4
3o Tryptophan sensitive clones were cultured in selective medium (Ura-/Lys-)
with
galactose as carbon source instead of glucose. In such medium, the GAL1
promoter
adjacent to the CEN4 in the pYAM4 vector will be induced. Activation of
transcription
from the GAL1 promoter should interfere with the CEN4 leading to non-
segregation of
the YAC DNA therefore increase the YAC DNA copy number per cell.
As shown in Fig 7, depending on the size and nature of the insert, human YAC
DNA
was amplified 3 to 5 fold. Although the amplification is not as high as that
achieved
with pCGS990, it helps to isolate more concentrated YAC DNA for transgenics.
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Introduction of an additional conditional promoter such as ADH2 adjacent to
the CEN4,
or of an additional selection gene, might improve the amplification further.
We have introduced the bacterial hygromycin B resistance gene under the
control of the
mouse Pgkl promoter into pYAM4. After retrofitting with pYAM4, isolated YAC
DNA can be introduced into mammalian cells such as embryonic stem (ES) cells,
which
is an alternative approach to microinjection of YAC DNA for making YAC
transgenic
animals (29).
1o If a genomic fragment (which is present in a YAC) is cloned into the CIaI-
NotI site of
pYAM4, the truncation of a large YAC and amplification of the shortened YAC
DNA
can be achieved in a single step.
COMBINATION OF INSERTION VECTOR AND AMPLIFICATION VECTOR
The results are presented in Figures 8 and 9. In this regard, Figures 8 and 9
are
photographs of gels.
The amplification of the YAC DNA can be seen from Fig 8 and 9. In this regard:
Lane
1 is un-amplified YAC DNA as present in original 35D8 YAC clone, lane 2 is
un-amplified YAC DNA in another YAC clone (132C6) containing the SERT gene,
and
lane 3 is amplified YAC DNA in 35D8/D6 subclone. The blot was hybridized with
genomic probes downstream (Fig.B) and upstream (Fig.9) of the SERT gene.
AMPLIFICATION AND PURIFICATION OF YAC DNA
The integration of the amplification vector pYAM4 into YAC clones 35D8 and
HSC7E526 greatly increased the yields of YAC DNA obtained. These results are
presented in Figure 11. In this regard, Figure 11 is a photograph of a gel
prepared by a
3o Southern blot and hybridised with a 32P-labelled pBR322 probe to detect YAC
sequences. In each lane, the hybridising band corresponding to YAC DNA is
arrowed
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Lane 1 is DNA from yeast containing YAC clone 35D8; Lane 2 is DNA from yeast
containing YAC clone 35D8/D6, in which the YAC has been modified by
integration of
the amplification vector pYAM4 and the insertion vector pYIV3; Lane 3 is the
isolated
YAC DNA from clone 35D8/D6 prior to microinjection. Lane 4 is DNA from yeast
s containing YAC clone HSC7E526/V 12, in which the YAC HSC7E526 has been
modified by integration of the amplification vector pYAM4 and the insertion
vector
pYIV3. Lane 5 is the isolated YAC DNA from clone HSC7E526/V 12 prior to
microinjection.
i o GENERATION OF YAC TRANSGENIC MICE
Modified YAC DNA was excised from a 1 % pulse-field agarose gel in 0.25 x TAE
buffer and concentrated into 4 % low melting point agarose. The gel slice
containing
YAC DNA was equilibrated with microinjection buffer (TE pH 7.0 with 0.1 M
NaCI)
t s and digested with gelase. YAC DNA was dialysed against the microinjection
buffer for
2 hours before injection into fertilised oocytes.
Two hundred and ninety-eight fertilised oocytes were injected with 35D8/D6 YAC
DNA
and 364 with HSC7E526/V 12. After transfer of injected oocytes into oviducts
of
2o pseudopregnant female mice, a total of 190 mice (28.7 % ) were born of
which 26
(13.70 carried YAC DNA as determined by PCR as shown below:
Table II. Survival rate of trausfered oocytes and YAC transgenic mice
YAC ConstructSize (kb)NEIT Born Died Transgenic
35D8/D6 500 298 97 6 18
HSC7E526/V 630 364 93 1 8
12
Total 662 190 7 26
(28.70 (13.7X0
2s N.E.LT: Number of oocytes injected and transferred
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PCR determination of the size of the integrated construct
The size of the integrated YAC 35D8/D6 and YAC HSC7E526/V I2 constructs in
each
transgenic founder animal were determined using two pairs of PCR primers (A
and H:
s Table III) to detect the two YAC vector arms and a series of PCR primer
pairs spanning
the SERT (B to G: Table III) and VIPR2 (I to L: Table III) genes respectively.
Table III. Primer Pairs Used For PCR
PrimerForward Primers (5' - Reverse Primers (5' -
Pair 3') 3')
A 34773 TTGACTGGAGCGAGGCGATGTTCG54774 TCTACACAGCCATCGGTCCAGACG
H 43084 GCGTCTAGGTGGCACCAGAATC43085 TCGCGCTTGTGTTCCCAGCTAC
C 44771 CTAGTGACTGACATTGCCTGG44772 TGTCCAGTCTATCTGCACATG
D 26375 AGTTCTGATGAGGCACGC26376 TTCATCACCTCCATCCACATCC
E 38127 TGGCATGCAATTGTAGTCTC38128 TTCTTTCCTTACTAAGTTGAGAACG
F 38125 GAATACCAGGTCACCACATGG38126 AAACCTTGCACAGGGTCTTG
G 38130 TGGCTTTAGTATTTTTCTTTGTTTT38131 GAAAAGTACTCCTCAGTAGGTTGAA
H 53537 TCTCCGAACAGAAGGAAGAACG53538 TGTTACTTCTTCTGCCGCCTGC
I 53525 CCACATACAGACTGATGAATC53526 AGCTGGAATTGGAACTCAGC.
J 53527 AGTGCCTAAAGGGTTATG53528 AAAGATGCTACACCTGAG
K 53531 CTCAATATCACACAACAGTG53532 GCTGAAAGGAAAACTGATTG
L 53533 CATCCCAGATTTGTATAG53534 ATAATCCCAAGAGGCAAG
20
SUBSTITUTE SHEET (RULE 26)
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The results are presented in Figures 12 and 13. In this regard, Figure 12
shows the size
of the integrated YAC DNA in transgenic founder animals carrying 35D8/D6 YAC
DNA. Figure 13 shows the size of integrated YAC DNA in transgenic founder
animals
carrying HSC7E526/V 12 YAC DNA. For each founder animal, the probable extent
of
s the transgene is indicated as a shaded bar, with pale circles indicating
presence of
markers, as determined by PCR. The location of these markers is indicated in
the
schematic diagram of the 35D8/D6 YAC construct (Figure 12) and HSC7E526/V 12
YAC (Figure 13) construct which are drawn above the markers.
1o Three independent transgenic founder mice carrying the intact YAC 35D8/D6
(A102.3,
A102.5, A105, Figure 12) and six carrying the intact YAC HSC7E526/V12 (A108,
A108.1, A108.2, A108.3, A108.5, A110: Figure 13) were identified. Thus, a
beneficial
number of mice born in this study carried intact YAC DNA.
1 s GERM LINE TRANSMISSION
An important feature of pYAM4 is that it does not necessarily have to contain
a
thymidine kinase (TK) gene which may cause male infertility. In the absence of
the TK
gene, we found that YAC transgenes were transmitted into the next generation
from both
2o male and female founders as determined by primer pair A, which is derived
from the
hygromycin resistance gene within pYAM4. Thus, preferably, the TK gene is not
present in the vector of the present invention.
Immunocytochemistry for beta -galactosidase
Animals were perfused transcardially with 4% paraformaldehyde in O.1M PBS.
Brains
were postfixed overnight in the same solution, then transferred into PBS next
day and kept
in that solution until cutting. Brains were infiltrated with 30% sucrose
overnight, then
25p,m thick sections were cut on a cryostat. Sections were collected in
multiwell plates
3o containing PBS and then processed for immunocytochemistry. Sections were
treated with
0.1% Triton X-100 and 0.02% H202 solution for 30 min, then rinsed two times
for 5 min
with PBS. This was followed by a blocking step with 2% solution of normal
donkey
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serum, for another half an hour. Sections were incubated in anti beta-
galactosidase primary
antibody (5'Prime3'Prime, Inc., diluted at 1:20000) for 48 hours. Biotinylated
donkey anti
rabbit IgG (from Jackson, diluted at 1:1000, for 60 min.), ABC Elite Kit (from
Vector,
diluted at 1:1000, for 60 min.) and NiDAB as chromogen were used to visualise
the
immunoreactive areas.
LacZ staining of adult transgenic mice expressing the YAC construct
HSC7E526/V12
Mice were anaesthetised with a lethal dose of sodium pentobarbitone and
briefly
perfused through the heart with 0.9 °~ sodium chloride solution to
remove blood followed
by a longer perfusion of the ice-cold fixative solution (4% paraformaldehyde
in O.1M
sodium phospahate buffer, pH 7.4). After perfusion with approximately 150-200
ml of
the fixative solution, the brains and internal organs were removed rapidly and
postfixed
in the same fixative for 2-4 hours at 4°C. Subsequently 2-5 mm coronal
slices of the
brain were cut and the brain and other organs were washed twice at room
temperature in
a detergent wash solution consisting of 2 mM Magnesium Chloride, O.OI k Sodium
Deoxycholate, and 0.02 % NP40 in phosphate-buffered saline (PBS); pH 7.4.
After
washing all tisues were transferred to a solution containing lmg/ml X-Gal in
SmM
2o potassium ferrocyanide, SmM potassium ferricyanide, 2mM Magnesium chloride,
0.019
Sodium deoxycholate, and 0.02 % NP40 in PBS, pH 7.4 and incubated overnight at
30°C. After staining, tissues were washed in PBS and cleared in 40% and
80 k glycerol
(v/v) in PBS and photographed.
2s The distribution of (3-galactosidase activity in transgenic mice was
consistent with the
published distribution of VIPR2 mRNA (Cagampang et al., 1998; Inagaki et al.,
1994;
Sheward et al., 1995; Usdin et al., 1994) and of binding sites for the
selective VIP2
receptor agonist Ro25-1553(Vertongen et al., 1997). In particular, high levels
of
expression of ~i-galactosidase were detected:
(i) In the suprachiasmatic nucleus {Figure 15a, 15b), where VIP and/or PACAP,
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acting through the VIP2 receptor, may play a role in the control of circadian
rhythms.
There is circadian variation of VIP immunoreactivity (Takahashi et al., 1989),
prepro
VIP mRNA (Albers et al., 1990; Glazer and Gozes, 1994; Gozes et al., 1989) and
of
VIP2 receptor mItNA (Cagampang et al., 1998) in the SCN and VIP (Piggies er
al.,
s 1995), VIP antagonists (Gozes et al., 1995) and VIP antisense
oligodeoxynucleotides
(Scarbrough et al., 1996) have been shown to disrupt circadian function.
(ii) In the pancreas (Figure Sc), where VIP and PACAP stimulate insulin
release by
interaction with the VIP2 receptor on the beta cell (Straub and Sharp, 1996).
Chemiluminescent assay for ~i-galactosidase in mouse tissues using the Tropix
Galacto-Light Plus kit.
Tissues from mice were dissected, frozen on dry ice and stored at -70oC. They
were
t5 thawed and homogenised immediately in 100-400,1 of cold lysis buffer (as
supplied in
the kit, with 0.2mM PMSF and S~,g/ml leupeptin added just before use). After
homogenisation, samples were centrifuged at 12000g for 10 min at 4°C.
An aliquot of
the supernatant was stored at -70°C for measurement of protein
concentration and the
rest was incubated at 48°C for 60 minutes to inactivate the endogenous
(3-galactosidase.
2o After centrifugation for 5 min at room temperature 201 of each sample were
used in the
assay. 200~c1 of Galacto-Light reaction buffer was added, inbubated for 60 min
at room
temperature and then 300~c1 of Accelerator was added and the sample counted in
a TD
20/20 luminometer (for 20 sec after 5 sec delay). Protein concentrations were
determined using the Bio- Rad assay and activity was expressed as light
units/min/mg of
2s protein.
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INSERTION VECTOR
5 Almost all YAC transgenic animals described to date have been made with
unmodified
YAC DNA.
Expression of the transgenes in these animals can only be assessed by in situ
hybridisation, Northern blotting, PCR and/or immunohistochemistry.
Introduction of a
reporter gene into the YAC DNA would simplify procedures for the detection of
transgene expression.
We have constructed a series of YAC modification vectors (pYIVI, pYIV2, pYIV3
and
pYIV4) which can be inserted into YACs after the translation initiation or
stop codon.
t 5 The vectors contain a lacZ reporter gene downstream of a viral internal
ribosome entry
site (IRES), so that a simple histochemical staining procedure can be used to
examine the
tissue distribution and regulation of the transgene. This approach provides a
more
complete picture of the pattern of expression of the transgene than standard
procedures
such as in situ hybridisation. One of the vectors (pYIV3 ) permits the HA
epitope tag
2o sequence (from influenza hemagglutinin) to be fused to the carboxyl
terminus of the gene
product of interest, so that the protein product of the transgene can be
detected using the
commercially available 12CA5 monoclonal antibody. pYIV4 contains IoxP elements
flanking the SV40 polyadenylation signal and the ADE2 gene. In transgenic
animals
generated using pYIV4, polyA and ADE2 sequences can be deleted using Cre
2s recombinase so that the transgene and IacZ reporter gene are flanked by the
authentic 3'-
untranslated region of the transgene.
In summation therefore, transgenic technology has played an important role in
the
understanding of gene function and regulation in vivo, and in creating animal
models of
3o human genetic diseases. The development of yeast artiftcial chromosome
(YAC)
technology has permitted the cloning of DNA segments thousands of kilobases in
size
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between two YAC vector arms, facilitating transfer of a whole gene and most
(if not all)
of the elements required for its faithful regulation into transgenic animals.
The analysis in transgenic animals of genes expressed in YACs can be greatly
facilitated
by the use of a reporter gene in accordance with the present invention for the
accurate
and sensitive detection of cellular sites of transcription.
AMPLIFICATION VECTOR
to Importance of YAC technology and YAC transgenesis
The development of the yeast artificial chromosome (YAC) technology has
permitted the
cloning of DNA segments thousands of kilobases in size between two YAC vector
arms.
YACs can be used to clone the complete sequences of large genes or gene
complexes
that exceed the size limit for cloning in conventional bacterial cloning
vectors such as
plasmids (10 kb), bacteriophage (15 kb), and cosmids (50 kb). Cloning of such
large
DNA fragments is essential for physical genome mapping (1) and to isolate
large genes
relevant to human genetic disease (2, 3). Although bacterial artificial
chromosome
(BAC) (4) and P1 artificial chromosome vectors (5) have a large cloning
capacity (up to
200 kb), it is relatively difficult to perform genetic manipulation in these
vectors. The
high efficiency of homologous recombination in yeast permits genetic
manipulations of
genes cloned in YAC vectors to be performed easily.
Transgenic technology has played an important role in the understanding of
gene
function and regulation in vivo, and in creating animal models of human
genetic diseases.
It is well recognised that transgenes containing genomic DNA with introns and
essential
regulatory sequences are expressed more appropriately in vivo than cDNA based
constructs (6-10). The use of YAC constructs to produce transgenic animals
facilitates
the presence and transfer of most (if not all) elements required for the
faithful regulation
of a gene and may avoid position effects related to the integration site,
which may lead
to low levels and, in some cases, aberrant patterns of gene expression in
transgenic
CA 02311282 2000-OS-26
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62
animals. However, the efficiency with which transgenic animals are produced
using
YAC DNA ( 1-5 % ) is lower than that using conventional vectors (approx. 10 qb
)
Importance of YAC amplification
YAC vectors (for example pYAC4) contain a yeast centromere, two telomeres, and
two
selective markers (TRPI and URA3). After incorporation of YAC DNA, yeast can
grow
in medium lacking the uracil and tryptophan. Under these conditions of
selection, YAC
clones are replicated along with the endogenous host chromosomes; only one
copy of
to YAC DNA is produced per cell. Using standard protocols, YAC DNA at a
concentration of 1 ~,g/ml can be isolated. However, there is a substantial
increase in
copy number if the YAC centromere is inactivated by induced transcription from
a GALL
or ADH2 promoter. This increase is thought to reflect the segregation bias of
the YAC
for the mother cells and loss of the daughter cells without the YAC from the
population
under selection.
The low e~ciency with which transgenic animals are produced using YAC DNA (1-
5°6)
compared to DNA from conventional vectors (approx. 10°k) is probably
caused by the
low concentration of YAC DNA available for injection. Also, conventional YACs
are
2o replicated in yeast at one copy per cell.
Therefore, assuming that 2 pl of S00 kb YAC at a concentration of 1 nghl is
injected
into a pronucleus, a fertilised egg would only receive 1 molecule of YAC DNA.
Amplification of YACs in yeast therefore provides a possible method for the
isolation of
2s more concentrated YAC DNA which should lead to more successful generation
of YAC
transgenic animals.
Advantages and disadvantages of the existing YAC amplification vectors pCGS966
and
pCGS990
To date two vectors which can be used to amplify YAC DNA have been reported:
pCGS966 (11) and pCGS990 (12). Both vectors include a conditional centromere
and a
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heterologous (herpes simplex virus) thymidine kinase (TK) gene. YAC DNA of
less than
600 kb is amplified efficiently (3 to 11 copies/cell). pCGS966 has been used
recently to
construct a number of new YAC libraries (13-15). However, when using this
vector for
the modification (retrofitting) of existing YACs, replacement of the left arm
occurs with
s very low frequency (0.5-2.5q6) (12). Most importantly, the expression of the
TK gene in
the testes of transgenic mice interferes with spermatogenesis and causes male
infertility
(16-22). This complication makes these vectors unsuitable for transgenic
studies.
Features of the novel YAC amplification vector pYAM4.
We have constructed a YAC amplification vector which has a number of
advantages:
1) it amplifies YAC DNA 3 to 5 fold.
~ 5 2) unlike existing vectors, it does not contain the herpes simplex virus
thymidine
kinase (TK) gene, which causes male infertility in transgenic mice.
3) it has much higher homologous recombination efficiency ( 13 °~ )
than existing
YAC amplification vectors.
4) it contains a selectable marker (hygromycin B resistance) which facilitates
the
transfer of YAC DNA into embryonic stem cells and other cell lines.
5) it can be used for targeted deletion of sequences cloned in YAC vectors.
6) Fusion of a NOI (such as the SERT gene) to a reporter gene (such as Lack
facilitates the determination of the sites/regions where the NOI is expressed
and the
testing of agents which may affect the expression pattern of the NOI.
">) Transgenic mice overexpressing the human VIPR2 gene together with the ~-
galactosidase reporter gene will facilitate the development of agents capable
of
influencing the activity of the VIP2 receptor in man. Two classes of agent
might be
CA 02311282 2000-OS-26
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identified: (i) agents regulating the expression of the human VIP2 receptor,
which could
be identified by their ability to influence ji-galactosidase activity in
transgenic mice and
(ii) agonists and antagonists of the human VIP2 receptor, for which the
transgenic mice
in which the human VIPR2 gene is expressed will provide an animal model.
Optimally,
s YAC transgenic animals, such as VIP2 receptor null ("knock out") mice, could
be bred
with a view to generating "humanised" animals in which the VIP2 receptor
displays
identical pharmacology to that seen in man.
By way of example, agents, acting in the suprachiasmatic nucleus, which are
capable of
influencing the activity of the VIP2 receptor, may prove useful in the
treatment of the
disturbances in circadian function. Such disturbances, which may lead to
impaired
physical and mental well-being, can occur through: (i) extremes in work
patterns (shift
work); (ii) travelling through many time zones (jet lag); (iii) in normal
ageing; and (iv)
in dementia. Such agents may also prove useful in the treatment of sleep
disorders,
t5 seasonal affective disorder (SAD), eating disorders and pre-menstrual
syndrome.
By way of further example, agents, acting in the pancreas, which are capable
of
regulating the expression of the human VIP2 receptor or agents acting as
agonists and
antagonists of the receptor may be useful in the treatment of diabetes.
In summation introduction of the IacZ reporter gene by using pYIVs, together
with
amplification of YAC DNA by using pYAM4 should greatly facilitate production
of
YAC transgenic animals and analysis of these animals in terms of expression,
regulation
and function of genes present in YAC DNA.
In these studies we present the construction of a series of YAC modification
vectors
(such as pYIV 1, pYIV2, pYIV3 and pYIV4) that contain a reporter gene (such as
the
lacZ reporter gene) to facilitate the study of the tissue distribution and
regulation of YAC
CA 02311282 2000-OS-26
WO 99/28449 PCT/CB98/03558-
transgenes. These vectors are likely to find widespread application in
transgenic
research.
In these studies we also present the construction of a YAC amplification
vector (such as
5 pYAM4) which has a number of advantages over previous vectors and is
suitable for the
amplification of YAC DNA for the creation of transgenic mice.
In these studies we also present the combined use of the above-mentioned
modification
vectors (such as pYIVI, pYIV2, pYIV3 and pYIV4) and the above-mentioned YAC
to amplification vector (such as pYAM4). The combined use of these vectors is
likely to
find widespread application in transgenic research.
For example, the vectors of the present invention - in particular the
insertion vectors of
the present invention - may be used to prepare other artificial chromosomes
(i.e.
t5 artificial chromosomes other than YACs), which may in turn be used to
prepare
transgenic organisms (including animals). In this alternative embodiment, the
above
mentioned statements of invention and description are still applicable but
wherein the
term YAC represents any suitable artificial chromosome, preferably a yeast
artificial
chromosome.
All publications mentioned in the above specification are herein incorporated
by
reference. Various modifications and variations of the described methods 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 or related fields are intended to be within
the scope of
the following claims.
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SEQ ID. NO. 1
5
1 ATCGATAAGC TTTAATGCGGTAGTTTATCA CAGTTAAATT GCTAACGCAG
TCAGGCACCG
61 TGTATGAAAT CTAACAATGCGCTCATCGTC ATCCTCGGCA CCGTCACCCT
GGATGCTGTA
121 GGCATAGGCT TGGTTATGCCGGTACTGCCG GGCCTCTTGC GGGATATCGT
CCATTCCGAC
181 AGCATCGCCA GTCACTATGGCGTGCTGCTA GCGCTATATG CGTTGATGCA
ATTTCTATGC
10 GCACCCGTTC TCGGAGCACTGTCCGACCGC TTTGGCCGCC GCCCAGTCCT
241 GCTCGCTTCG
301 CTACTTGGAG CCACTATCGACTACGCGATC ATGGCGACCA CACCCGTCCT
GTGGATCAAT
361 TCCCT'TTAGT ATAAATTTCACTCTGAACCA TCTTGGAAGG ACCGGTAATT
ATTTCAAATC
421 TCTTTTTCAA TTGTATATGTGTTATGTTAT GTAGTATACT CTTTCTTCAA
CAATTAAATA
481 CTCTCGGTAG CCAAGTTGGTTTAAGGCGCA AGACTTTAAT TTATCACTAC
GGAATTCCGT
15 AATCTTGAGA TCGGGCGTTCGATCGCCCCG GG
541
SEQ ID. NO. 2
20LOCUS SERT.DOC 2889 BP DS-DNA ENTERED 11/24/98
BASE COUNT672 A 753 C 722 G 742 T 0 OTHER
CON~HENT 1 - 85 axon 1A
86 - 182 axon 1B (some splice variants do not include
this axon)
25 183 - 648 axon 2
649 - 783 axon 3
784 - 1003 axon 4
1004 - 1142 axon 5
1143 - 1277 axon 6
30 1278 - 1381 axon 7
1382 - 1509 axon 8
1510 - 1622 axon 9
1623 - 1754 axon 10
1755 - 1854 axon 11
35 1855 - 1955 axon 12
1956 - 2123 axon 13
2124 - 2889 axon 14
180 - 2198 open reading frame encoding SERT
ORIGIN -
401 ACAGCCAGCG CCGCCGGGTG CCTCGAGGGC GCGAGGCCAG CCCGCCTGCC
CAGCCCGGGA
61 CCAGCCTCCC CGCGCAGCCT GGCAGGTCTC CTGGAGGCAA GGCGACCTTG
CTTGCCCTCT
121 ATTGCAGAAT AACAAGGGGC TTAGCCACAG GAGTTGCTGG CAAGTGGAAA
GAAGAACAAA
181 TGAGTCAATC CCGACATATC AATCCCGACG ATAGAGAGCT CGGAGGTGAT
CCACAAATCC
241 AAGCACCCAG AGATCAATTG GGATCCTTGG CAGATGGACA TCAGTGTCAT
TTACTAACCA
45301 GCAGGATGGA GACGACGCCC TTGAATTCTC AGAAGCAGCT ATCAGCGTGT
GAAGATGGAG
361 AAGATTGTCA GGAAAACGGA GTTCTACAGA AGGTTGTTCC CACCCCAGGG
GACAAAGTGG
421 AGTCCGGGCA AATATCCAAT GGGTACTCAG CAGTTCCAAG TCCTGGTGCG
GGAGATGACA
481 CACGGCACTC TATCCCAGCG ACCACCACCA CCCTAGTGGC TGAGCTTCAT
CAAGGGGAAC
541 GGGAGACCTG GGGCAAGAAG GTGGATTTCC TTCTCTCAGT GATTGGCTAT
GCTGTGGACC
50601 TGGGCAATGT CTGGCGCTTC CCCTACATAT GTTACCAGAA TGGAGGGGGG
GCATTCCTCC
661 TCCCCTACAC CATCATGGCC ATTTTTGGGG GAATCCCGCT CTTTTACATG
GAGCTCGCAC
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721 TGGGACAGTA CCACCGAAAT GGATGCATTT CAATATGGAG GAAAATCTGC CCGATTTTCA
781 AAGGGATTGG TTATGCCATC TGCATCATTG CCTTTTACAT TGCTTCCTAC TACAACACCA
841 TCATGGCCTG GGCGCTATAC TACCTCATCT CCTCCTTCAC GGACCAGCTG CCCTGGACCA
901 GCTGCAAGAA CTCCTGGAAC ACTGGCAACT GCACCAATTA CTTCTCCGAG GACAACATCA
S 961 CCTGGACCCT CCATTCCACG TCCCCTGCTG AAGAATTTTA CACGCGCCAC GTCCTGCAGA
1021 TCCACCGGTC TAAGGGGCTC CAGGACCTGG GGGGCATCAG CTGGCAGCTG GCCCTCTGCA
1081 TCATGCTGAT CTTCACTGTT ATCTACTTCA GCATCTGGAA AGGCGTCAAG ACCTCTGGCA
1141 AGGTGGTGTG GGTGACAGCC ACCTTCCCTT ATATCATCCT TTCTGTCCTG CTGGTGAGGG
1201 GTGCCACCCT CCCTGGAGCC TGGAGGGGTG TTCTCTTCTA CTTGAAACCC AATTGGCAGA
1261 AACTCCTGGA GACAGGGGTG TGGATAGATG CAGCCGCTCA GATCTTCTTC TCTCTTGGTC
1321 CGGGCTTTGG GGTCCTGCTG GCTTTTGCTA GCTACAACAA GTTCAACAAC AACTGCTACC
1381 AAGATGCCCT GGTGACCAGC GTGGTGAACT GCATGACGAG CTTCGTTTCG GGATTTGTCA
1441 TCTTCACAGT GCTCGGTTAC ATGGCTGAGA TGAGGAATGA AGATGTGTCT GAGGTGGCCA
1501 AAGACGCAGG TCCCAGCCTC CTCTTCATCA CGTATGCAGA AGCGATAGCC AACATGCCAG
IS 1561 CGTCCACTTT CTTTGCCATC ATCTTCTTTC TGATGTTAAT CACGCTGGGC TTGGACAGCA
1621 CGTTTGCAGG CTTGGAGGGG GTGATCACGG CTGTGCTGGA TGAGTTCCCA CACGTCTGGG
1681 CCAAGCGCCG GGAGCGGTTC GTGCTCGCCG TGGTCATCAC CTGCTTCTTT GGATCCCTGG
1741 TCACCCTGAC TTTTGGAGGG GCCTACGTGG TGAAGCTGCT GGAGGAGTAT GCCACGGGGC
1801 CCGCAGTGCT CACTGTCGCG CTGATCGAAG CAGTCGCTGT GTCTTGGTTC TATGGCATCA
2O 1861 CTCAGTTCTG CAGGGACGTG AAGGAAATGC TCGGCTTCAG CCCGGGGTGG TTCTGGAGGA
1921 TCTGCTGGGT GGCCATCAGC CCTCTGTTTC TCCTGTTCAT CATTTGCAGT TTTCTGATGA
1981 GCCCGCCACA ACTACGACTT TTCCAATATA ATTATCCTTA CTGGAGTATC ATCTTGGGTT
2041 ACTGCATAGG AACCTCATCT TTCATTTGCA TCCCCACATA TATAGCTTAT CGGTTGATCA
2101 TCACTCCAGG GACATTTAAA GAGCGTATTA TTAAAAGTAT TACCCCAGAA ACACCAACAG
25 2161 AAATTCCTTG TGGGGACATC CGCTTGAATG CTGTGTAACA CACTCACCGA GAGGAAAAAG
2221 GCTTCTCCAC AACCTCCTCC TCCAGTTCTG ATGAGGCACG CCTGCCTTCT CCCCTCCAAG
2281 TGAATGAGTT TCCAGCTAAG CCTGATGATG GAAGGGCCTT CTCCACAGGG ACACAGTCTG
2341 GTGCCCAGAC TCAAGGCCTC CAGCCACTTA TTTCCATGGA TTCCCCTGGA CATATTCCCA
2401 TGGTAGACTG TGACACAGCT GAGCTGGCCT ATTTTGGACG TGTGAGGATG TGGATGGAGG
30 2461 TGATGAAAAC CACCCTATCA TCAGTTAGGA TTAGGTTTAG AATCAAGTCT GTGAAAGTCT
2521 CCTGTATCAT TTCTTGGTAT GATCATTGGT ATCTGATATC TGTTTGCTTC TAAAGGTTTC
2581 ACTGTTCATG AATACGTAAA CTGCGTAGGA GAGAACAGGG ATGCTATCTC GCTAGCCATA
2641 TATTTTCTGA GTAGCATATA GAATTTTATT GCTGGAATCT ACTAGAACCT TCTAATCCAT
2701 GTGCTGCTGT GGCATCAGGA AAGGAAGATG TAAGAAGCTA AAATGAAAAA TAGTGTGTCC
35 2761 ATGCAAGCTT GTGAGTCTGT GTATATTGTT GTTTCAGTGT ATTCTTATCT CTAGTCCAAT
2821 ATTTTGGGCC CATTACAAAT ATATGAATTC CCCAAATTTT TCTTACATTA ACAAATTCTA
2881 CCAACTCAA
4o SEQ ID. NO. 3
LOCUS hVIP2.DOC 3944BP DS-DNA ENTERED 11/23/98
BASE 795 A 1159C 1154 G 836 T 0 OTHER
COUNT
45 C0I4~IENT1 - 238 axon1
239 - 338 axon2
339 - 446 axon3
447 - 544 axon4
545 - 642 axon5
50 643 - 784 axon6
785 - 935 axon7
936 - 996 axon8
997 - 1066axon9
1067 - axon10
1158
55 1159 - axon11
1288
1289 - axon12
1330
1331 - axon13
3944
188 - 1504openreading frame encoding VIP2 receptor
ORIGIN -
60 1 GTGCATTGAG
CGCGCTCCAG
CTGCCGGGAC
GGAGGGGGCG
GCCCCCGCGC
TCGGGGCGCT
61 CGGCTACAGC TGCGGGGCCC GAGGTCTCCG CGCACTCGCT CCCGGCCCAT GCTGGAGGCG
121 GCGGAACCGC GGGGACCTAG GACGGAGGCG GCGGGCGCTG GGCGGCCCCC GGCACGCTGA
181 GCTCGGGATG CGGACGCTGC TGCCTCCCGC GCTGCTGACC TGCTGGCTGC TCGCCCCCGT
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241 GAACAGCATT CACCCAGAAT GCCGATTTCA TCTGGAAATA CAGGAGGAAG
AAACAAAATG
301 TGCAGAGCTT CTGAGGTCTC AAACAGAAAA ACACAAAGCC TGCAGTGGCG
TCTGGGACAA
361 CATCACGTGC TGGCGGCCTG CCAATGTGGG AGAGACCGTC ACGGTGCCCT
GCCCAAAAGT
421 CTTCAGCAAT TTTTACAGCA AAGCAGGAAA CATAAGCAAA AACTGTACGA
GTGACGGATG
S 481 GTCAGAGACG TTCCCAGATT TCGTCGATGC CTGTGGCTAC AGCGACCCGG
AGGATGAGAG
541 CAAGATCACG TTTTATATTC TGGTGAAGGC CATTTATACC CTGGGCTACA
GTGTCTCTCT
601 GATGTCTCTT GCAACAGGAA GCATAATTCT GTGCCTCTTC AGGAAGCTGC
ACTGCACCAG
661 GAATTACATC CACCTGAACC TGTTCCTGTC CTTCATCCTG AGAGCCATCT
CAGTGCTGGT
721 CAAGGACGAC GTTCTCTACT CCAGCTCTGG CACGTTGCAC TGCCCTGACC
AGCCATCCTC
781 CTGGGTGGGC TGCAAGCTGA GCCTGGTCTT CCTGCAGTAC TGCATCATGG
CCAACTTCTT
841 CTGGCTGCTG GTGGAGGGGC TCTACCTCCA CACCCTCCTG GTGGCCATGC
TCCCCCCTAG
901 AAGGTGCTTC CTGGCCTACC TCCTGATCGG ATGGGGCCTC CCCACCGTCT
GCATCGGTGC
961 ATGGACTGCG GCCAGGCTCT ACTTAGAAGA CACCGGTTGC TGGGATACAA
ACGACCACAG
1021 TGTGCCCTGG TGGGTCATAC GAATACCGAT TTTAATTTCC ATCATCGTCA
ATTTTGTCCT
1S 1081 TTTCATTAGT ATTATACGAA TTTTGCTGCA GAAGTTAACA TCCCCAGATG
TCGGCGGCAA
1141 CGACCAGTCT CAGTACAAGA GGCTGGCCAA GTCCACGCTC CTGCTTATCC
CGCTGTTCGG
1201 CGTCCACTAC ATGGTGTTTG CCGTGTTTCC CATCAGCATC TCCTCCAAAT
ACCAGATACT
1261 GTTTGAGCTG TGCCTCGGGT CGTTCCAGGG CCTGGTGGTG GCCGTCCTCT
ACTGTTTCCT
1321 GAACAGTGAG GTGCAGTGCG AGCTGAAGCG AAAATGGCGA AGCCGGTGCC
CGACCCCGTC
1381 CGCGAGCCGG GATTACAGGG TCTGCGGTTC CTCCTTCTCC CGCAACGGCT
CGGAGGGCGC
1441 CCTGCAGTTC CACCGCGGCT CCCGCGCCCA GTCCTTCCTG CAAACGGAGA
CCTCGGTCAT
1501 CTAGCCCCAC CCCTGCCTGT CGGACGCGGC GGGAGGCCCA CGGTTCGGGG
CTTCTGCGGG
1561 GCTGAGACGC CGGCTTCCTC CTTCCAGATG CCCGAGCACC GTGTCGGGCA
GGTCAGCGCG
1621 GTCCTGACTC CGTCAAGCTG GTTGTCCACT AAACCCCATA CCTGGAATTG
GAGTCGTGTT
2S 1681 GTCATTGACT CGATTTAAAC TCCAGCATTT AGATAATCTT GTGCAAAATG
TGTTTCAGCC
1741 GTATAGTGGA TCCACTTTTT TTTTTTTTTT TTTTTGAGAC GGAGTCTTGC
TCTGTCGCCC
1801 AGGCTGGAGT GCAGTGGCCT GATCTCTGCT CCCTGCAAGC TCCGCCTCCC
GGGTTCACGC
1861 CATTCTCCTG CCTCAGCCTC CCATAGCTGG GACTACAGGC GCCCGCCAAC
ACGCCTGGCT
1921 AATTTTTTGT ATTTTTAGTA GAGACAGGGT TTCACCATGT TAGCCAGGAT
GGTCTCGATC
1981 TCCTGACCTC GTGATGGGCC CGCCTCGGCC TCCCAAAGTG CTGGGATTAA
GGCGTGAGCC
2041 ACTGCGCCCG GCCCAAGAGA ATAGGGGAGC CAAGGAGGAA ATGTGGAAAC
GCAGTTGTGT
2101 GGCCCAGCAC GAGCCTGGGC GACCACCGGG TGACATCCGT CCCACATCAG
GGCGGCCTCC
2161 CAGGTCCCAT AAGGGTAGCC CCCTCATCTG CAGGACAGAG GGAAGCCAGT
CAGGGCCCCC
2221 CCGGACGTTA GGACCAGGAG AAATCAACAG GAGGGCAGCC CGTCCTCTCT
CTTGGGGCGC
3S 2281 CCACCCGGCC CGGCTGAGCC CTGCCCCACC CAACTCCACA GGGCTGTTTT
GCCTCCCCAC
2341 GGAAGGCGGG CTGAGGAGAC AACCAGATCA GGAGAGCAAG GTCATGAAGG
AGGGGACCTC
2401 TCCACACAGG TGTTCCGTGG GACCCTCAGC AGCTCTGGCT CTGCCTCAGG
AGGTCACCTG
2461 CCGCCCTGTG GGAGCCGCAG AGCCTGACGC TCAGCCCCAG GCCAGCTGCG
GCCAGGCCTG
2521 CGGGCCCCTG GTGATGGGGT TACGTGGGGT GCGGGATACA GCTGAGTGGG
AACCGGAAAC
2581 CTATTCTCTT TTTAACAAAA ATAATCTTAG GATAAGAATT ATTTTAACAA
CATATAAAAC
2641 TGTTTCAAGC CCTCCTCCCC AGAGCTGGCG CTCAGCAGCC CTAGCGGCTG
CTCCTTCAGG
2701 CGAAGGGTGG TTTGCAGATG TGGGGAGGGT GTCTGGGGAC GTTGCTGAGC
TGGCTGCAGA
2761 AGGGTGGGGA TATCAGGGCA CAGTCTCCAT GTGTGTGCCA AGCCCTGGCC
CCCACAGCGC
2821 TCGATGGACC TCAGCAAGCT GCCCAGCCCT GGCCCAGGTG CCCCGACTGT
GGGACTCAGT
4S 2881 TGTTCTGAGC ACATTTGACT CCACTTTTCC TTTAAAAATG AATGTCTTGT
TCCTGTGCAT
2941 TGGTGGCATC ACAGACCCCA GCTGGGGCGC GATGTCAAAG GTCGGGACAG
CTGTGCCGGG
3001 AGGCAGCCAC AGGGAAGCTC ACACATCCTG TCAGTGTCAC CTTGGTTTGC
AAAACCCATA
3061 TCCCCGGTAA AATGAGGCCG GACAGAGGGG CTGTTAGGAC AGCAAAGCAG
CAGTGTCCAG
3121 AGACCCCTCA ATCCCCAAAG GTCCGCACCC TGTCCTGCAC ACCCTGGGCC
ACGCCGGCCA
SO 3181 CACCCCTCTG CTGCAACAAG CTCATCCCTG GACTTCTGGG AGAATGAACC
CGAGGTTGGT
3241 TTGGGGAGAC AGGTGAGGCG GTTGGATCTA CAGAACAACC CACCATTTCT
GGGGGCCGCA
3301 GAGGATCCAT CACAGACGGA TACTGGGGAG TAAACGGCCC AGGCCAGGTG
CCCAGGAAAG
3361 GACGGCTGAG CATGTGGAGC GAGAGGGAGG CAGGTGGACG CTGCAGACCC
CAGGTTCAGT
3421 GCGGCCCCTC GGCTGTTCCT CCCCTGTAGG GTTTGGACAG ACCCACCCCC
AGCCTTGCCC
SS 3481 AGCTTTCAAA GGACAAAAGG GAGCATCCCC CACCTACTCT CAGGTTTTTG
AGGAAACAAA
3541 GATTTGTGGT AACTGAAGGT GTTGGGTCAG TGGCCAGGTG CCGACACTGA
GCTGTGACCC
3601 AGAGGGGACG CTGAGGAAGT GGGCGTGAGT GGACATGTCA GGTGGTTACC
AGGCACTGGT
3661 TGTTGATGGT CGGTGGTTGG GTGTGGGCAG TCATCAGTCA TCAGGTGTGC
TCAGGGGACA
3721 ATCTCCCCTC AACCGCACAT GTGCCACTGT TCAGCGGAGC TGACTGGTTT
CTCCTGGTAG
60 3781 AGGGCCGGCT GTATCCTGAC AGATGCCTGG TGAGCAGGGG AAGCAGGACC
CAGTGGTCAA
3841 CAGGTGTCTT TAACTGTCAT TGTGTGTGGA ATGTCGCAGA CTCCTCCACG
TGGCGGGAAT
3901 GAGCTGTGTA AATACTTCAA TAAAGCCTGA TCTCACATCT GCAA
CA 02311282 2000-OS-26
WO 99/28449 PCT/GB98/03558
73
SEQ ID NO. 4
GAATTCCGCC CCTCTCCCTC CCCCCCCCCT AACGTTACTG GCCGAAGCCG
CTTGGAATAA GGCCGGTGTG CGTTTGTCTA TATGTTATTT TCCACCATAT
TGCCGTCTTT TGGCAATGTG AGGGCCCGGA AACCTGGCCC TGTCTTCTTG
ACGAGCATTC CTAGGGGTCT TTCCCCTCTC GCCAAAGGAA TGCAAGGTCT
GTTGAATGTC GTGAAGGAAG CAGTTCCTCT GGAAGCTTCT TGAAGACAAA
CAACGTCTGT AGCGACCCTT TGCAGGCAGC GGAACCCCCC ACCTGGCGAC
AGGTGCCTCT GCGGCCAAAA GCCACGTGTA TAAGATACAC CTGCAAAGGC
GGCACAACCC CAGTGCCACG TTGTGAGTTG GATAGTTGTG GAAAGAGTCA
AATGGCTCTC CTCAAGCGTA TTCAACAAGG GGCTGAAGGA TGCCCAGAAG
GTACCCCATT GTATGGGATC TGATCTGGGG CCTCGGTGCA CATGCTTTAC
ATGTGTTTAG TCGAGGTTAA AAAACGTCTA GGCCCCCCGA ACCACGGGGA
CGTGGTTTTC CTTTGAAAAA CACGATGATA AGCTTGCCAC AACCATG
IS
(this sequence is also presented as Figure 10).
CA 02311282 2000-OS-26
WO 99/28449 _ ~4 PCT/GB98/03558'
.apphcanrs or agcnc's tile p/3330.W0 CTH I In~utonat appltcatn
reference number 1
tYDtC~TtOi~S REL.~TtvC TO ~ DEPOSITED dItCROORC.~NISrt
(PCT Rule I3bts)
.~. The indications made below
relate to the microuraanism
referred to in the description
on page ~7 . line 15
B. IDE.VTIFtC~1T10N OF DEPOSIT
Further deposiu are identified
on an additional sheet o
iVame of dcpusitarv, institution
The National Collections of
Industrial and Marine Bacteria
Limited (NCIMti)
.lddrcss of depository institution
/including postal colt and
tonetrv, ) .
23 St Machar Drive
Aberdeen
A82 1 RY
United Kingdom
O;ue of deposit Accession Number
24 November 1997
NCI1~ 40907
C. A00(TIOVAL INDIC.1T101VS
/lsaw blank ijnot applieab(t)
This information is continued
on an additional sheet
In respect of those designations
in which a European patent
is sought, and any
other designated state having
equivalent legislation, a sample
of the deposited
microorganism will only be made
available either until the
publication of the
mention of the grant of the
patent or after twenty years
from the date of filino
_
if the application has been
refused or withdrawn or is
deemed to be withdrawn,
only by the issue of such a
sample to an expert nominated
by the person requestin
the sample. (Rule 28(a) EPC)
D. DESICYATED STATES FOR WNICEI
l, IDIC.ITIOiYS dRE MADE Iijths
irtditattons ors not jot all
designated States/
E. SEPARATE FURIVISEItYC OF
IYDICAT10NS Iltaw blcnk ijnot
applicable)
The indications listed below
will be submitted co the Incerctstionsl
Burau lacer (Specij
trtJtt ~Clldal nantft OjfJlt
IIIGfKaQOItf Gg. :Iceariaut
'
~
.Vumoer ojOtpa~stt
7
For receiving Office use only For Intemaaonal Bureau use only
his sheet was received with the international application a This sheet wu
received by the lntemuional Bureau on:
2 7 NOVEir~BFR 1998
Authorized oiFcerP. J. ~~~ p3~/~9S Au~orixed officer
Form PCTIRO/13~t (July 1992)
CA 02311282 2000-OS-26
WO 99/28449 ~5 PCT/GB98/03558
.aooncart ; ur :4:nt i :;.: ; tntr..suwnat acancauun
':re:_~.ta,urnee: P3330.W0 CTH i
I,YD(C.~TIOt~S REL.aTI~C TO .a DEPOSITED :vItCROORCa~IS~t
(PCT Rule uors)
a. Tac mdtuttons mate oc:ow rctate :u the mtc: aur~antxm ret:~ to m the
uexenpnon
on page 27 n"~ 16
B. IDE~T1FTC.aTtO~ OF DEPOSIT
Funhcr deaostu are ttJenuried
on art ardtuonat she~: Q
~amc of ccoustt_ . msnwnon
The vational C~Llections of
tndustrial and Marine Bacteria
Limited (NCIMB)
~ .addtxsf of ceeostta. i mfntnuon
rtnciudrrtq ppsrat cacs ana
courtrrvl
I 23 St Machar Drive
abe:Ceen
a82 1PY
United Kingoom
I
_
Dilte ul CepOftt ,umoe:
24 November 1997 NCIMB 40908
C. ADDITIOnrtL WDIC.1TIOIVS
!leave oiwric t/rtnr aoplreablei
This iniormsuon is tronetnued
on an seditiunaJ she.: a
tn respect of those designations
in which can patent is sought,
and any
other designated state havinc
equivalent legislation, a samol_
of the deoasited
microorganism will only be mace
available either until the
puolication of the
mention of the grant of the
oat_nt cr after twenty years
from the dat_ of filino
if the application nas been
refused or withdrawn or is
deemeo to be withdrawn,
only by the LSSUe o. sucn a
sample t(1 all OttnOe~ nmwin~raA
h". rne ser:..
the samo:e. ;Rule 29(a) E?r) . ___
~ 0. OESiC.WTED ST.aTES FOR WHICH I~DIC.~TiO~S .aRE rt.lDE /i/:hr rnoaortons
ars rtor jor all c'str;narrdStortsr
1
i E. SEPaR.~TE Ft;R.YISHIYC OF IVDIC.aTIOYS IJeave oienK:jnor eoaueeoiu
t ne mcte..t.ons as,ea ae:ow wul oe suomtetec to :ae Into :atsonal e3ureau
rate: rzec:rp:ne;er~r,ct~o~rne utc»:»o~se;.. ;accsstton i
i ~ Boer ~j Cexnr 7
I
v
I
I
=or rece: vtng Ofie: use onto =or inte-suonu e3ureau uss onr~
:us stsr_: ~was rr.::vea ~~run the :nte~auonu aoaueuton (~ ,'ii: snc:: ~~as
:::e:v:a av :ne tats.-..a::onu 3u :au oe:
2 7 t~~0y~i;~~=:~ seog
.autnort: : ar'ic:: Autnort::a ori:a:
P~TR~c~ s.IZ.gs
Font)?C;'~ROIt3.t(July 19921
CA 02311282 2000-OS-26
WO 99/28449 ~6 PCT/GB98/03558-
~pouc_.::.:.~ . -:, :... P . 0 CTH ::n~:::unu::puca:un
::c~:::e:-.ra-:- !3330 w
IVOICaTt0:~5 REL.aTtaG TO.a OEPOStTEO ,~ItCROORG~~IS~t
(PCT Rule l Jors)
A. T'.:e tnwue:ons rts ce:ov
eesate :u ;nc nsrc:out~:atsn
re:..--.-_, :u m the uaenpnvn
on osqe 27 . true 17
Il. IDE~TIFtC.aTiO,Y OF OEPOStT
FutAe: :rostu u: :ee.-.ut:e:
on as actauonat saes: Q
~atete at ee:usst_; .nsuwuon
The v3tional Collections aP
tnduscciai and Marine 6actecia
Limited (vCIMB)
i .a,atsns of ar_csvt:r! rnstttuuon
rrncawrrrq power toes :na eowunr
E 23 St 'lacnar D: ive
~bO:C!.~
I
i Uniteo K:ngcom
I
Oats ut c~os,c A~asron W met:
24 November 1997 NCIMB x0909
C. a001T(O~ al. iVOiC.aTtO'tS
Itcsa oitrea r/rrr sooltcsa(a
This utiorituton a cant:aa~
on :.s :euruunat stir..
In cesoect cf those designations
in which a Eurooesn patent
is sought. and any
other cesicnated sate navino
eavivalent legislation, a satpole
oP the oeoosited
micc:crranism will only oe maCe
available either until the
puolicaticn of the
men d=n c the grant c,' the
oatenc ~_~ after raenty years
P:nm cne date aP filing
if t-e ape::=aticn has teen
refuseo c: .rithdrawn o: is
Ce!meo :: be withdrawn.
~ on!y ~v c~. issue cP suGt
a samole :~ an exoec= ~ominacea
~y cne person reouestinc
:ht s3npi=. ~~ule 2~ti) :fir!
D. DESIG'.1TEI) S;.iTES FOR WtitCH IOICATTOsYS ARE 51.10E Iil:J,c rreweanons
sn nor jor all etsr~areeSrortsr
I
j E. SEP.aR.aT='L:RYISItt,tG OF I~DiCAT10~5 Ite_~e owni rr nor a=aue::ia
,
ins mea~::ans :a:e: :c:ow W rrr oe suomtstsc ~ :.~te snc -:::gnu 3ureau :z:»
:»earr ~e;rrsr~ r~crr of u~ ~==w~i t.;.. :;cxsmn
o,~r ai Ct_-es:: ';
I
I
i
.
T--- ~ .. ...s~.mns ptT . as omy =or ln:er.:n:::..~.: =::.-:=a v:: :nr~ --
_.:.s mre: ~..as :-s:v:c ~rrrtn use :a:-_.-.sauona~ r : oi: snr.: ~cu .-:~~s=
:v we :.~.::.-..~.... :: 3z::~ :e:
sccne_.on
21 MOYEi~;~:~ 1°98
.a~i~.or::a .7:~~::
j~ 3.~Z.(~g - ~ auusonxM agree:
~oApP'"dOriiaiirlv t99Z1
CA 02311282 2000-OS-26
WO 99/28449 ~~ PtV'T/GB98103558-
j aooi~c_::: or :: .-.:s :::. p/3330.W0 CTH , m~:.u:unu:coucsi:
u~
.~=rs.-.ee .~.un::
:
tVDtC.~TtOVS REL.aTt'IC TO .> DEPOSITED vttCROORC:1~1S,~1
1PCT Rate l3irtst
a. T~c tnu:uc:cnx ."z:: ae:ow rc:ace w u:c m,v:.-our:an:m r::'-_.~.:p :u m ine
i:escapaun
I an pace 27 I,ne 18
I B. IOE~TtFfC.>TtOS OF DEPOSIT F::.hr. a::osiu ar: :ccan~ an s.~s ,ua:utonst
sar: O
~~aia: ui ce:usu_ : :nsatutson I
The vatio~ai C~11=ctions of Industrial and htacine 8actecia limited (NCtMB)
I .~'swi:::Sf Ot aQOSit3.~! ,n3ittuGOn rytaci:r;~ aOt:Ct :DCl :rttt COU,Itn~1
23 St ~lacnac n: ine
~be:~san
~ :_=v
Uniced K:nCCOfi1
O:W of aWOx,t Act:sxxton ~umoe:
24 November 1997 NCI14B 40910
C. a00fTtOn~L I~OIC.>TtOVS Ih=yr oin,rs tj ~r,r aao(ie=dltl T.Sis inionat:on
i::ant:nu:d ort an sG~itiunal she:
In c~soect ~f those designations in which a Eucaoean patent a sougnt, and any
ot!"ec cesi~nated state havino eauivalent legislation, a samoie of the
deposited
micc:cccanis~ will only be mace availabl~_ either until the ouolication oP the
menc:cn o° t!~e grant of the patent c: after twenty years fccm cne date
of filing
', :,' the accl:=atlCn nas bean ;.fused c: .ritndcawn oc a deemed =c be
Withdrawn,
only cv c-.e issue e. suc.~. a sample to an expec= ~ominaced by :ne person
reouestin~
the sarao:=. rule ?3;4) ~?~)
~ D. OESICN.1TED ST.>TES FOR WHICH IsDIC.IT(OrS aRE V1AOE /i/:irs vwaeesaans
an nor r'or all dtti;nettd Srattst
I
I
i
E. SEP.>R.>TE FCR.YISHi:iC OF t~D(C.aT10n5llr:rt picnic y'not got=cn.-.at I
~'. ae mc:c~:ons n: ac :e:ow w,H oe sucm,r:: :o :ae iias.~..at:onai ~uresu
ia:~ :.-tr:r~:re;ersr~ r.~lrt oru~ m= ass r.;.. :ac:miort I
'. yt~tr ~f ~t?G. t ~, i
i
.
I
/ =:r :e::rnn; Ofr:c-. .use on,Y =ar ir.::.~..a::onu 3caai: a. on: ~ -~~i
y( ; :~.a tats: was r:::r,:a .rttn ;ne :na.-.:a::onat s::uearton (~ ', ii:
tote: ~>rss r::: v~ :~~ .-s l:::e-a::una 3u: =a :r:
Z ~ novE~,~3a Isss
.auG.or:::: a:T::-
.7 7 Autaor:x:: at::c::
Form?'T.,"ZOlti~(luy t99=)
CA 02311282 2000-OS-26
WO 99/28449 PCT/GB98/03558
_ 78
~ooucs:c s or .ys.-r s ;h' p/3330.W0 CTH I m~wuau aooucauun
~:rcrsic: nunce.~
tvOtC~TtOnS REL.aTtvG TO .~ DEPOStTED W tCROORG~:ilSV1
(PCT Rule 136ts1
T:~e mmuaons ramie 6e:ow reeste
w ;ne mtcrourg:msm re.'e-'eri
w m cne >iesenpuun
on oagc 27 . line 19
B. IOE~TIFIC.aTtON OF pEPOSIT
furtha oeposus are idenutied
on aer addiuonal slt~ o
,Vame of tiepuatt_.~i ulsututton
The wational Collections of
Industrial and Marine Bacteria
Limited (NCIMB)
.adrtrsss of dr_ostta:y msucuuon
hnc:udmq posrce case enu eoanrryl
t 23 St Macnar Orive
Aberan
I .a8? ,,y
United Kingdom
I
Oate w scposu Accas;on W moo
24 Novealber 1997 NCIhB 40906
C. ADOITtON~L I;IOlCATIOiYS
Ilrave olnnir ynrx ooplteablri
This inlortaarroo is cnounurl
an an muitriuoal sheer Q
In respect oP those designations
in which a European patent
a sought, and any
other desicnated state havin4
eauivalent legislation, a sample
of the deposited
microorganism will only be made
available either until the
publication of the
mention of the grant of the
patent or after twenty years
from the date of filing
if the application has been
refused or withdrawn ar is
deemed to be withdrawn,
only by the issue of sucn a
sample to an exoerc nominates
by the person ceouestinq
j the samoie. ;Rule ?9(0) EPC
-l)
0. OESIGrJITED STATES FOR WHICH
IrDiC.lTtOi'15 .1RE VIADE h/:Hr
tnatectrons are not Ior all
disesnertd Stottsl
I
E. SEPARATE FURNISfitYC OF I~D(CAT10V5
Ilscve otcnit /nor tmoucaoli)
I Tile me;cstsons;:s.e. oetow
wed oe suomtaee to :ae lnr:~rartonst
Bureau are: ticecgiuayerircenemrsw~u~e:c~:onrs.3..
v rs:eron
a unrosr of Ccaaea ')
,~,/ ?u recmmns Otlic: use omY .-.-'or Inte~.:auocu 3uresu use onr~
~'va me-_: was rr_aveo ~.nrn me :nre:rauona; aoouescton C ; oi: sne:: wu
:::::v:. :v ~.-o ;-r..-..s::onm 3ur:;u an:
2 7 HOVEtr;BER 1998
.author:; = ot':c::
.~uteonus ottic::
n 3.2.9
Fanu P="..:ZOII 3a (July 199'.1
