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Patent 2385162 Summary

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(12) Patent Application: (11) CA 2385162
(54) English Title: COMPOSITIONS AND METHODS FOR ALTERING GENE EXPRESSION
(54) French Title: COMPOSITIONS ET METHODES POUVANT MODIFIER L'EXPRESSION GENIQUE
Status: Dead
Bibliographic Data
(51) International Patent Classification (IPC):
  • C12N 15/54 (2006.01)
  • A01K 67/027 (2006.01)
  • C12N 1/21 (2006.01)
  • C12N 5/10 (2006.01)
  • C12N 9/10 (2006.01)
  • C12N 15/63 (2006.01)
  • C12N 15/67 (2006.01)
  • C12N 15/85 (2006.01)
  • C12N 15/90 (2006.01)
(72) Inventors :
  • FODOR, WILLIAM L. (United States of America)
  • RAMSOONDAR, JAGDEECE J. (United States of America)
(73) Owners :
  • ALEXION PHARMACEUTICALS, INC. (United States of America)
(71) Applicants :
  • ALEXION PHARMACEUTICALS, INC. (United States of America)
(74) Agent: OSLER, HOSKIN & HARCOURT LLP
(74) Associate agent:
(45) Issued:
(86) PCT Filing Date: 2000-10-02
(87) Open to Public Inspection: 2001-04-05
Availability of licence: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): Yes
(86) PCT Filing Number: PCT/US2000/027065
(87) International Publication Number: WO2001/023541
(85) National Entry: 2002-03-28

(30) Application Priority Data:
Application No. Country/Territory Date
60/156,953 United States of America 1999-09-30

Abstracts

English Abstract




Disclosed are novel compositions and methods useful for modulating expression
of a target gene in a cell by insertion of exogenous DNA sequence into the
target gene. The compositions and methods of the invention are useful for
generation of knockout animals including mammals.


French Abstract

L'invention concerne de nouvelles compositions et méthodes pouvant moduler l'expression d'un gène cible dans une cellule, par insertion d'une séquence d'ADN exogène dans le gène cible. Les compositions et méthodes de l'invention sont utiles pour la production d'animaux à gène inactivé, notamment de mammifères.

Claims

Note: Claims are shown in the official language in which they were submitted.



We Claim:

1. A method of modulating the expression of a eukaryotic gene in a cell,
comprising
transfecting said cell with a nucleic acid construct, said construct
comprising a first
construct sequence homologous to a first gene sequence, a sequence encoding a
selectable
marker, and a second construct sequence homologous to a second gene sequence,
wherein said
first and second gene sequences independently comprise at least a portion of
one or more intron
regions of said eukaryotic gene, and

integrating said selectable marker into said eukaryotic gene,
wherein expression of said selectable marker results in modulation of
expression of said
eukaryotic gene in said cell.

2. The method of claim 1, wherein said first construct sequence and said
second construct
sequence are each homologous to at least a portion of an intron region of the
gene.

3. The method of claim 1, wherein said sequence encoding a selectable marker
is integrated
into said eukaryotic gene by homologous recombination, wherein said first
construct sequence
recombines with said first gene sequence and said second construct sequence
recombines with
said second gene sequence to insert the selectable marker into the gene.

4. The method of claim 1, further comprising screening said cell for
expression of said
selectable marker.

5. The method of claim 2, wherein said first construct sequence and said
second construct
sequence are homologous to different regions from within the same intron of
the gene.


49


6. The method of claim 2, wherein said first construct sequence and said
second construct
sequence are homologous to regions of different introns.

7. The method of claim 1, wherein said selectable marker gene is an antibiotic
resistance
gene.

8. The method of claim 1, wherein said sequence encoding a selectable marker
is a
nucleotide sequence which, when expressed, confers a phenotype selected from
the group
consisting of ampicillin resistance, kanamycin resistance, gentecin
resistance, neomycin
resistance, puromycin resistance, hygromycin b resistance, thymidine kinase
activity, tryptophan
synthetase activity, adenine phosphribosyltransferase activity, dihydrofolate
reductase activity,
and histidinol dehydrogenase, anthocyanin, bets-glucuronidase and luciferase.

9. The method of claim 8, wherein said sequence encoding a selectable marker
confers
neomycin resistance or puromycin resistance.

10. The method of claim 1, wherein said eukaryotic gene is selected from the
group
consisting of genes encoding B7.3, P-selectin, E-selectin, ICAM-1, ICAM-2,
VCAM-1, CD28,
CD80, CD86, CD154, major histocompatibility complex class I .beta.-2-
microglobulin, invariant
chain, caspase-1 caspase-3, and Gal .alpha.(1,3) galactosyl transferase.

11. The method of claim 10, wherein said eukaryotic gene encodes Gal
.alpha.(1,3) galactosyl
transferase.

12. The method of claim 11, wherein said Gal .alpha.(1,3) galactosyl
transferase gene is a porcine
gene.


50



13. The method of claim 12, wherein said first construct sequence and said
second construct
sequence are independently selected from homologous regions of the intron
selected from the
group consisting of intron 3, intron 4, intron 5, intron 6, intron 7, intron
8, and intron 9 of the
porcine Gal .alpha.(1,3) galactosyl transferase gene.

14. The method of claim 13, wherein intron 4 has the nucleotide sequence of
nucleotides
4938-11716 in Figure 1.

15. The method of claim 13, wherein intron 5 has the nucleotide sequence of
nucleotides
11753-13748 in Figure 1.

16. The method of claim 13, wherein intron 6 has the nucleotide sequence of
nucleotides
13810-14358 in Figure 1.

17. The method of claim 13, wherein intron 7 has the nucleotide sequence of
nucleotides
14463-21627 in Figure 1.

18. The method of claim 13, wherein intron 8 has the nucleotide sequence of
nucleotides 21766-
27048 in Figure 1.

19. The method of claim 13, wherein said first construct sequence and said
second construct
sequence are homologous to different regions within the same intron of the
eukaryotic gene.

20. The method of claim 19, wherein said intron is intron 3 of the porcine Gal
.alpha.(1,3)
galactosyl transferase gene.

21. The method of claim 13, wherein said first construct sequence and said
second construct
sequence are homologous to different introns of porcine Gal .alpha.(1,3)
galactosyl transferase gene.


51


22. The method of claim 21, wherein said first construct sequence is upstream
of said second
construct sequence.

23. The method of claim 21, wherein said first construct intron region is
homologous to an
intron 3 region and said second construct intron region is homologous to an
intron 4 region of
porcine Gal .alpha.(1,3) galactosyl transferase.

24. The method of claim 2, wherein said sequence encoding a selectable marker
is a
promoterless gene.

25. The method of claim 2, wherein said sequence encoding a selectable marker
further
comprises a promoter.

26. The method of claim 25, wherein said promoter is a phoshoglycerate kinase
(PGK)
promoter.

27. The method of claim 2, wherein said sequence encoding a selectable marker
is
transcribed in the opposite orientation relative to the orientation of said
eukaryotic gene.

28. The method of claim 27, wherein said sequence encoding a selectable marker
further
comprises a promoter sequence.

29. The method of claim 1, wherein said cell is selected from the group
consisting of a
fibroblast, epithelial cell, endothelial cell, transgenic embryonic
fibroblast, embryonic stem cell,
and primordial germ cell.


52


30. The method of claim 2, wherein said cell is a porcine cell.

31. The method of claim 2, wherein said construct further comprises an AG
dinucleotide
splice acceptor site.

32. The method of claim 2, wherein said construct further comprises a GT
dinucleotide splice
donor site.

33. A nucleic acid construct comprising a first construct sequence homologous
to a first gene
sequence, a sequence encoding a selectable marker, and a second construct
sequence
homologous to a second gene sequence, wherein said first and second gene
sequences
independently comprise at least a portion of one or more intron regions of a
eukaryotic gene.

34. The nucleic acid construct of claim 33, further comprising an AG
dinucleotide splice
acceptor site.

35. The nucleic acid construct of claim 33, further comprising a GT
dinucleotide splice donor
site.

36. The nucleic acid construct of claim 33, further comprising a Kozak
consensus sequence.

37. The nucleic acid construct of claim 33, wherein said sequence encoding a
selectable
marker is a nucleotide sequence, which when expressed, confers a phenotype
selected from the
group consisting of ampicillin resistance, kanamycin resistance, gentecin
resistance, neomycin
resistance, puromycin resistance, hygromycin b resistance, thymidine kinase
activity, tryptophan
synthetase activity, adenine phosphribosyltransferase activity, dihydrofolate
reductase activity,
and histidinol dehydrogenase, anthocyanin, bets-glucuronidase and luciferase.


53



38. The nucleic acid construct of claim 37, wherein said sequence encoding a
selectable
marker confers puromycin resistance or neomycin resistance.

39. The nucleic acid construct of claim 33, wherein said eukaryotic gene is
selected from the
genes encoding B7.3, P-selectin, E-selectin, ICAM-1, ICAM-2, VCAM-1, CD28,
CD80, CD86,
CD154, major histocompatibility complex class I .beta.-2-microglobulin,
invariant chain, caspase-1,
caspase-3, and Gal .alpha.(1,3) galactosyl transferase.

40. The nucleic acid construct of claim 39, wherein said eukaryotic gene is
porcine Gal
.alpha.(1,3) galactosyl transferase.

41. The nucleic acid construct of claim 40, wherein said first construct
sequence and said
second construct sequence are independently selected from homologous regions
of the intron
selected from the group consisting of intron 3, intron 4, intron 5, intron 6,
intron 7, intron 8, and
intron 9 of the porcine Gal .alpha.(1,3) galactosyl transferase gene.

42. The nucleic acid construct of claim 41, wherein intron 4 has the
nucleotide sequence of
nucleotides 4938-11716 in Figure 1, intron 5 has the nucleotide sequence of
nucleotides 11753-
13748 in Figure 1, intron 6 has the nucleotide sequence of nucleotides 13810-
14358 in Figure 1,
intron 7 has the nucleotide sequence of nucleotides 14463-21627 in Figure 1,
and intron 8 has the
nucleotide sequence of nucleotides 21766-27048 in Figure 1.

43. The nucleic acid construct of claim 41, wherein said first construct
sequence and said
second construct sequence are homologous to different regions within the same
intron of the
eukaryotic gene.


54



44. The nucleic acid construct of claim 43, wherein said intron is intron 3 of
the porcine Gal
.alpha.(1,3) galactosyl transferase gene.

45. The nucleic acid construct of claim 41, wherein said first construct
sequence and said
second construct sequence are homologous to different introns of porcine Gal
a(1,3) galactosyl
transferase gene.

46. The nucleic acid construct of claim 45, wherein said first construct
sequence is
homologous to an intron 3 region and said second construct sequence is
homologous to an intron
4 region of porcine Gal .alpha.(1,3) galactosyl transferase.

47. A cell transfected with the nucleic acid construct of claim 33.

48. A cell transfected with the nucleic acid construct of claim 41.

49. A cell transfected with the nucleic acid construct of claim 44.

50. A cell transfected with the nucleic acid construct of claim 46.

51. A bacterial cell transformed with the nucleic acid construct of claim 33.

52. A bacterial cell transformed with the nucleic acid construct of claim 41.

53. A bacterial cell transformed with the nucleic acid construct of claim 44.

55. A bacterial cell transformed with the nucleic acid construct of claim 46.

56. A nucleotide sequence of intron 4 of the Gal .alpha.(1,3) galactosyl
transferase gene having
nucleotides 4938-11716 in Figure 1.


55



57. A nucleotide sequence of intron 5 of the Gal .alpha.(1,3) galactosyl
transferase gene having
nucleotides 11753-13748 in Figure 1.

58. A nucleotide sequence of intron 6 of the Gal .alpha.(1,3) galactosyl
transferase gene having
nucleotides 13810-14358 in Figure 1.

59. A nucleotide sequence of intron 7 of the Gal .alpha.(1,3) galactosyl
transferase gene having
nucleotides 14463-21627 in Figure 1.

60. A nucleotide sequence of intron 8 of the Gal .alpha.(1,3) galactosyl
transferase gene having
nucleotides 21766-27048 in Figure 1.

61. A lambda phage clone derived from a porcine genomic library comprising at
least a portion of
the Gal .alpha.(1,3) galactosyl transferase gene, wherein the lambda phage
clone is selected from the group
consisting of pgGT, lambda 1, lambda 2, lambda 4-1 and lambda 8-2.

62. A method of making a transgenic mammal comprising transfecting a nuclear
donor cell with
the nucleic acid construct of claim 33, selecting for transfected cells
comprising the nucleic acid of the
construct, introducing said selected cells into an embryo, impregnating said
embryo into an
appropriate host mammal, and generating offspring from said impregnated host
mammal.

63. A method of making a transgenic mammal comprising transfecting a nuclear
donor cell with
the nucleic acid construct of claim 44, selecting for transfected cells
comprising the nucleic acid of the
construct, introducing said selected cells into an embryo, impregnating said
embryo into an
appropriate host mammal, and generating offspring from said impregnated host
mammal.


56


64. A method of making a transgenic mammal comprising transfecting a nuclear
donor cell with
the nucleic acid construct of claim 46, selecting for transfected cells
comprising the nucleic acid of the
construct, introducing said selected cells into an embryo, impregnating embryo
into an appropriate
host mammal, and generating offspring from said impregnated host mammal.

65. A transgenic mammal made according to the method of claim 62.

66. A transgenic mammal made according to the method of claim 63.

67. A transgenic mammal made according to the method of claim 64.

68. A method of reducing transplant rejection comprising transfecting a
nuclear donor cell with
the nucleic acid construct of claim 32, selecting for transfected cells
comprising the nucleic acid of the
construct, introducing said selected cells into an embryo, impregnating embryo
into an appropriate
host mammal, generating offspring from said impregnated host mammal,
harvesting cells, tissue, or
organs from said offspring, and transplanting said harvested cells, tissue, or
organs into a patient in
need thereof.

69. A method of reducing transplant rejection comprising transfecting a
nuclear donor cell with
the nucleic acid construct of claim 44, selecting for transfected cells
comprising the nucleic acid of the
construct, introducing said selected cells into an embryo, impregnating embryo
into an appropriate
host mammal, generating offspring from said impregnated host mammal,
harvesting cells, tissue, or
organs from said offspring, and transplanting said harvested cells, tissue, or
organs into a patient in
need thereof.

70. A method of reducing transplant rejection comprising transfecting a
nuclear donor cell with
the nucleic acid construct of claim 46, selecting for transfected cells
comprising the nucleic acid of the
construct, introducing said selected cells into an embryo, impregnating embryo
into an appropriate


57


host mammal, generating offspring from said impregnated host mammal,
harvesting cells, tissue, or
organs from said offspring, and transplanting said harvested cells, tissue, or
organs into a patient in
need thereof.

71. The nucleic acid construct of claim 43, further comprising a nucleic acid
sequence
encoding a gene which is toxic to said eukaryotic cell

72. The nucleic acid construct of claim 71, wherein said gene which is toxic
to said
eukaryotic cell is the ricin A toxin gene.


58

Description

Note: Descriptions are shown in the official language in which they were submitted.



CA 02385162 2002-03-28
WO 01/23541 PCT/US00/27065
COMPOSITIONS AND METHODS FOR ALTERING GENE EXPRESSION
TECHNICAL FIELD
The present invention is directed generally to the biological sciences,
including
recombinant genetics and immunology. More particularly, there are described
herein non-human
knockout animals, preferably mammals, in which the expression of one or more
genes has been
altered. Also provided herein are xenograft transplants in which the
expression of one or more
genes has been modulated to prevent or reduce the likelihood of rejection by
the transplant
recipient.
BACKGROUND
The immune response of mammals, including humans, against invading pathogens,
toxins, and other foreign substances involves many specialized cells that act
together.
Lymphocytes are a class of white blood cells responsible for the specificity
of the immune
system. Two important classes of lymphocytes are T cells and B cells. T cells
develop in the
thymus, and are responsible for cell mediated immunity. There are many types
of specialized T
cells, such as for example, helper T cells (which enhance the activity of
other types of white
blood cells), suppressor T cells (which suppress the activity of other white
blood cells), and
cytotoxic T cells (which kill cells). B cells develop in the bone marrow and
exert their effect by
producing and secreting antibodies.
A key to the coordinated immune response is complement, which, as described in
U.S.
Patent No. 5,679,345, is involved in the pathogenesis of tissue injury
observed in many
immunologically mediated diseases, such as systemic lupus, erythematosis,
rheumatoid arthritis,


CA 02385162 2002-03-28
WO 01/23541 PCT/LJS00/27065
and immune-hemolytic anemia. Complement is also involved in rejection of
transplanted organ
grafts. Complement is responsible for much of the tissue injury in
transplantation due to
inflammatory conditions resulting from rejection or superimposed by infection,
ischemia, and
thrombosis of vessels in the graft, as well as tissue injury due to
inflammation from similar
causes in patients who have not received an organ transplant. In particular,
complement attack
on cells is central to the rapid onset phase of immune mediated graft
rejection (hyperacute
rejection), where complement activation and subsequent tissue damage occur
within hours.
Graft rejection may occur through a number of different mechanisms, with the
time
course of rejection being characteristic of the particular mechanism. Early
rejection (hyperacute
rejection), occurring within minutes or hours of transplantation, involves
complement activation
by components that are present at the time of the transplant operation.
Activation may occur via
the classical pathway by preformed antibodies that are reactive with the
"foreign" or non-self
markers of the graft or via the alternative pathway in response to tissue
damage in the graft as a
result of, for example, ischemic damage to the organ during storage before
transplantation.
Acute rejection occurs days to weeks after transplantation, and is caused by
sensitization of the
host to the foreign tissue that makes up the graft. Once the host's immune
system has identified
the transplanted tissue as foreign, all the resources of the immune system are
marshaled against
the graft, including both specific (antibody and T cell-dependent) responses
and non-specific
(phagocytic and complement-dependent) responses. Chronic rejection will
usually only occur
when the graft recipient is immune-suppressed. Then the graft may survive long
enough for
tissue to undergo changes which ultimately affect survival of the graft. Such
changes include
hyperplasia and tissue hypertrophy, and endothelial cell damage leading to
narrowing of the
vascular lumen and potentially impairing the oxygen supply of the graft
tissue.
2


CA 02385162 2002-03-28
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Xenograft rejection of pig tissue is triggered by natural human antibodies
that recognize
carbohydrate xeno-antigens, such as Gal a(1,3) galactose, which is expressed
on pig endothelial
cells that line blood vessels. Weiss, Science, 285(20):1221-1222 (August 20,
1999). U.S. Patent
No. 5,821,117 describes inhibiting xenotransplant rejection by disrupting the
wild type porcine
Gal a(1,3) galactosyl transferase gene with a cloned mutant porcine Gal a(1,3)
galactosyl
transferase sequence specifically within an exon of the wild type gene. The
resultant mutant
gene does not encode a functional galactosyl transferase, with the expected
result that rejection
of the transplanted xenograft by the patient's immune system is avoided.
In such so called "knockout" mammals, expression of an endogenous gene has
been
altered (typically, suppressed) through genetic manipulation. Preparation of
knockout mammals
typically has required introducing into an undifferentiated cell type (termed
an embryonic stem
cell) a nucleic acid construct to suppress expression of a target gene. This
cell is introduced and
integrated into a mammalian embryo. The embryo is implanted into a foster
mother for the
duration of gestation. For example, Pfeffer et al. (Cell, 73:457-467 [1993])
describe mice in
which the gene encoding the tumor necrosis factor receptor p55 has been
disrupted by mutation
utilizing homologous recombination. The mice showed a decreased response to
tumor necrosis
factor signaling. Fung-Leung et al. (Cell, 65:443-449 [1991]; J. Exp. Med.,
174:1425-1429
[1991]) describe knockout mice lacking expression of the gene encoding CDB.
These mice were
found to have a decreased level of cytotoxic T cell response to various
antigens and to certain
viral pathogens such as lymphocytic choriomeningitis virus.
Typical prior methods, however, describe manipulation of an exon region of the
target
gene. There is thus a need in the art for new and improved methods for
modulating gene
3


CA 02385162 2002-03-28
WO 01/23541 PCT/US00/27065
expression in animals including mammals, particularly for overcoming xenograft
transplant
rejection. It is to these, as well as other, important ends that the following
is addressed.
SUMMARY
It has been discovered that the expression of a particular gene in an animal
may be
modulated by introducing into the genomic DNA of the animal a new DNA sequence
that results
in the disruption of at least some portion of the DNA sequence of the gene to
be modulated. The
methods described herein are of general utility for altering gene expression
in animals including
mammals. In contrast to prior methods, it has surprisingly been found that
gene expression may
be suppressed in part or in total by inserting new DNA sequence into the
intron of the target
genomic DNA.
The versatility of the methods described herein for generating "knockout"
animals is
illustrated by the following general description of a preferred embodiments,
including the
examples. It is to be understood that while the remaining discussion is
directed largely to the
utility of Gal a(1,3)galactosyl transferase knockout pigs, the utility of the
methods described
herein is not limited to solely this protein. Rather, the following discussion
is provided merely
for exemplification of their versatility and preferred use.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the nucleotide sequence of introns of the porcine Gal a(1,3)
galactosyl
transferase gene from within intron 3 to the end of intron 8. Dashes indicate
nucleotides within
an exon region. Thus, nucleotide sequence numbering represents the number of
bases in the
entire porcine Gal a(1,3) galactosyl transferase gene relative to nucleotide
position 1 of the insert
isolated from the lambda-2 phage clone.
4


CA 02385162 2002-03-28
WO 01/23541 PCT/US00/27065
FIG. 2 shows a schematic representation of the gene targeting vector used for
inactivation
of the porcine Gal a(1,3) galactosyl transferase gene (see Example 1). This
vector is designed to
contain a sequence with homology to the 5' region of intron 3 of the Gal
a(1,3) galactosyl
transferase gene, a promoterless neomycin phosphotransferase gene engineered
to contain multiple
stop codons (engineered exon), an engineered splice acceptor site, the 5'
region of intron 4 sequence
for splicing the engineered exon to the downstream exon 4, and a sequence with
homology to the 3'
region of intron 3 to aid with annealing to the porcine Gal a(1,3) galactosyl
transferase gene. Arrows
indicate location of primers used for PCR.
FIG. 3 shows the nucleotide sequence of the gene targeting vector used for
inactivation of
the porcine Gal a(1,3) galactosyl transferase gene (see Example 1). This
vector is designed to
contain (A.) a sequence with homology to the 5' region of intron 3 of the Gal
a(1,3) galactosyl
transferase gene, (B.) an intron 4 splice acceptor sequence, (C.) a
promoterless neomycin
phosphotransferase gene engineered to contain multiple stop codons (engineered
exon), (D.) an intron
4 splice donor signal sequence, and (E.) a 3' intron 3 sequence to aid with
annealing to the porcine Gal
a(1,3) galactosyl transferase gene. All underlined sequences correspond to
restriction sites in the
primer sequences. Bold type indicates primer regions used for PCR. Normal type
indicates PCR
fragment sequences.
FIG. 4 shows the nucleotide sequence for the neomycin phosphotransferase gene
(the
neomycin resistance gene). Bold type indicates the location of gene start and
stop codons. The
underlined sequence corresponds to primer sequences. Nucleotides which are
capitalized are
within the coding region of this gene.
FIG. 5 shows the nucleotide sequence of the puromycin/bovine growth hormone
poly A.
The underlined sequences correspond to the puromycin gene start codon.


CA 02385162 2002-03-28
WO 01/23541 PCT/tJS00/27065
FIG. 6 shows a schematic representation of the gene targeting vector used for
inactivation
of the porcine Gal a(1,3) galactosyl transferase gene (see Example 2). This
vector is designed to
contain a sequence with homology to the Gal a(1,3) galactosyl transferase gene
3' intron 3
sequence including the 3' intron splice acceptor sequence, a Kozak consensus
sequence, a
promoterless puromycin gene engineered to contain a bovine growth hormone poly
A sequence
(engineered exon), and a sequence with 5' intron 4 sequence homology including
the 5' intron splice
donor sequence. Arrows indicate location of primers used for PCR.
FIG. 7 shows the nucleotide sequence of the gene targeting vector shown
schematically
in FIG. 6 (see Example 2). The underlined sequences correspond to the primer
sequences used.
Bold type indicates the intron regions used for homology. The AG and GT splice
consensus
sequences at the 3' end of intron 3 and the 5' end of intron 4 are in upper
case.
FIG. 8 shows the nucleotide sequence of the ricin A toxin gene. Nucleotides
which are
capitalized are within the coding region of this gene.
FIG. 9 shows a schematic representation of the collision construct used for
inactivation of
the porcine Gal a(1,3) galactosyl transferase gene (see Example 3). This
vector is designed to
contain a sequence with homology to the Gal a(1,3) galactosyl transferase gene
3' intron 3
sequence including the 3' intron splice acceptor sequence, a reverse
orientation puromycin gene
engineered to contain a bovine growth hormone poly A sequence under the
control of a
phosphoglycerate kinase (PGK) promoter, and a sequence with 5' intron 4
sequence homology
including the 5' intron splice donor sequence, and a ricin A toxin gene under
the control of a
cytomegalovirus (CMV) promoter and containing a SV40 poly A sequence located
outside the
regions of homology. Arrows indicate location of primers used for PCR.
6


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DESCRIPTION OF THE PREFERRED EMBODIMENTS
Technical and scientific terms used herein have the same meanings as commonly
understood by one of ordinary skill in the art, unless otherwise defined
herein. Although any
methods and materials similar or equivalent to those described herein may be
used in the practice
or testing of the described methods, preferred methods, devices, and materials
are now described.
All publications mentioned herein are incorporated herein by reference for the
purpose of
describing and disclosing the cell lines, vectors, and methodologies which are
reported in the
publications which might be used in connection herewith.
As used herein and in the appended claims, the singular forms "a," "an," and
"the" are
intended to include the plural reference unless the context clearly dictates
otherwise. Thus, for
example, reference to "a host cell" is intended to include a plurality of such
host cells, reference
to "an antibody" is intended as a reference to one or more antibodies and
equivalents thereof
known to those skilled in the art, and so forth. It is to be understood that
the appended claims are
not limited to the particular methodology, protocols, cell lines, vectors, and
reagents described,
which those of skill will appreciate may vary. It is also to be understood
that the terminology
used herein is for the purpose of describing particular embodiments only, and
is not intended to
limit the scope of the present invention, which is to be limited only by the
appended claims.
The term "knockout" refers to the modulation of the expression of at least a
portion of a
protein encoded by the target gene. The term "knockout construct" refers to a
nucleic acid
sequence that is designed to modulate a protein encoded by endogenous DNA
sequences in a
cell. The nucleic acid sequence used as the knockout construct is typically
comprised of DNA
from some portion of the gene or genes (including, but not limited to, the
exon sequence, intron
7


CA 02385162 2002-03-28
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sequence, and/or promoter sequence) to be modulated and a sequence marker used
to disrupt and
select for the presence of the knockout construct in the cell. The nucleic
acid sequence of the
knockout construct is inserted into a cell, and integrates with the genomic
DNA of the cell in
such a position so as to prevent or interrupt protein expression from the
native gene. Such
insertion usually occurs by homologous recombination (i.e., regions of the
knockout construct
that are homologous to endogenous DNA sequences hybridize to each other when
the knockout
construct is introduced into the cell and recombines so that the knockout
construct is
incorporated into the corresponding position of the endogenous DNA).
The knockout construct nucleic acid sequence may comprise a full or partial
sequence of
one or more exons and/or introns of the gene to be modulated, a full or
partial promoter sequence
of the gene to be modulated, or combinations thereof. In one embodiment of the
invention, the
nucleic acid sequence of the knockout construct comprises a first nucleic acid
sequence region
homologous to a first nucleic acid sequence region of the gene to be
modulated, and a second
nucleic acid sequence region homologous to a second nucleic acid sequence
region of the gene to
be modulated. The orientation of the knockout construct should be such that
the first nucleic
acid sequence is upstream of the second nucleic acid sequence and the sequence
marker should
be therebetween.
A suitable nucleic acid sequence regions) should be selected so that there is
homology
between knockout construct sequences) and the gene of interest. Preferably,
the knockout
construct sequences are isogenic sequences with respect to the target
sequences. The nucleic
acid sequence region of the knockout construct may correlate to any region of
the gene provided
that it is homologous to the gene. A nucleic acid sequence is considered to be
"homologous" if it
is at least about 90% identical, preferably at least about 95% identical, or
most preferably, about
8


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100% identical to the nucleic acid sequence. Furthermore, the 5' and 3'
nucleic acid sequences
flanking the selectable marker should be sufficiently large to provide
complementary sequence
for hybridization when the knockout construct is introduced into the genomic
DNA of the target
cell. For example, homologous nucleic acid sequences flanking the selectable
marker gene
should be at least about 500 bp, preferably, at least about 1 kilobase (kb),
more preferably about
2-4 kb, and most preferably about 3-4 kb in length. In a preferred embodiment,
both of the
homologous nucleic acid sequences flanking the selectable marker gene of the
construct should
be should be at least about 500 bp, preferably, at least about 1 kb, more
preferably about 2-4 kb,
and most preferably about 3-4 kb in length.
Another suitable DNA sequence includes cDNA sequence provided the cDNA is
sufficiently large. Each of the flanking nucleic acid sequences used to make
the construct is
preferably homologous to one or more exon and/or intron regions, and/or a
promoter region.
Each of these sequences is different from the other, but may be homologous to
regions within the
same exon and/or intron. Alternatively, these sequences may be homologous to
regions within
different exons and/or introns of the gene. Preferably, the two flanking
nucleic acid sequences of
the knockout construct are homologous to two sequence regions of the same or
different introns
of the gene of interest. In addition, it is preferred that isogenic DNA is
used to make the
knockout construct of the present invention. Thus, the nucleic acid sequences
obtained to make
the knockout construct are preferably obtained from the same cell line as that
being used as the
target cell.
In accordance with the present invention, the integration of the knockout
construct
nucleic acid sequence into at least one gene of interest results in the
modulation of the expression
of the gene product. "Modulating" the expression of a gene includes
suppressing the expression
9


CA 02385162 2002-03-28
WO 01/23541 PCT/L1S00/27065
of the gene, disrupting the expression of the gene, eliminating the expression
of the gene, altering
the expression of the gene, or decreasing the expression of the gene relative
to expression of the
wild-type gene. Preferably, the integrated knockout construct results in
reduced protein function
relative to native protein function. Most preferably, the integrated knockout
construct results in
the production of a non-functional protein. Complete or absolute non-
functionality of the protein
is not required.
The phrases "disruption of the gene" and "gene disruption" refer to insertion
of a nucleic
acid sequence into at least one region of the native DNA sequence (usually one
or more exons or
one or more introns) and/or the promoter region of a gene so as to modulate
expression of that
gene in the cell as compared to the wild-type or naturally occurring sequence
of the gene. By
way of example, a nucleic acid construct may be prepared containing a DNA
sequence encoding
an antibiotic resistance gene which is inserted between the DNA sequence
complementary to the
target gene DNA sequence (promoter and/or coding region) to be disrupted. When
this nucleic
acid construct is then transfected into a cell, the construct will integrate
into the genomic DNA
either randomly or into the target gene by homologous recombination. It has
been found that
selection for drug resistant cells in the population of transfectants enhance
the probability of
obtaining a homologous gene knockout. Thus, many progeny of the cell will no
longer express
the gene, or will express it at a decreased level, as the DNA is now disrupted
by the antibiotic
resistance gene.
In some instances, such as, for example, where the methods described herein
are used to
produce cells, tissues or organs suitable for xenotransplant into humans, it
may not be necessary
to completely eliminate the production of functional protein. Rather, it will
be satisfactory to
reduce the production of functional protein only to a level that will, in
conjunction with other


CA 02385162 2002-03-28
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therapeutic regimens, prevent or reduce the patient's immune response and the
likelihood of
rejection. Thus, for example, a knockout achieved according to the methods
described herein
may preferably reduce the biological activity of the polypeptide normally
encoded therefrom by
at least about 70%, preferably at least about 80%, relative to the unmutated
gene.
The knockout construct may be inserted into any suitable target cell for
integration into
its genomic DNA that may be maintained in culture. Suitable cells include, but
are not limited
to, fibroblast, epithelial cell, endothelial cell, transgenic embryonic
fibroblast, embryonic stem
cell, and primordial germ cell. In one embodiment, the knockout construct is
inserted into an
embryonic stem cell (ES cell) and is integrated into the ES cell genomic DNA.
ES cells
comprising the integrated knockout construct are then injected into, and
integrate with, a
developing mouse embryo. In another embodiment, the knockout construct is
inserted into a
nuclear transfer donor cell. Suitable nuclear transfer donor cells include
fibroblasts, epithelial
cells, and cumulous cells. In this embodiment, the knockout construct is
inserted into the nuclear
transfer donor cell, and the donor cells comprising the knockout construct are
fused with an
enucleated oocyte. The resultant fused oocyte is then transferred to a
surrogate female.
Furthermore, where the target cell is intended to be used to produce a
knockout mammal, it is
preferred that the target cell be derived from the same species as the
knockout mammal to be
generated. Thus, for example, pig embryonic stem cells or pig fibroblasts will
usually be used
for generation of knockout pigs.
The nucleic acid sequence of the knockout construct may be integrated into the
genomic
DNA of the host cell using any suitable method. In one preferred embodiment,
integration is
achieved by the process of homologous recombination. Homologous recombination
has been
described previously, for example, in Kucherlpati et al. (1984) Proc. Natl.
Acad. Sci. USA
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WO 01/23541 PCT/US00/27065
81:3153-3157; Kucherlapati et al. (1985) Mol. Cell. Bio. 5:714-720; Smithies
et al. (1985)
Nature 317:230- 234; Wake et al. (1985) Mol. Cell. Bio. 8:2080-2089; Ayares et
al. (1985)
Genetics 111:375-388; Ayares et al. (1986) Mol. Cell. Bio. 7:1656- 1662; Song
et al. (1987)
Proc. Natl. Acad. Sci. USA 84:6820-6824; Thomas et al. (1986) Cell 44:419-428;
Thomas and
Capecchi ( 1987) Cell 51:503- 512; Nandi et al. ( 1988) Proc. Natl. Acad. Sci.
USA
85:3845-3849; and Mansour et al. (1988) Nature 336:348-352, which are herein
incorporated by
reference. Furthermore, various aspects of using homologous recombination to
create specific
genetic mutations in embryonic stem cells and to transfer these mutations to
the germline have
been described. (Evans and Kaufman ( 1981 ) Nature 294:154-146; Doctschman et
al., ( 1987)
Nature 330:576-578; Thomas and Capecchi ( 1987) Cell 51:503-512; Thompson et
al. ( 1989)
Cell 56:316-321.) In homologous recombination, DNA fragments between two DNA
molecules
are exchanged during crossover at the site of the homologous nucleic acid
sequences. Thus,
crossover would occur between the knockout construct and eukaryotic gene at
the site of
homology within the 5' region of the first nucleic acid sequence of the
construct (homologous to
the first nucleic acid region of the gene of interest). A second crossover
event would occur in the
3' region of the construct homologous to the second nucleic acid region of the
gene of interest.
As a result, the sequence information between these two regions of the
knockout construct would
be inserted into the gene of interest in the host cell's genomic DNA.
The methods described herein may be used to produce a mammal in which one,
two, or
more genes have been knocked out. Such mammals may be generated by repeating
the
procedures set forth herein for generating each knockout construct, or by
breeding two
mammals, each with a different single gene knocked out, to each other, and
screening for those
with the double knockout genotype.
12


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The term "marker sequence" or "selectable marker" refers to a nucleic acid
sequence that
is used as part of the knockout construct to modulate the expression of the
gene of interest, and
as a means to identify those cells that have incorporated the knockout
construct into the genome.
The selectable marker may be any sequence that serves these purposes. For
example, the
selectable marker may encode a protein that confers a detectable trait on the
cell, such as an
antibiotic resistance gene, or an assayable enzyme not typically found in the
target cell. The
selectable marker gene may be any nucleic acid sequence that is detectable
and/or assayable,
which is used to recover transformed cell lines. One having skill in the art
will be capable of
determining suitable selectable markers for use in the present invention. For
example, suitable
selectable markers include, but are not limited to, (3-lactamase (ampicillan
resistance),
kanamycin resistance, gentecin resistance, puromycin-N-acetyl-transferase,
hygromycin b-
phosphotransferase, thymidine kinase, and tryptophan synthetase. For example,
the herpes
simplex virus thymidine kinase (tk) (Wigler, M. et al. ( 1977) Cell 11:223-32)
or adenine
phosphoribosyltransferase (aprt) (Lowy, I. et al. (1980) Cell 22:817-23)
genes, which may be
employed in tk or aprt cells, respectively, may be used as the selectable
marker. Also,
antimetabolite, antibiotic or herbicide resistance may be used as the basis
for selection; for
example, dihydrofolate reductase (dhfr), which confers resistance to
methotrexate (Wigler, M. et
al. ( 1980) Proc. Natl. Acad. Sci. 77:3567-70); neomycin phosphotransferase
(npt), which confers
resistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin, F. et
al (1981) J. Mol.
Biol. 150:1-14). Additional selectable genes have been described and include,
for example,
tryptophan synthetase (trpB), which allows cells to utilize indole in place of
tryptophan, or
histidinol dehydrogenase (hisD), which allows cells to utilize histidinol in
place of histidine
(Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047- 51).
Recently, the
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use of visible markers has gained popularity with such markers as
anthocyanins, beta-
glucuronidase (GUS), and luciferase and its substrate luciferin, being widely
used not only to
identify transformants, but also to quantify the amount of transient or stable
protein expression
attributable to a specific vector system (Rhodes, C. A. et al. (1995) Methods
Mol. Biol.
55:121-131). In the present invention, it is preferred that the selectable
marker gene is an
antibiotic resistance gene, such as the neomycin resistance gene or puromycin
resistance gene.
Moreover, when the selectable marker encodes a protein, it may also contain a
promoter
that regulates its expression, or require expression from an endogenous
promoter, preferably the
target gene promoter. Thus, the selectable marker gene may be operably linked
to its own
promoter or be promoterless. The selectable marker gene may be inserted into
the knockout
construct without its own promoter attached as it may be transcribed using the
promoter of the
gene to be suppressed. In addition, the marker gene may have a polyA signal
sequence attached
to the 3' end of the gene, which serves to terminate transcription of the gene
and process the
transcript with the addition of adenine residues at the 3' end to stabilize
the mRNA.
In one embodiment a target gene (e.g., Gal a(1,3) galactosyl transferase) is
modulated by
insertion of an engineered exon or active gene within an intron of the target
gene. In this
embodiment, the target gene is prevented from being translated by insertion of
an in-frame,
promoterless engineered exon (e.g., an antibiotic resistance gene) that
contains multiple stop codons
within an intron of the target gene. Using this 'promoter-trap' strategy, the
engineered exon is spliced
in frame upstream of the exon comprising the start codon. This results in the
expression of the drug
resistance gene prior to the gene of interest and concomitantly inhibits
expression of the target gene
due to the presence of multiple stop codons downstream of the drug resistance
gene. As described
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herein, any gene that confers survival of the targeted cells under appropriate
selection conditions may
be used as the engineered exon.
Using the 'promoter trap' strategy, a gene targeting construct is designed
which contains
a sequence with homology to an intron sequence of the target gene (e.g., the
intron 3 sequence of
the Gal a(1,3) galactosyl transferase gene), a downstream intron splice
acceptor signal sequence
comprising the AG dinucleotide splice acceptor site (e.g., the intron 4 splice
acceptor signal sequence
of the Gal a(1,3) galactosyl transferase gene), a promoterless selectable
marker engineered exon (e.g.,
drug resistance gene) engineered to contain multiple stop codons, the intron
splice donor signal
sequence comprising the GT dinucleotide splice donor site (e.g., the intron 4
splice donor sequence of
the Gal a(1,3) galactosyl transferase gene) for splicing the engineered exon
to the immediate
downstream exon (e.g., exon 4 of the Gal a(1,3) galactosyl transferase gene),
and additional sequence
with homology to the intron sequence of the target gene (e.g., intron 3
sequence homology of the Gal
a(1,3) galactosyl transferase gene ) to aid with annealing to the target gene.
It will be appreciated that
the method may be used to target any intron within target gene of interest.
In another embodiment, the 'promoter trap' strategy is used to modulate target
gene
expression by replacing an endogenous exon with an in-frame, promoterless
engineered exon (e.g.,
an antibiotic resistance gene). The engineered exon is spliced in frame and
results in the
expression of the drug resistance gene and concomitant inhibited expression of
the full-length target
gene.
This 'promoter trap' gene targeting construct may be designed to contain a
sequence with
homology to the target gene 3' intron sequence upstream of the start codon
(e.g., the Gal a(1,3)
galactosyl transferase gene 3' intron 3 sequence), the upstream intron splice
acceptor sequence
comprising the AG dinucleotide splice acceptor site (e.g., the intron 3 splice
acceptor sequence), a


CA 02385162 2002-03-28
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Kozak consensus sequence, a promoterless selectable marker gene containing
e.g., a poly A
termination sequence (the engineered exon), a splice donor sequence comprising
the GT dinucleotide
splice donor site from a intron region downstream of the start codon (e.g.,
the 5' intron 4 splice donor
sequence), and a sequence with 5' sequence homology to the downstream intron
(e.g., 5' intron 4). It
will be appreciated that the method may be used to target any exon within the
Gal a( 1,3) galactosyl
transferase gene or any other gene of interest. A representative construct
useful for targeting the pig
Gal a(1,3) galactosyl transferase gene was deposited with the American Type
Culture Collection
(ATCC) on 28 September 2000 with accession number and is described herein in
Example 2.
In yet another embodiment, the selectable marker may be inserted into the
knockout
construct in a reverse orientation to the targeted gene. In this embodiment, a
strong promoter is
used with the selectable marker all in the reverse orientation, which drives
transcription in the
reverse direction and therefore, modulates the expression of the targeted
gene. The target gene is
modulated using a "collision construct" to insert an active gene in place of
an exon and at least part of
the flanking introns, including the splice donor and splice acceptor sites.
The inserted gene, such as a
selectable marker gene, is under the control of a highly active promoter such
as the phosphoglycerate
kinase I (PGK) gene promoter, such that transcription of this gene causes the
termination of
transcription of the endogenous gene (Rosario et al., (1996) Nat.
Biotech.l4:1592-1596). The
selectable marker gene is further engineered to contain a transcription
termination sequence. Insertion
of the engineered gene may be made to replace any exon, within any intron, or
portions thereof to
result in a truncated transcript which modulates the expression of a
functional target gene product. It
will be appreciated that this method may be used to target any intron or exon
of interest of the target
gene. Positive selection for transfected cells in which the construct has been
integrated may be
accomplished via expression of the selectable marker gene. As described
herein, it will be appreciated
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that any selection marker gene that confers survival of the targeted cells
under appropriate selection
conditions may be driven by the strong PGK promoter. Additionally, a toxin
gene (e.g., Ricin A
toxin) is preferably engineered into the collision construct inserted to
eliminate random
integration events. A representative collision construct useful for targeting
the pig Gal a(1,3)
galactosyl transferase gene has been deposited with the ATCC on 28 September
2000 with accession
number and is described herein in Example 3.
The integrated selectable marker nucleic acid in the cell is capable of
modulating the
expression of the gene of interest. Expression of the selectable marker allows
for selection of the
cells which comprise the integrated sequence. Modulation of the expression of
the gene of
interest is accomplished by disruption of the endogenous gene by an engineered
exon in forward
or reverse orientation with the endogenous gene.
The term "animal," as used herein, is intended to include any multicellular
eukaryotic
organism, preferred among which are mammals. When used in the context of a
xenograft donor,
the term "mammal" preferably includes, but is not limited to, pigs, sheep,
goats, cows, deer,
rabbits, hamsters, rats, mice, horses, cats, dogs, and the like. Preferably,
humans are excluded.
The term "progeny" refers to any and all future generations derived or
descending from a
particular mammal, i.e., a mammal containing a knockout construct inserted
into its genomic
DNA. Thus, progeny of any successive generation are included herein such that
the progeny, the
Fl, F2, F3 generations, and so on indefinitely, are included in this
definition.
The terms "immunomodulate" and "immunomodulation" refer to changes in the
level of
activity of any components of the immune system as compared to the average
activity of that
component for a particular species. Thus, as used herein, immunomodulation
refers to an
increase or a decrease in activity. Preferably, in accordance with the present
invention, the
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integration of the selectable marker into the gene of interest results in a
decreased immune
response, when the host cell is introduced to a patient. Immunomodulation may
be detected by
assaying the level of antibody reactivity, complement activity, B cells, any
or all types of T cells,
antigen presenting cells, and any other cells believed to be involved in
immune function.
Additionally or alternatively, immunomodulation may be detected by evaluating
the level of
expression of particular genes believed to have a role in the immune system,
the level of
particular compounds such as cytokines (interleukins and the like) or other
molecules that have a
role in the immune system, and/or the level of particular enzymes, proteins,
and the like that are
involved in immune system functioning.
The target gene to be knocked out may be any gene, provided that at least some
sequence
information on the DNA to be disrupted is available to use in the preparation
of both the
knockout construct and the screening probes. It is not necessary that the
entire genomic
sequence of the target gene be known in order to use the methods described
herein.
The target gene to be knocked out preferably will be a gene that is expressed
in mature
and/or immature T cells and/or B cells. It is a further preference that the
target gene is expressed
in target antigen presenting cell, target endothelial cell, target neuronal
cell, or any target cell that
may be attacked by the humoral or cellular immune system of the recipient. The
target gene is
further preferably involved, either directly or indirectly, in the activation
pathway during
inflammation or immunosuppression responses by the immune system, and does not
result in
lethality when knocked out. In accordance with the present invention,
expression of target genes
may advantageously be altered according to the methods described herein to
produce
xenotransplant cells, tissues and organs for use in humans, in order to reduce
or prevent immune
response and rejection by the patient.
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Thus, in accordance with the present invention, any gene may be used provided
that it
can undergo homologous recombination and the expression of which may be
modulated by
insertion of the knockout construct of the present invention. Suitable genes
include, but are not
limited to, B7.3, P-selectin, E-selectin, ICAM-l, ICAM-2 or VCAM-l, CD28,
CD80, CD86,
CD154, major histocompatbility complex class I,13-2-microglobulin, invariant
chain (Ii),
caspase-l, caspase-3, and Gal a(1,3) galactosyl transferase gene. This list is
not intended to be
exhaustive. One having ordinary skill in the art would be capable of
ascertaining suitable genes
to be modulated. Preferably, the gene is implicated in the immunoresponse
system of a patient.
More preferably, the target gene is a porcine target gene selected from the
group consisting of
CD 80, CD 86, B7.3, P-selectin, E-selectin, ICAM-1, ICAM-2 or VCAM-1. A
presently
preferred porcine target gene is the Gal a(1,3) galactosyl transferase gene.
The DNA sequence to be used to knock out a selected gene may be obtained using
methods well known in the art such as those described by Sambrook et al.
(Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y. (1989)).
Such methods include, for example, screening a genomic library with a cDNA
probe encoding at
least a portion of the same gene in order to obtain at least a portion of the
genomic sequence.
Alternatively, if a cDNA sequence is to be used in a knockout construct, the
cDNA may be
obtained by screening a cDNA library with oligonucleotide probes or antibodies
(where the
library is cloned into an expression vector). If a promoter sequence is to be
used in the knockout
construct, synthetic DNA probes may be designed for screening a genomic
library containing the
promoter sequence. Another method for obtaining the DNA to be used in the
knockout construct
is to manufacture the DNA sequence synthetically, using a DNA synthesizer.
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In another embodiment, porcine genomic DNA encoding the Gal a(1,3) galactosyl
transferase gene is isolated from a lambda phage clone library. A pig genomic
library is screened
using a cDNA corresponding to exon 4 of the Gal a(1,3) galactosyl transferase
gene. Phage are
screened and unique clones, which contain exon 4 sequences are isolated using
standard library
screening methods. (Sambrook et al.) Clones obtained by this procedure contain
inserts 15-40 kb
in length. These clones, were designated pgGT, lambda l, lambda 2, lambda 4-1
and lambda 8-
2. Five vectors comprising unique, overlapping nucleotide sequences which span
the entire the
pig Gal a(1,3) galactosyl transferase gene from within intron 3 through intron
8 have been
deposited with the ATCC: ( 1 ) a 1.6 kb insert within intron 3 of the extreme
5' end of the 18.275
kb lambda-2 phage clone, (2) a 6.7 kb Hind>ZI fragment spanning intron 3 to
intron 4 of the
18.275 kb lambda-2 phage clone, (3) a 4 kb HindIll fragment following the 6.7
kb fragment 2 of
the 18.275 lambda-2 phage clone, (4) a 6 kb HindllI-SaII fragment at the 3'
most portion of the
18.275 lambda-2 phage clone, and (5) a 13 kb fragment of the lambda-2 phage
clone spanning
exon 7 to exon 9. These five vectors were deposited with ATCC on 29 September
2000 with
accession numbers , respectively. Subclones of the various inserts were
used to generate the claimed intron sequences from within intron 3 to intron 8
as provided in
Figure 1. These sequences may be used to determine regions of sequence
homology in design of
targeting constructs for modulation of the pig Gal a(1,3) galactosyl
transferase gene.
The DNA sequence encoding the knockout construct is preferably generated in
sufficient
quantity for genetic manipulation and insertion the target cell. Amplification
may be
accomplished by known methods, such as by placing the sequence into a suitable
vector and
transforming bacterial or other cells that may rapidly amplify the vector, by
PCR amplification,
or by synthesis with a DNA synthesizer.


CA 02385162 2002-03-28
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The DNA sequence to be used in producing the knockout construct is digested
with a
restriction enzyme selected to cut at a locations) such that a new DNA
sequence encoding a
selectable marker gene may be inserted in the proper position within this DNA
sequence. The
proper position for a selectable marker gene insertion is that which will
serve to modulate
expression of the native gene. This position will depend on various factors
such as the restriction
sites in the sequence to be cut, and whether, for example, an intron sequence,
an exon sequence
or a promoter sequence is (are) to be modulated. In other words, the precise
location of insertion
of the selectable marker into the DNA sequence is that which will result in
the modulation of
promoter function or of synthesis of the native exon. For example, the
knockout construct may
be engineered to insert the selectable marker entirely within a single intron
of the target gene. In
this manner, the first nucleic acid sequence would comprise a region of the
selected intron
upstream from the second nucleic acid sequence and the second nucleic acid
sequence would be
selected comprising a region of the selected intron located downstream of the
first nucleic acid
sequence. The selectable marker would be introduced between the first and
second nucleic acid
sequences. When the construct is then introduced to the cell, the construct
nucleic acid sequence
is integrated into the target gene and the selectable marker is inserted
entirely within the targeted
intron.
Similarly, the construct may be engineered to insert the selectable marker
within any
desired and suitable region of the gene provided that expression is modulated.
For example, the
construct may be engineered to insert the selectable marker between two
adjacent introns and
thereby completely remove an endogenous exon of the target gene, to span over
a region
comprising at least a portion of an intron and at least a portion of an
adjacent intron of the
targeted gene, to span over a region comprising at least a portion the
promoter for the targeted
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WO 01/23541 PCT/US00/27065
gene to an adjacent intron, to span over a region encompassing more than one
targeted gene, and
combinations thereof.
After the genomic DNA sequence has been digested with the appropriate
restriction
enzymes, the selectable marker gene is ligated into the genomic DNA sequence
using methods
well known to the skilled artisan and described in Sambrook et al., supra. The
ends of the DNA
fragments to be ligated must be compatible; this is achieved by either cutting
all fragments with
enzymes that generate compatible ends, or by blunting the ends prior to
ligation. Blunting is
done using methods well known in the art, such as for example by the use of
Klenow fragment
(DNA polymerase I) to fill in sticky ends.
The ligated knockout construct may be introduced directly into the target
cell, or it may
first be placed into a suitable vector for amplification prior to insertion.
Preferred vectors are
those that are rapidly amplified in bacterial cells such as the pBluescript II
SK vector
(Stratagene, San Diego, Calif.) or pGEM7 (Promega Corp., Madison, Wis.).
In another embodiment of the invention, embryonic stem (ES) cells are used as
the target
cell for their ability to integrate into and become part of the germ line of a
developing embryo so
as to create germ line transmission of the knockout construct. Thus, any ES
cell line that is
believed to have this capability is suitable for use herein. For example, one
mouse strain that is
has been used for production of ES cells is the 129J strain. The cells are
cultured and prepared
for DNA insertion using methods well known to the skilled artisan such as
those set forth by
Robertson (Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.
J. Robertson,
ed. IRL Press, Washington, DC (1987)), Bradley et al. (Current Topics in
Devel. Biol., 20:357-
371 ( 1986)), and Hogan et al. (Manipulating the Mouse Embryo: A Laboratory
Manual, Cold
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CA 02385162 2002-03-28
WO 01/23541 PCT/LTS00/27065
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1986)), all of which
are incorporated
by reference herein.
Insertion of the knockout construct into the target cells may be accomplished
using a
variety of transfection methods well-known in the art. For example, suitable
transfection
methods include electroporation, microinjection, and calcium phosphate
treatment (see Lovell-
Badge, in Robertson, ed., supra). A preferred method of transfection is
electroporation. If the
cells are to be electroporated, the targeted cells and knockout construct DNA
are exposed to an
electric pulse using an electroporation machine and following the
manufacturer's guidelines for
use. After electroporation, the cells are allowed to recover under suitable
incubation conditions.
The cells are then screened for the presence of the knockout construct.
Each knockout construct DNA to be introduced into the cell must first be
linearized if the
knockout construct has been inserted into a vector. Linearization is
accomplished by digesting
the DNA with a suitable restriction endonuclease selected to cut only within
the vector sequence
and not within the knockout construct sequence.
For introduction of the DNA sequence, the knockout construct DNA is added to
the
target cells under appropriate conditions for the insertion method chosen.
Where more than one
construct is to be introduced into the target cell, DNA encoding each
construct may be
introduced simultaneously or one at a time.
Screening may be done using methods known in the art or combinations thereof.
Where
the selectable marker gene is an antibiotic resistance gene, the cells are
cultured in the presence
of an otherwise lethal concentration of the antibiotic. Those cells that
survive have presumably
integrated the knockout construct. If the selectable marker gene is other than
an antibiotic
resistance gene, the genomic DNA of the target cell may be extracted from the
cells using
23


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standard methods such as those described by Sambrook et al., supra. The DNA
may then be
probed on a Southern blot with a probe or probes designed to hybridize only to
the selectable
marker sequence. If the selectable marker gene is a gene that encodes an
enzyme whose activity
may be detected (e.g., beta-galactosidase), the enzyme substrate may be added
to the cells under
suitable conditions, and an appropriate assay for enzymatic activity may be
conducted. In
addition, the genomic DNA may be amplified by polymerase chain reaction (PCR)
with probes
specifically designed to amplify DNA fragments of a particular size and
sequence (i.e., only
those cells containing the knockout construct in the proper position will
generate DNA fragments
of the proper size). PCR may be used in detecting the presence of homologous
recombination
(Kim and Smithies, (1988) Nucleic Acid Res. 16:8887-8903; Joyner et al (1989)
Nature
338:153-156). Primers may be used which are complementary to a sequence within
the
construct and complementary to a sequence outside the construct and at the
target locus. In this
way, one may only obtain DNA duplexes having both of the primers present in
the
complementary chains in which homologous recombination has occurred. By
demonstrating the
presence of the primer sequences or the expected size sequence, the occurrence
of homologous
recombination is supported.
Upstream and/or downstream from the target gene knockout construct may be
inserted a
gene which provides for identification of whether a double crossover has
occurred. For this
purpose, any suitable marker may be used for as described herein. Preferably,
the selectable
marker used to identify double crossovers is different than the selectable
marker used to identify
the integration of the target gene knockout construct. In one preferred
embodiment, the herpes
simplex virus thymidine kinase gene is employed, since the presence of the
thymidine kinase
gene may be detected by the use of nucleoside analogs, such as Acyclovir or
Gancyclovir, for
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their cytotoxic effects on cells that contain a functional HSV-tk gene. The
absence of sensitivity
to these nucleoside analogs indicates the absence of the thymidine kinase gene
and, therefore,
where homologous recombination has occurred, a double crossover event has also
occurred.
The knockout construct may be integrated into several locations in the target
cell genome,
and may integrate into a different location in each cell's genome, due to the
occurrence of
random insertion events. Notwithstanding random multiple integration sites,
the desired location
of the insertion is in a complementary position to the DNA sequence to be
knocked out. It has
been found that less than about 1-5°Io of the targeted cells that take
up the knockout construct
will actually integrate the knockout construct in the desired location.
Identification of those cells
with proper integration of the knockout construct is described herein.
In one embodiment of the present invention, suitably transfected target cells
containing
the knockout construct in its proper location are inserted into an embryo.
Insertion may be
accomplished in any suitable method known in the art. Preferably, the cells
are introduced into
the embryo by microinjection. Most preferably, the cells are ES cells for
injection into mouse
embryos. For microinjection, about 10-30 cells are collected into a micropipet
and injected into
embryos that are at the proper stage of development to integrate the
transfected cell into the
developing embryo. The suitable stage of development for injecting into the
embryo is prior to
the formation of the germinal layer of the developing embryo as one having
ordinary skill in the
art may readily determine. Preferably, the embryo is in the early blastocyst
stage. By way of
example, mice embryos may be introduced to the transfected cells in about 3.5
days. The
embryos are obtained by perfusing the uterus of pregnant females by methods
known to the
skilled artisan (e.g., Bradley (in Robertson, ed., supra)). Preferably, the
embryos are male.


CA 02385162 2002-03-28
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After the transfected target cell having proper integration of the target gene
has been
introduced into the embryo, the embryo is implanted into the uterus of a
pseudopregnant foster
mother. While any foster mother may be used, selection of the foster mother is
based upon its
ability to breed and reproduce well, and to care for its young. Such foster
mothers are typically
prepared by mating with vasectomized males of the same species. The stage of
the
pseudopregnant foster mother is important for successful implantation, and is
species dependent.
For mice, this stage is about 2-3 days pseudopregnant.
In another embodiment, the suitable transfected target cells are nuclear
transfer donor
cells. Nuclear transfer donor cells may be virtually any somatic cell type and
include fibroblasts,
epithelial cells, cumulus cells, etc. Nuclear transfer donor cells are
cultured in vitro and targeted
using the constructs and techniques described herein via homologous
recombination. Cells are
grown in the appropriate medium to allow for selection of cells comprising the
having properly
integrated the knockout construct. PCR may also be done for confirmation of
correctly targeted
integration. Thereafter, an unfertilized oocyte of an animal is enucleated
using known methods.
The enucleated unfertilized oocyte is then fused to the selected knockout
nuclear transfer donor
cell. Fusion may be conducted by electrical stimulation, chemical stimulation,
insertion by
injection, or other known methods. The fused product is then cultured,
assessed for viability and
transferred to a surrogate recipient female. For reference and methods, see
e.g., Campbell et al.
(1996) Nature 380:64; Wilmut et al. (1997) Nature 385:810; WO00/25578;
W097/07669;
W099/36510; WO00/42174; W099/53751; W099/45100, which are incorporated herein
by
reference.
Offspring or progeny that are born to the foster mother or surrogate recipient
female are
screened (e.g., by PCR) for genomic DNA comprising the knockout construct.
This step is
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particularly important for selecting for progeny of foster mothers that
carried embryos in which
the transfected target cell was injected. On the other hand, the progeny of
surrogate recipient
females that carned the transfected target cell fused with the enucleated
unfertilized oocyte will
typically have the knockout construct inserted into its genome.
Any suitable selection method may be used. For example, if a coat color
selection
strategy has been used, the offspring may be screened for a coat color
indicative of proper
integration of the targeted gene into the offspring. Other methods include
obtaining DNA from
the offspring and screening for the presence of the knockout construct using
Southern blots
and/or PCR as described herein. Other means of identifying and characterizing
the knockout
offspring include the use of Northern blots and Western blots. For example,
Northern blots may
be used to probe the mRNA for the presence or absence of transcripts encoding
either the gene
knocked out, the marker gene, or both. In addition, Western blots may be used
to assess the level
of expression of the gene knocked out in various tissues of these offspring by
probing the
Western blot with an antibody against the protein encoded by the gene knocked
out, or an
antibody against the marker gene product, where this gene is expressed. In
situ analysis (such as
fixing the cells and labeling with antibody) and/or fluorescence activated
cell sorting (FACS)
analysis of various cells from the offspring may be conducted using suitable
antibodies to look
for the presence or absence of the knockout construct gene product.
Offspring that appear to contain the integrated knockout construct in its
genome may then
be out-crossed to generate multiple offspring if they are believed to carry
the knockout construct
in their germ line to generate Fl offspring heterozygous for the knockout
construct. Fl's will
then be crossed to generate homozygous knockout animals.
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The heterozygotes may then be crossed with each other to generate homozygous
knockout offspring. Homozygotes may be identified by any screening method as
described
herein. For example, the homozygotes may be identified by Southern blotting of
equivalent
amounts of genomic DNA from the host animals) that is (are) the product of
this cross, as well
as host animals that are known heterozygotes and wild-type host animals.
Probes to screen the
Southern blots may be designed as set forth herein.
The knockout mammals described herein will have a variety of uses depending on
the
gene or genes that have been modulated. Where the targeted gene or genes
modulated encode
proteins believed to be involved in immunosuppression or inflammation, the
knockout mammal
may be used to screen for drugs useful for immunomodulation, i.e., drugs that
either enhance or
inhibit these activities. Screening for useful drugs may involve administering
the candidate drug
over a range of dosages to the knockout mammal, and assaying at various time
points for
immunomodulatory effects of the drug on the immune disorder being evaluated.
Such assays
may include, for example, looking for increased or decreased T and B cell
levels, increased or
decreased immunoglobulin production, increased or decreased levels of chemical
messengers
such as cytokines (e.g., interleukins and the like), andlor increased or
decreased levels of
expression of particular genes involved in the immune response.
For example, patients undergoing chemotherapy often experience
immunosuppression. It
would be desirable to activate the immune system of such individuals by
administering to the
patient a therapeutic agent capable of producing such an effect. A knockout
mammal as
described herein could be used to screen a variety of compounds, either alone
or in combination,
to determine whether partial or total restoration or activation of the immune
response results.
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Similarly, the same strategy could be applied to find compounds that would be
useful in
suppressing the inflammatory response observed in many patients with
arthritis, or useful in
suppressing the autoimmune phenomenon observed in patients with rheumatoid
arthritis and
lupus. In addition, mammals may be useful for evaluating the development of
the immune
system, and for studying the effects of particular gene mutations.
In a preferred embodiment, the knockout mammals described herein are used for
xenograft transplantation into human patients. The xenograft tissue may be
from any mammal,
preferably a pig. The xenotransplanted tissue may be in the form of an organ
including, for
example, a kidney, a heart, a lung, or a liver. Xenotransplant tissue may also
be in the form of
parts of organs, cell clusters, and glands including, for example, lenses,
pancreatic islet cells,
skin, corneal tissue, and the like.
In yet another aspect of the present invention, the target gene is the Gal
a(1,3) galactosyl
transferase gene in pigs. The Gal a( 1,3) galactosyl transferase is an
attractive target for
knockout in the pig. This enzyme is responsible for the addition of a
carbohydrate residue,
Gal a(1,3) Gal, that is recognized by human IgM and IgG antibodies in pig-to-
human
xenotransplanted tissue and leads to subsequent hyperacute rejection. Knockout
pigs, which lack
the Gal a(1,3) galactosyl transferase gene, may thus potentially serve as a
rich source for
xenotransplanted organs. Nucleic acid sequences encoding Gal a(1,3) galactosyl
transferase and
mutants thereof are disclosed. Preferably, the nucleotide sequence encodes pig
Gal a(1,3)
galactosyl transferase. Nucleotide sequences may be in the form of DNA, RNA or
mixtures
thereof. Nucleotide sequences or isolated nucleic acids may be inserted into
replicating DNA,
RNA or DNA/RNA vectors (as are well known in the art), such as plasmids, viral
vectors, and
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the like (Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor
Laboratory Press, NY, Second Edition 1989).
Nucleotide sequences encoding Gal a(1,3) galactosyl transferase may include
promoters,
enhancers and other regulatory sequences for expression, transcription and
translation. Vectors
encoding such sequences may include restriction enzyme sites for the insertion
of additional
genes and/or selection markers, as well as elements necessary for propagation
and maintenance
of vectors within cells.
Targeting constructs comprising nucleotide sequences, and mutants thereof, of
the Gal
a(1,3)galactosyl transferase are particularly preferred as they may be used to
inactivate wild type
Gal a(1,3) galactosyl transferase genes according to the methods of the
present invention.
Mutant Gal a(1,3) galactosyl transferase nucleotide sequences include, but are
not limited to,
nucleotide deletions, insertions, substitutions and additions to wild type Gal
a(1,3) galactosyl
transferase, such that the resultant mutant does not encode a functional
galactosyl transferase.
These nucleotide sequences may be utilized in the methods of modulating
expression of
galactosyl transferase of the present invention. In this manner, mutant
sequences are recombined
with wild type genomic sequences in the target cells.
In a most preferred embodiment, knockout pigs are produced in which the
Gal a( 1,3)galactosyl trasferase gene produces a non-functional protein. By
producing a non-
functional protein, the human antibody that would otherwise bind to the Gal
a(1,3)Gal epitope
expressed on the xenotransplanted tissue does not bind, so that immune
responses which give
rise to tissue rejection are prevented. In this embodiment, any knockout
construct capable of


CA 02385162 2002-03-28
WO 01/23541 PCT/US00/27065
modulating the interaction between antibodies directed to the Gala(1,3)
galactosyl transferase
linkage may be used.
EXAMPLES
The following examples are for purposes of illustration only, and are not
intended to limit
the scope of the disclosure or claims.
EXAMPLE 1
Inactivation of the Gal a(1,3) galactosyl transferase gene by insertion of an
engineered active
gene in the form of an engineered exon within intron 3.
In this example, the Gal a( 1,3) galactosyl transferase protein is prevented
from being
translated by insertion of an in-frame, promoterless engineered exon (e.g., an
antibiotic resistance
gene) that contains multiple stop codons within an intron of the Gal a(1,3)
galactosyl transferase gene.
Using this 'promoter-trap' strategy, the engineered exon is spliced in frame
upstream of exon 4 of the
Gal a(1,3) galactosyl transferase gene. This results in the expression of the
drug resistance gene prior
to the gene of interest and concomitantly inhibits expression of the
transferase gene due to the
presence of multiple stop codons downstream of the drug resistance gene. As
described herein, any
gene that confers survival of the targeted cells under appropriate selection
conditions may be used as
the engineered exon, including, but not limited to, ampicillin, kanamycin,
genticin, neomycin
phosphopotransferase, puromycin-N-acetyl-transferase, hygromycin b-
phosphotransferase, thymidine
kinase, and tryptophan synthetase. The present example employs neomycin.
A gene targeting construct is designed which contains a sequence with homology
to the
Gal a(1,3) galactosyl transferase gene 5' intron 3 sequence, an intron 4
splice acceptor signal
sequence, a promoterless neomycin phosphotransferase gene engineered to
contain multiple stop
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CA 02385162 2002-03-28
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codons (engineered exon), the intron 4 splice donor sequence for splicing the
engineered exon to the
downstream exon 4, and additional intron 3 sequence homology to aid with
annealing to the porcine
Gal a(1,3) galactosyl transferase gene. Although this example describes
targeting intron 3, it will be
apppreciated that the method may be used to target any intron within the Gal
a( 1,3) galactosyl
S transferase gene or any other gene of interest. A sequence listing of the
introns in the Gal a( 1,3)
galactosyl transferase gene (from within intron 3 to the end of intron 8) is
provided in Figure 1. A
schematic diagram of the targeting vector and corresponding nucleotide
sequence are shown in
Figures 2 and 3.
The gene targeting construct is generated by ligating 5 distinct DNA fragments
(1-5
below) together to form the final gene targeting construct using standard
molecular biology
techniques well known to those skilled in the art. The PCR reactions use the
ELONGASE
Enzyme Mix (Life Technologies, Gaithersburg, MD) according to the
manufacturer's
instructions. In the present example, a 50 u1 final reaction volume is used,
with 2 u1 of DNA
template, 1 u1 of ELONGASE Enzyme Mix, 60 mM Tris-S04 (pH9.1) lBmM (NH4)2S04,
1.2
mM MgS04, 200mM dNTP mix, 10% DMSO and 200nM of each primer. The reaction is
hot
started at 95°C for 1 minute and followed by 30-40 cycles in a standard
PCR thermocycler
(GeneAmp PCR System 2400; PE Applied Biosystems, Foster City, CA).
1. A polymerase chain reaction (PCR) product consisting of intron 3 sequences
as listed in
Figures 1 and 3, nucleotide numbers 10-4020, is generated using standard PCR
conditions for
long range PCR of genomic fragments. Primers used include a 5 ' primer
containing a NotI
restriction site and intron 3 sequences 10-23 (GGCGGCCGCAGGCCTCACTGGCC); and a
3'
primer containing a SaII restriction site and sequences homologous to intron 3
sequences 3999-
4020 (GGTCGACGGATGCTGGGTGGAATAACAGG), where underlined sequence
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WO 01/23541 PCT/US00/27065
indicates restriction sites and bold type indicates homology to endogenous
sequences. An
additional guanine nucleotide is added to the 5' end of all probes in this
example to balance out 1
by deletions that sometimes occur during cloning.
2. A PCR product is generated consisting of intron 4, the 3' splice sequence
(the pyrimidine
rich lariot and Gal a(1,3) galactosyl transferase intron 3 dinucleotide
acceptor sequences) and
196 bases 5' flanking the ag dinucleotide acceptor site (nucleotides 11521-
11716 in Figures 1
and 3). Primers used include a 5' primer containing a SaII restriction site
(GGTCGACCCACCGTTTGATCTGAG); and a 3' primer containing a EcoRI restriction
site
and the complementary strand homologous to the pyrimidine rich lariot and Gal
a(1,3)
galactosyl transferase dinucleotide acceptor sequences
(GGAATTCCTAAAAGCAAATGGAAATAAAAACATATC), where underlined sequences
indicate restriction sites and bold type indicates sequences with homology to
the endogenous
sequence.
3. A PCR product consisting of a neomycin resistance gene (Genbank Accession
#AF081957; Figure 4) is generated using a 5' primer containing an EcoRI
restriction site,
and homology to the neomycin resistance gene, including the ATG start codon
(GGAATTCAATGGATCCCCACCATGGG); and a 3' primer containing a Hind>ZI restriction
site and complementary strand sequences to the 3' coding region of the
neomycin gene,
including the natural stop codon followed by two additional engineered stop
codons
(GAAGCTTCGGCTATTACTAAGTAGTGGATATCC), where underlined sequences
indicate restriction sites and bold type indicates sequences with homology to
the endogenous
sequence (see Figures 3 and 4).
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4. A PCR product is generated containing the 5' splice donor sequences for
intron 4 of the
Gal a(1,3) galactosyl transferase gene, corresponding to sequences 4938-5173
in the claimed
sequence comprising intron 4 (Figures 1 and 3). Primers used include a 5'
primer containing a
IiindIll site and sequence identity to intron 4, sequences 4938-4962,
including the Gal a(1,3)
galactosyl transferase dinucleotide splice site (GAAGCTTGTAATTATGAAACATGATG);
and a 3' primer containing a PstI site and complementary strand sequence from
intron 4
corresponding to nucleotide numbers 5152-5173 and includes multiple stop
codons
(GCTGCAGCCACAGGTCACGGCAATGCGG); where underlined sequences indicate
restriction sites and bold type indicates sequences with homology to the
endogenous sequence.
5. A PCR product containing 1150 nucleotides of intron 3, corresponding to
nucleotides
4024-4826 of the claimed sequence (Figures 1 and 3). Primers used include a 5'
primer
containing a PstI site and sequences 4024-4050 of the claimed sequence
(GCTGCAGCCCTCTTCAACTACAATTTCATGCAGC); and a 3' primer containing a XhoI
restriction site and complementary strand sequences to 4801-4826 of the
claimed sequence
(GCTCGAGAGAAAATTAGATTAAATACACCCAGAG);
where underlined sequences indicate restriction sites and bold type indicates
sequences with
homology to the endogenous sequence.
Each PCR fragment (steps 1-5) is separately amplified. A single PCR fragment
is cloned
into the pCR2.l vector (Invitrogen , San Diego, CA) according to the
manufacturer's ligation
instructions. The recombinant plasmid DNA is transformed into a suitable
bacterial host
(Invitrogen, San Diego, CA). The bacteria are cultured and plasmid DNA is
isolated. Plasmid
DNA with the correct insert, as determined by restriction analysis and
sequence analysis, is used
to construct the final product.
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Following PCR fragment amplification, a series of ligations is performed to
clone the
final construct in the bacterial plasmid pBS SK+ (Stratagene, La Jolla, CA).
a. The HindllI-PstI fragment from the intron 4 PCR product (step 4) and the
PstI-XhoI 3'
homology fragment from intron 3 (step 5 above) are ligated to a pBS SK+ vector
DNA following
digestion with HindllI and XhoI. The 3 DNA fragments are mixed in equal molar
ratios and
incubated in the presence of T4 DNA ligase (New England Biolabs, Beverly, MA)
according to
the manufacturer's recommendations. Following ligation, the recombinant
plasmid DNA is
transformed into a suitable bacterial host (DH IOB, Life Technologies,
Gaithersburg, MD). The
bacteria are cultured, and plasmid DNA is isolated. Plasmid with the correct
insert, as
determined by restriction analysis and sequence analysis, is used to construct
the final product.
b. The resulting plasmid (step Sa) is digested with HindI)I and EcoRI and
ligated with the
HindllI-EcoRI Neomycin resistance gene fragment (step 3), that has been
previously digested
with HindIII and EcoRI. The resulting recombinant plasmid DNA is transformed
into a suitable
bacterial host (DH l OB, Life Technologies, Gaithersburg, MD). The bacteria
are cultured, and
plasmid DNA is isolated. Plasmid with the correct insert, as determined by
restriction analysis
and sequence analysis, is used to construct the final product.
c. The resulting plasmid (step Sb) is digested with EcoRI and NotI and ligated
to the SaII-
EcoRI intron 4-3'splice fragment (step 2) previously digested with SaII and
EcoRI and the intron
3 4 kb NotI-SaII fragment (step 1) previously digested with NotI and SaII. The
3 DNA
fragments are incubated in equal molar ratios in the presence of T4 DNA ligase
(New England
Biolabs, Beverly, MA) according to the manufacturer's recommendations.
Following ligation,
the recombinant plasmid DNA is transformed into a suitable bacterial host
(DHlOB, Life


CA 02385162 2002-03-28
WO 01/23541 PCT/US00/27065
Technologies, Gaithersburg, MD). The bacteria are cultured, and the
recombinant plasmid DNA
is isolated.
This final construct is used to transfect porcine embryonic fibroblasts,
transgenic pig
fibroblasts, or porcine embryonic stem cells, or porcine primordial germ
cells. Cell clones that
are resistant to neomycin are screened by PCR to determine the site of
integration. A primer
located in the region of intron 4 not incorporated into the final construct
(complementary strand
of 5407-5427; GGACAATGGCAACATGGCAGG; see Figures 1 and 3) is used in
combination
with the 5' neomycin gene primer (step 3). Only targeted insertions yield the
appropriate sized
PCR fragment. All other integration events produce a negative result.
Cell clones with a targeted insertion are then used to generate transgenic
animals using
nuclear transfer techniques, or in the case of the stem cells, used to inject
into developing
blastocysts and produce chimeric offspring.
EXAMPLE 2
Inactivation of the Gal a(1,3) galactosyl transferase gene by replacement of
exon 4 with an active
gene in the form of an engineered exon.
In this example, the Gal a(1,3) galactosyl transferase protein was prevented
from being
translated by replacing an endogenous exon (exon 3) with an in-frame,
promoterless engineered
exon (an antibiotic resistance gene) that contained a bovine growth hormone
poly A sequence
attached to the 3' end of the gene, which served to terminate transcription of
the engineered exon.
The engineered exon was spliced in frame, so as to take advantage of the
endogenous promoter
typically used by the Gal a(1,3) galactosyl transferase gene ('promoter-trap'
strategy). This resulted
in the expression of the drug resistance gene and concomitantly inhibited
expression of the full-length
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CA 02385162 2002-03-28
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Gal a( 1,3) galactosyl transferase gene. As described herein, any gene that
confers survival of the
targeted cells under appropriate selection conditions may be used as the
engineered exon, including,
but not limited to, ampicillin, kanamycin, genticin, neomycin
phosphopotransferase, puromycin-N-
acetyl-transferase, hygromycin b-phosphotransferase, thymidine kinase, and
tryptophan synthetase.
The present example utilizes puromycin.
A gene targeting construct was designed which contained a sequence with
homology to
the Gal a(1,3) galactosyl transferase gene 3' intron 3 sequence, an intron 3
splice signal sequence
(splice acceptor sequence), a Kozak consensus sequence, a promoterless
puromycin N-acetyl
transferase gene linked to a bovine growth hormone poly A sequence (bpoly A)
(engineered exon),
the 5' intron 4 splice signal sequence (splice donor sequence), and a sequence
with 5' intron 4
sequence homology. Exon 4 of the Gal a(1,3) galactosyl transferase gene codes
for ATG start codon
and the N-terminal portion of the protein. Although this example describes
targeting introns 3 and 4,
it will be appreciated that the method may be used to target any exon within
the Gal a( 1,3) galactosyl
transferase gene or any other gene of interest. A sequence listing of the
introns in the Gal a(1,3)
galactosyl transferase gene (from within intron 3 to the end of intron 8) is
provided in Figure 1.
The gene targeting construct was generated by ligating two distinct DNA
fragments
together to form the final gene targeting construct using standard molecular
biology techniques
well known to those skilled in the art. The first DNA fragment was obtained
from the 3' end of
intron 3 containing the 3' splice sequence (the pyrimidine-rich branch site
used in forming the
lariot during splicing and the AG dinucleotide splice acceptor sequence). The
second DNA
fragment was obtained from the 5' end of intron 4 containing the GT
dinucleotide splice donor
sequence: The fragments were ligated into the pBluescript vector containing a
Kozak consensus
sequence in-frame with the coding sequence of a promotorless puromycin gene
linked to the
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CA 02385162 2002-03-28
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bovine growth hormone poly A sequence (Figure 5) to form the final gene
construct. A
schematic diagram of the targeting vector and corresponding nucleotide
sequence are shown in
Figures 6 and 7. Additionally, this construct has been deposited with ATCC on
28 September
2000 with accession number
1. Generation of the first DNA fragment.
The first DNA fragment was a polymerase chain reaction (PCR) product
consisting of
intron 3 sequence as shown in Figure 7 (nucleotide numbers 235-4851, positions
relative to
nucleotide position 1 of the insert isolated from the lambda phage clone) and
generated using
standard PCR conditions as described by Randolf et al., (1996) for long range
PCR of genomic
fragments. The 5' primer, consisting of intron 3 sequences 235-260, was
5'-AAGATTATAAATAGCCTCGTGTCAGG-3'. The 3' reverse primer sequence was
complementary to sequence 4827-4851 at the extreme 3' end of intron 3 and
containing the AG
splice acceptor site, and was 5'-CTCCTGGGAAAAGAAAAGGAGAAGG-3'.
PCR reaction conditions to generate the 4.616 kb intron 3 sequence were
performed using
the ELONGASE Enzyme Mix (Life Technologies, Gaithersburg, MD) according to
manufacturer's conditions. In the present example, a 50 ~.1 final reaction
volume was used, with
2 u1 of DNA template, lul of the ELONGASE Enzyme Mix, 60 mM Tris S04 (pH 9.1),
18 mM
(NH4)2S04, 1.2 mM MgS04, 200 mM dNTP mix, 10% DMSO and 200 nM of each primer.
The
reaction was hot started at 95°C for 1 min, followed by 30-40 cycles in
a standard PCR machine
(e.g., Gene Amp PCR Systems 2400; PE Applied Biosystems, Foster City, CA).
2. Preparation of the PCR2.1 cloning vector.
The Not I site of the PCR2.1 cloning vector (Invitrogen, San Diego, CA) was
destroyed
to avoid carrying over a second Not I site into the final construct. The Not I
site was unique and
38


CA 02385162 2002-03-28
WO 01/23541 PCT/US00/27065
used to linearize the final plasmid construct. The PCR2.1 vector was digested
with Not I and the
overhangs filled-in using the Klenow enzyme (Roche Molecular Biochemicals,
Indianapolis, IN)
according to the manufacturer's specifications. The plasmid was re-ligated
using T4 DNA ligase
(New England Biolabs, Beverly, MA) according to the manufacturer's
recommendations.
Plasmid DNA was transformed into a suitable bacterial host (Top 10 F',
Invitrogen, San Diego,
CA). The bacteria were cultured, and plasmid DNA was isolated and incubated
with Not I
enzyme to confirm loss of this site by restriction analysis.
3. Insertion of the first DNA fragment into the PCR2.1 vector.
Following PCR, the 4.616 kb fragment was ligated into the modified PCR2.1
vector
using T4 DNA ligase according to the manufacturer's specifications. Plasmid
DNA was
transformed into a suitable bacterial host (e.g.,Top 10 F', Invitrogen, San
Diego, CA). The
bacteria are cultured and plasmid DNA is isolated. Plasmid with the correct
insert in the proper
orientation, as determined by restriction analysis and sequence analysis, was
used to construct
the final product.
4. Preparation of the second DNA fragment.
A 2.084 kbp PCR product consisting of the intron 4 homology sequence
containing the
GT dinucleotide donor consensus splice sequence was constructed using standard
PCR
conditions as described above in step 1. The 5' primer consisting of sequence
4938-4961 at the
extreme 5' end of intron 4 was 5'-GTAATTATGAAACATGATGAAATG-3'. The 3' primer
was homologous to the complementary strand of intron 4 at position 6997-7021
and has the
sequence 5'-AGCCAGCGCTTACTAAGTACGTTGC-3'
5. Insertion of the second DNA fragment into the PCR2.1 vector.
39


CA 02385162 2002-03-28
WO 01/23541 PCT/US00/27065
Following PCR, the 2.084 kb fragment was ligated into the pCR2.1 vector from
Invitrogen (San Diego, CA) using the manufacturer's ligation conditions.
Following ligation, the
recombinant plasmid DNA was transformed into a suitable bacterial host (Top 10
F', Invitrogen,
San Diego, CA). The bacteria were cultured, and plasmid DNA was isolated.
Plasmid with the
correct insert, and orientation in the plasmid, as determined by restriction
analysis and sequence
analysis, was used to construct the final product.
6. Preparation of a synthetic oligonucleotide linker sequence.
A synthetic oligonucleotide linker containing a Kozak consensus sequence and
relevant
restriction enzyme sites was prepared for in-frame cloning of the promoterless
puromycin gene:
Xho I ~ Kozak seq.--- ~ HpaI ~ Hind III ~ Bgl II ~ Sal ~ Eco RV ~ Eco RI
TCGAGCCACCATGGTTAACAAGCTTAGATCTGTCGACGATATCG
CGGTGGTACCAATTGTTCGAATCTAGACAGCTGCTATAGCTTAA
7. Assembly of the gene targeting construct.
The following ligations were performed to generate the final construct in the
bacterial
plasmid pBS KS+ (Strategene , La Jolla, CA). The final construct is
illustrated in Figure 6:
a. The oligonucleotide linker containing the Kozak consensus sequence (step 6)
was ligated
to the pBS KS+ vector DNA following digestion with Xho I and Eco RI. Ligation
was carned
out using at least a 3:1 molar ratio of linker to vector in the presence of T4
DNA ligase (New
England Biolabs, Beverly, MA) according to the manufacturer's recommendations.
Following
ligation, the recombinant plasmid was transformed into a suitable bacterial
host (XL1-Blue
MRF', Strategene , La Jolla, CA). The bacteria were cultured, and plasmid DNA
was isolated.
Restriction enzyme analysis was performed to confirm successful ligation using
unique


CA 02385162 2002-03-28
WO 01/23541 PCT/US00/27065
restriction sites within the linker (Bgl II or Hpa 1). This plasmid containing
the linker was then
used to construct the final product.
b. The resulting "mother" plasmid (step 7a) was then digested with Eco RV and
Spe I to
clone in the 3' arm of the targeting construct. The 2.084 kb PCR fragment
cloned into the PCR
2.1 vector (step 5) was digested with Eco RV and Spe I, isolated away from
vector DNA by
agarose gel electrophoresis and purified. The 2.084 kb fragment was ligated
between the EcoRV
and Spe I sites of the mother plasmid (step 7a). Ligation was carried out
using a 3:1 molar ratio
of insert to vector in the presence of T4 DNA ligase (New England Biolabs,
Beverly, MA)
according to the manufacturer's recommendations. Following ligation, the
recombinant plasmid
was transformed into a suitable bacterial host ( XL1-Blue MRF', Strategene ,
La Jolla, CA). The
bacteria were cultured, and plasmid DNA was isolated. Plasmid with the correct
insert, as
determined by restriction analysis and sequence analysis, was then used to
construct the final
product.
c. The next fragment cloned into the mother plasmid (step 7b) was the cassette
containing
the promoterless puromycin gene coding sequence with the bovine growth hormone
gene polyA
signal sequence attached to its 3' end following the TGA stop codon (Figure
5). The PGK
puromycin bpolyA plasmid (used as a positive control for puromycin resistance
of transformed
cells) was digested with Hind III and Xho I. The puromycin bpolyA fragment was
separated
away from the rest of the vector DNA containing the PGK promoter by
electrophoresis on a
0.7% agarose gel and purified. The mother plasmid (step 7b) was digested with
Hind III and Sal
I. The Hind III/Xho I puromycin bpolyA cassette was ligated to the Hind III
and Sal I sites of the
mother plasmid. Ligation was carried out using a 3:1 molar ratio of insert to
vector in the
presence of T4 DNA ligase (New England Biolabs, Beverly, MA) according to the
41


CA 02385162 2002-03-28
WO 01/23541 PCT/US00/27065
manufacturer's recommendations. Following ligation, the recombinant plasmid
was transformed
into a suitable bacterial host (XL1-Blue MRF', Strategene, La Jolla, CA). The
bacteria were
cultured, and plasmid DNA was isolated. Plasmid with the correct insert, as
determined by
restriction analysis and sequence analysis, was used to construct the final
product.
d. The final cloning step involved ligating the 5' arm of the construct, which
was the 4.616
kb intron 3 insert from the PCR2.1 vector (step 3). The PCR2.1 vector (step 3)
was digested
with Kpn I and Xho I. The 4.616 kb PCR fragment was isolated away from vector
DNA by
agarose gel electrophoresis and purified. The 4.616 kb Kpn I/Xho I insert was
ligated into the
mother plasmid (step 7c) that was digested with Kpn I and Xho I. Ligation was
can ied out using
equimolar ratio of insert to vector in the presence of T4 DNA ligase (New
England Biolabs,
Beverly, MA) according to the manufacturer's recommendations. Following
ligation, the
recombinant plasmid was transformed into a suitable bacterial host ( XL1-Blue
MRF',
Strategene, La Jolla, CA). The bacteria were cultured, and plasmid DNA was
isolated by
standard molecular biology techniques. Plasmid with the correct insert, as
determined by
restriction analysis and sequence analysis, was used as the final product.
e. The final construct may be used to transfect porcine embryonic fibroblasts,
transgenic
porcine fibroblasts, or porcine embryonic stem cells, or porcine primordial
germ cells. Cell
clones that are resistant to puromycin may be screened by PCR to determine the
site of
integration by methods well known to those of skill in the art. A primer
located in a region of
intron 4, which is not incorporated into the final construct, may be used in
combination with a 5'
puromycin gene primer. Only targeted insertions will yield the appropriate
size PCR fragment.
All other integration events will produce a negative result.
42


CA 02385162 2002-03-28
WO 01/23541 PCT/US00/27065
Cell clones with a targeted insertion may then be used to generate transgenic
animals
using nuclear transfer techniques, or in the case of stem cells, used to
inject into developing
blastocysts and produce chimeric offspring.
Example 3
Inactivation of the Gal a( 1,3) galactosyl transferase gene by replacement of
exon 4 with an a reverse
orientation active gene.
In this example, the Gal a(1,3) galactosyl transferase gene was functionally
inactivated by
using a "collision construct" to insert an active gene in place of an exon and
at least part of the
flanking introns, including the splice donor and splice acceptor sites. The
inserted gene is under the
control of a highly active promoter such as the phosphoglycerate kinase I
(PGK) gene promoter, such
that transcription of this gene causes the termination of transcription of the
endogenous gene (Rosario
etal., (1996) Nat. Biotech.14:1592-1596). Exon 4 of the Gal a(1,3) galactosyl
transferase gene codes
for ATG start codon and the N-terminal portion of the protein. Thus, the
insertion was made to
replace exon 4 as well as a portion of the flanking introns 3 and 4, resulting
in a truncated transcript
that did not code for a functional enzyme. Although this example describes
targeting introns 3 and 4,
this method could be used to target any introns within the Gal a( 1,3)
galactosyl transferase gene or
any other gene of interest. A sequence listing of the introns in the Gal
a(1,3) galactosyl transferase
gene (from within intron 3 to the end of intron 8) is provided in Figure 1.
In this example, the PGK promoter was inserted driving the expression of the
puromycin
resistance gene with the bovine growth hormone poly A (bpolyA) transcription
termination
sequence. This gene replaced the Gal a(1,3) galactosyl transferase exon 4 as
well as a portion of the
flanking intron 3 and 4 sequences by standard homologous recombination
techniques utilizing intron 3
43


CA 02385162 2002-03-28
WO 01/23541 PCT/I1S00/27065
and 4 sequences for homology flanking the inserted gene. Intron 3, which
separates exons 3 and 4, is
greater than 5 kb in length, and a construct was built such that there was at
least about 4.6 kb of
homologous sequence on one end of the gene. Intron 4, which separates exons 4
and 5 is about 6.8 kb
in length, and the construct was built such there is at least about 2.2 kb of
homologous sequence on
the other end of the gene. Positive selection for transfected cells in which
the construct has been
integrated was accomplished via expression of the puromycin resistance gene.
As described herein, it
will be appreciated that any selection marker gene that confers survival of
the targeted cells under
appropriate selection conditions may be driven by the strong PGK promoter.
Additionally, a toxin
gene was inserted to eliminate random integration events.
The collision construct was generated using standard molecular biology
techniques well
known to those skilled in the art. The 4.616 kb intron 3 homology fragment and
the 2.084 kb
intron 4 homology fragment were generated using PCR and cloned into the PCR2.1
cloning
vector as described in Example 2, steps 1-5 above for the replacement
targeting construct. The
generation of the collision construct first involved ligating the 2.084 kb
intron 4 homology
fragment into the pBS KS+ vector as the 3' arm of the collision construct,
followed by the PGK-
puromycin-bovine polyA cassette in the opposite orientation to the coding
sequence of the GT
gene. The 4.616 kb intron 3 homology fragment, as the 5' arm, was cloned in
next. This
generated the targeting construct for homologous recombination. The ricin A
toxin gene was
also added to the plasmid outside the region of homology, which will
effectively kill a
percentage of the cells in which random integration has occurred. The ricin A
toxin gene was
PCR amplified and cloned based upon the published sequence (Figure 8). A
schematic diagram
of the final construct is shown in Figure 9. Additionally, this collision
construct has been
deposited with ATCC on 28 September 2000 with accession number
44


CA 02385162 2002-03-28
WO 01/23541 PCT/US00/27065
1. The 2.084 kb 3' arm of the construct (intron 4 homology fragment) was the
first fragment
to be ligated into the pBluscript cloning vector, which was modified to
contain the XhoI - EcoRI
linker (see Example 2, step 6) within its multiple cloning site (pBS KS+). The
ligation of the
linker into the vector is described above in Example 2, step 7a, and ligation
of the 2.084 kb 3'
arm into the Eco RV and Spe I sites of the vector is described above in
Example 2, step 7b.
2. The next step involved ligation of the PGK puromycin bovine polyA cassette
into the
pBS KS+ vector, which contained the 2.084 kb 3' arm. The PGK-puro-bPA cassette
was
digested with Eco RI which was immediately 5' of the PGK promoter. The Eco RI
overhangs
were blunted by filling in with Klenow enzyme (Roche Molecular Biochemicals,
Indianapolis,
IN) using the manufacturer's specifications. The PGK-puro-bPA cassette was
then released
from the vector by digestion with Xho I, which was immediately 3' of the
bovine polyA
sequence. The blunted PGK-puro-bPA-Xho I cassette was separated from vector
DNA by
agarose gel electrophoresis and purified. The pBS KS+ vector (step 1 ) was
digested with Hpa I
and Xho I, and the blunted PGK-puro-bPA-Xho I fragment was ligated between the
Hpa I and
Xho I sites of the vector. Following ligation, the recombinant plasmid was
transformed into a
suitable bacterial host (XL1-Blue MRF', Strategene, La Jolla, CA). The
bacteria were cultured,
and plasmid DNA was isolated. Plasmid with the correct insert, as determined
by restriction
analysis and sequence analysis, was then used as the final product.
3. The 4.616 kb intron 3 homology fragment was ligated into the pBS KS+ mother
plasmid
(step 2) and represented the 5' arm of the collision construct. Isolation of
this fragment from the
PCR2.1 cloning vector and ligation into the Kpn I and Xho I sites of the
mother plasmid is
described above in Example 2, step 7d.


CA 02385162 2002-03-28
WO 01/23541 PCT/US00/27065
4. The ricin A toxin gene (Figure 8) was inserted into a commercially
available mammalian
expression vector, e.g., pcDNAI/Amp (Invitrogen). The insert was then excised
with the CMV
promoter and the SV40 poly A site and cloned into the Not I site of the
recombinant plasmid by
blunt end ligation, following Klenow fill in reactions on both the insert and
vector.
S 5. Following ligation, the recombinant plasmid DNA was used to transform a
suitable
bacterial host (XLI-blue, Strategene). The bacteria were cultured, and plasmid
DNA isolated.
This final construct DNA was then linearized with Kpn I (a unique enzyme site
in the plasmid
MCS outside of the construct sequence). Linearized plasmid may be used to
transfect porcine
embryonic fibroblasts, transgenic porcine fibroblasts, or porcine embryonic
stem cells, or porcine
primordial germ cells. Cell clones resistant to puromycin may be screened by
PCR to determine
the site of integration by methods well known to those of skill in the art. A
primer located in the
region of intron 4 not incorporated into the final construct may be used in
combination with a 5'
puromycin gene primer. Only targeted insertions yield the appropriate size PCR
fragment. All
other integration events produce a negative result.
Cell clones with a targeted insertion may then be used to generate transgenic
animals
using nuclear transfer techniques, or in the case of stem cells, used to
inject into developing
blastocysts and produce chimeric offspring.
EXAMPLE 4
Isolation of porcine genomic DNA encoding the Gal a(1,3) galactosyl
transferase gene from a
Lambda phage clone library.
In this example, a pig genomic library was screened using a cDNA corresponding
to exon
4 of the Gal a( 1,3) galactosyl transferase gene using molecular biology
techniques that are well
46


CA 02385162 2002-03-28
WO 01/23541 PCT/US00/27065
known to those skilled in the art (e.g., Sambrook et al., supra). A pig
genomic library (Clontech, Palo
Alto, CA) was obtained and screened with a PCR fragment derived from exon 4 of
the porcine Gal
a(1,3) galactosyl transferase gene. Exon 4 was labeled with 32P dCTP using the
Random Prime Kit
(Stratagene, La Jolla, CA) according to the manufacturer's instructions.
Approximately 4
million phage forming units were screened and unique clones that contain exon
4 sequences as
determined by Southern blotting were isolated. Clones obtained by this
procedure contained
inserts 15-40kb in length. These clones, designated pgGT, lambda 1, lambda 2,
lambda 4-1 and
lambda 8-2. Five vectors comprising unique, overlapping nucleotide sequences
which span the
entire the pig Gal a(1,3) galactosyl transferase gene from within intron 3
through intron 8 have
been deposited with the ATCC: ( 1 ) a 1.6 kb insert within intron 3 of the
extreme 5' end of the
18.275 kb lambda-2 phage clone, (2) a 6.7 kb HindIll fragment spanning intron
3 to intron 4 of
the 18.275 kb lambda-2 phage clone, (3) a 4 kb HindllI fragment following the
6.7 kb fragment 2
of the 18.275 lambda-2 phage clone, (4) a 6 kb HindllI-SaII fragment at the 3'
most portion of
the 18.275 lambda-2 phage clone, and (5) a 13 kb fragment of the lambda-2
phage clone
spanning exon 7 to exon 9. These five vectors were deposited with ATCC on 29
September 2000
with accession numbers , respectively. Subclones of the
various inserts were used to generate the claimed intron sequences from within
intron 3 to intron
8 as provided in Figure 1 using molecular biology techniques well-known to
those skilled in the
art (see e.g., Sambrook et al., supra). These sequences may be used to
determine regions of
sequence homology in design of targeting constructs for modulation of the pig
Gal a(1,3)
galactosyl transferase gene
Although the compositions and methods provided herein have been set forth in
detail, one
skilled in the art will recognize that numerous changes and modifications may
be made, and that
47


CA 02385162 2002-03-28
WO 01/23541 PCT/I1S00/27065
such changes and modifications may be made without departing from the spirit
and scope
thereof.
48

Representative Drawing
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Administrative Status

Title Date
Forecasted Issue Date Unavailable
(86) PCT Filing Date 2000-10-02
(87) PCT Publication Date 2001-04-05
(85) National Entry 2002-03-28
Dead Application 2005-10-03

Abandonment History

Abandonment Date Reason Reinstatement Date
2004-10-04 FAILURE TO PAY APPLICATION MAINTENANCE FEE

Payment History

Fee Type Anniversary Year Due Date Amount Paid Paid Date
Application Fee $300.00 2002-03-28
Maintenance Fee - Application - New Act 2 2002-10-02 $100.00 2002-09-24
Registration of a document - section 124 $100.00 2003-03-13
Maintenance Fee - Application - New Act 3 2003-10-02 $100.00 2003-09-29
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
ALEXION PHARMACEUTICALS, INC.
Past Owners on Record
FODOR, WILLIAM L.
RAMSOONDAR, JAGDEECE J.
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
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