U.S. patent application number 10/054365 was filed with the patent office on 2002-09-12 for transfection, storage and transfer of male germ cells for generation of trangenic species.
Invention is credited to Readhead, Carol W., Winston, Robert.
Application Number | 20020129398 10/054365 |
Document ID | / |
Family ID | 22065360 |
Filed Date | 2002-09-12 |
United States Patent
Application |
20020129398 |
Kind Code |
A1 |
Winston, Robert ; et
al. |
September 12, 2002 |
Transfection, storage and transfer of male germ cells for
generation of trangenic species
Abstract
A composition for in vivo transfection of vertebrate male germ
cells comprises a nucleic acid or transgene, and a gene delivery
system, and optionally a protective internalizing agent, such as an
endosomal lytic agent, a virus or a viral component, which is
internalized by cells along with the transgene and which enhances
gene transfer through the cytoplasm to the nucleus of the male germ
cell. A pharmaceutical preparation and a transfer kit utilize the
composition. A method for introducing a polynucleotide into
vertebrate male germ cells comprises the administration of the
composition to a vertebrate. A method for isolating or selecting
transfected cells utilizes a reporter gene, and a method for
administering transfected male germ cells utilizes male germ cells
which have been transfected in vitro.
Inventors: |
Winston, Robert; (London,
GB) ; Readhead, Carol W.; (Pasadena, CA) |
Correspondence
Address: |
Edward G. Poplawski, Esq.
Sidley & Austin
A Partnership including Professional Corporation
555 West Fifth Street
Los Angeles
CA
90013-1010
US
|
Family ID: |
22065360 |
Appl. No.: |
10/054365 |
Filed: |
November 12, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10054365 |
Nov 12, 2001 |
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09191920 |
Nov 13, 1998 |
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6316692 |
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60065825 |
Nov 14, 1997 |
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Current U.S.
Class: |
800/21 ; 435/456;
435/458 |
Current CPC
Class: |
C12N 2510/00 20130101;
C12N 2799/022 20130101; A61P 15/08 20180101; A01K 2227/10 20130101;
C12N 15/8509 20130101; A01K 2267/01 20130101; A61K 35/12 20130101;
A01K 2267/025 20130101; A61K 48/00 20130101; A01K 2267/03 20130101;
A01K 2227/105 20130101; C12N 2799/021 20130101; A61P 43/00
20180101; C12N 2510/02 20130101; A01K 2217/05 20130101; A01K
2267/02 20130101; A01K 67/0275 20130101; C12N 5/061 20130101; A01K
67/027 20130101 |
Class at
Publication: |
800/21 ; 435/456;
435/458 |
International
Class: |
A01K 067/027; C12N
015/86; C12N 015/88 |
Claims
What is claimed:
1. An in vivo method of incorporating a polynucleotide into a male
vertebrate's germ cells, comprising administering to a male
vertebrate's gonads a transfection mixture comprising at least one
polynucleotide encoding a desired trait or product, and at least
one transfecting agent, and optionally a genetic selection marker,
and under conditions effective to reach the vertebrate's germ cells
or precursors thereof; and allowing the polynucleotide encoding a
desired trait or product to be taken up by, and released into, the
germ cells or precursors thereof.
2. The method of claim 1, further comprising allowing the
incorporation of the released polynucleotide into the genome of the
germ cells.
3. The method of claim 1 wherein the transfecting agent is selected
from the group consisting of liposomes, viral vectors,
transferrin-polylysine enhanced viral vectors, retroviral vectors,
lentiviral vectors, and uptake enhancing DNA segments, or comprises
a mixture of any members of said group.
4. The method of claim 3, wherein the transfecting agent comprises
a viral vector selected from the group consisting of retroviral
vectors, adenoviral vectors, transferrin-polylysine enhanced
adenoviral vectors, human immunodeficiency virus vectors,
lentiviral vectors, Moloney murine leukemia virus-derived vectors,
mumps vectors, and virus-derived DNAs that facilitate
polynucleotide uptake by and release into the cytoplasm of germ
cells, or comprises an operative fragment of- or mixture of any
members of said group.
5. The method of claim 1, wherein the transfecting agent comprises
an adenovirus vector having endosomal lytic activity, and the
polynucleotide is operatively linked to the vector.
6. The method of claim 1, wherein the transfecting agent comprises
a lipid transfecting agent.
7. The method of claim 1, wherein the transfecting agent further
comprises a male-germ-cell-targeting molecule.
8. The method of claim 7, wherein the male-germ-cell-targeting
molecule is specific for targeting spermatogonia, and is a c-kit
ligand.
9. The method of claim 1, where the transfection mixture further
comprises an immunosuppressing agent.
10. The method of claim 9, wherein the immunosuppressing agent is
selected from the group consisting of cyclosporin and
corticosteroids, and the agent is administered systemically.
11. The method of claim 1, wherein the transfection mixture is
administered by injection.
12. The method of claim 11, where injection comprises percutaneous
injection into the vertebrate's testis.
13. The method of claim 1, wherein the transfection mixture is
administered into the vertebrate's testis.
14. The method of claim 13, wherein the transfection mixture is
directly administered into the vertebrate's vas efferens.
15. The method of claim 13, wherein the transfection mixture is
directly administered into a seminiferous tubule of the
vertebrate's testis.
16. The method of claim 1, wherein the transfection mixture is
directly administered into the rete of the vertebrate's testis.
17. The method of claim 1, wherein the vertebrate is a mammal.
18. The method of claim 17, wherein the mammal is a human.
19. The method of claim 17, wherein the mammal is selected from the
group consisting of human and non-human primates, farm mammals, and
marine mammals.
20. The method of claim 19, wherein the farm mammal is selected
from the group consisting of swine, equines, ovines and
bovines.
21. The method of claim 1, wherein the vertebrate is a bird
selected from the group consisting of ducks, geese, turkeys and
chickens.
22. The method of claim 1, wherein the vertebrate is selected from
the group consisting of wild and domesticated vertebrates.
23. A gene therapy method, comprising the method of claim 1,
wherein the polynucleotide encoding a desired trait or product is
derived from the same species as the male vertebrate.
24. A non-human transgenic vertebrate produced by the method of
claim 1, or progeny thereof, wherein the polynucleotide encoding a
desired trait or product is derived from any genome.
25. The non-human transgenic vertebrate of claim 24, comprising
native germ cells carrying in their genome at least one xenogeneic
polynucleotide.
26. The non-human transgenic vertebrate of claim 25, wherein the
polynucleotide comprises at least one biologically functional
gene.
27. The non-human transgenic vertebrate of claim 24, being a
male.
28. The progeny resulting from breeding the non-human transgenic
vertebrate of claim 27, with a female of the same species.
29. A non-human vertebrate, carrying in its germ cells at least one
xenogeneic polynucleotide sequence, said non-human vertebrate being
obtained by breeding the vertebrate of claim 24, or progeny
thereof, with a member of the opposite sex of the same species, and
selecting the bred progeny for the presence of the transfected
xenogeneic polynucleotide.
30. The non-human vertebrate of claim 29, which is selected from
the group consisting of mammals and birds.
31. The non-human vertebrate of claim 30, which is a mammal
selected from the group consisting of humans and non-human
primates, canines, felines, swine, farm and marine mammals,
pachyderms, equines, murine, ovines and bovine, or a bird selected
from the group consisting of ducks, geese, turkeys and
chickens.
32. The vertebrate of claim 31, wherein the mammal is selected from
the group consisting of wild and domesticated mammals.
33. The vertebrate of claim 31, wherein the mammal is a farm or
marine animal.
34. The vertebrate of claim 30, wherein the mammal is selected from
the group consisting of a bull and a pig, and the bird is a
chicken.
35. A germ cell, obtained from the vertebrate of claim 25.
36. Vertebrate male germ cells, obtained by a method comprising the
method of claim 1; raising the transfected male vertebrate; and
collecting male germ cells produced by the male vertebrate.
37. The vertebrate male germ cells of claim 36, wherein the method
for obtaining them further comprises breeding the transfected
vertebrate to produce progeny, and then collecting the germ cells
produced by a male progeny.
38. Vertebrate semen, comprising the germ cell of claim 35.
39. Vertebrate semen, comprising the germ cells obtained from the
vertebrate of claim 25.
40. A method of producing a non-human vertebrate animal line
comprising native germ cells carrying in their genome at least one
xenogeneic polynucleotide, comprising breeding of the vertebrate of
claim 25, with a member of the opposite sex of the same species;
and selecting progeny for the presence of said polynucleotide.
41. A method of isolating or selecting a male germ cell transfected
with at least one polynucleotide encoding a desired trait or
product and at least one genetic selection marker, comprising the
method of claim 1, wherein the transfection mixture comprises at
least one genetic selection marker; and isolating or selecting a
transfected male germ cell with the aid of the genetic selection
marker.
42. A method of transferring maturing male germ cells transfected
with at least one polynucleotide encoding a desired trait or
product to the testis of a recipient male vertebrate, comprising
isolating or selecting maturing male germ cells carrying at least
one polynucleotide encoding a desired trait or product and at least
one polynucleotide encoding a genetic selection marker, from a
donor male vertebrate by the method of claim 41; administering the
germ cells, thus isolated or selected, to a testis of a recipient
male vertebrate; and allowing the administered germ cells to lodge
in a seminiferous tubule of the recipient male vertebrate.
43. A method of transferring autologous germ and support cells to
the testis of a vertebrate, comprising the method of claim 42,
wherein the donor vertebrate is the same as the recipient
vertebrate.
44. The method of claim 41, further comprising the step of
incorporating into the genome of the germ cell the polynucleotide
encoding a desired trait or product.
45. The method of claim 41, wherein the transfected male germ cell
comprises an undifferentiated male germ cell.
46. The method of claim 41, wherein transfection is conducted under
conditions of temperature of about 25.degree. C. to about
38.degree. C.
47. The method of claim 41, wherein the transfecting agent is
selected from the group consisting of liposomes, viral vectors,
transferrin-polylysine enhanced viral vectors, retroviral vectors,
lentiviral vectors, and other uptake enhancing DNA segments, or
comprises a mixture of any members of said group.
48. The method of claim 47, wherein the transfecting agent
comprises a viral vector selected from the group consisting of
retroviral vectors, adenoviral vectors, transferrin-polylysine
enhanced adenoviral vectors, human immunodeficiency virus vectors,
lentiviral vectors, Moloney murine leukemia virus-derived vectors,
mumps vectors, and virus-derived DNAs that facilitate
polynucleotide uptake by and release into the cytoplasm of germ
cells, or said transfecting agent comprises an operative fragment
of- or mixture of any members of said group.
49. The method of claim 47, wherein the transfecting agent
comprises an adenovirus vector having endosomal lytic activity, and
the polynucleotide is operatively linked to the vector.
50. The method of claim 41, wherein the polynucleotide encoding a
desired trait or product is in the form of a complex with a viral
vector.
51. The method of claim 41, wherein the transfecting agent
comprises a lipid transfecting agent.
52. The method of claim 42, wherein the transfecting agent further
comprises an agent selected from the group consisting of a
male-germ-cell-targeting molecule and at least one genetic
selection marker.
53. The method of claim 52, wherein the male-germ-cell-targeting
molecule is specifically targeted to spermatogonia and comprises a
c-kit ligand; and the genetic selection marker comprises a gene
encoding a detectable product. expression of said gene being driven
by a spermatogonia-specific promoter, said promoter being selected
from the group consisting of c-kit promoter, b-Myb promoter,
c-raf-1 promoter, ATM (axataia-telangiectasia) promoter, RBM
(ribosome binding motif) promoter, DAZ (deleted in azoospermia)
promoter, XRCC-1 promoter, HSP 90 (heat shock gene) promoter, and
FRMI (from fragile X site) promoter.
54. The method of claim 41, wherein the vertebrate is a mammal.
55. The method of claim 54, wherein the mammal is a human.
56. The method of claim 54, wherein the mammal is selected from the
group consisting of human and non-human primates and farm and
marine mammals.
57. The method of claim 42, wherein the polynucleotide encoding a
desired trait or product is derived from the same species of
vertebrate as the recipient vertebrate.
58. The method of claim 42, wherein the vertebrate is selected from
the group consisting of wild and domesticated vertebrates.
59. The method of claim 41, wherein the polynucleotide encoding a
desired trait or product is derived from a mammal selected from the
group consisting of human and non-human primates, canines, felines,
swines, farm mammals, pachyderms, marine mammals, equines, murine,
ovine and bovine, or from a bird selected from the group consisting
of ducks, geese, turkeys and chickens.
60. The method of claim 59, wherein the polynucleotide is derived
from a human.
61. A non-human transgenic vertebrate, comprising native germ cells
carrying in their genomes at least one xenogeneic polynucleotide,
said transgenic vertebrate being the recipient male vertebrate of
the method of claim 42, or progeny thereof.
62. The non-human transgenic vertebrate of claim 61, wherein the
polynucleotide comprises at least one biologically functional
gene.
63. The non-human transgenic vertebrate of claim 62, being a
male.
64. The non-human transgenic vertebrate of claim 63, harboring
native male germ cells transfected with a xenogeneic
polynucleotide.
65. The progeny resulting from breeding the non-human transgenic
vertebrate of claim 63 or progeny thereof, with a female of the
same species.
66. A non-human vertebrate, carrying in its germ cells at least one
xenogeneic polynucleotide sequence, obtained by breeding the
vertebrate of claim 61 or progeny thereof, with a member of the
opposite sex of the same species, and selecting the bred progeny
for the presence of the transfected xenogeneic polynucleotide.
67. The non-human vertebrate of claim 66, which is selected from
the group consisting of mammals and birds.
68. The non-human vertebrate of claim 67, which is a mammal
selected from the group consisting of humans and non-human
primates, canines, felines, swine, farm and marine mammals,
pachyderms, equines, murine, ovines and bovine, and a bird selected
from the group consisting of ducks, geese, turkeys and
chickens.
69. The non-human vertebrate of claim 67, which is a bird selected
from the group consisting of ducks, geese, turkeys and
chickens.
70. The non-human vertebrate of claim 67, wherein the mammal is a
farm or marine mammal.
71. The non-human vertebrate of claim 68, wherein the mammal is a
bull.
72. The non-human vertebrate of claim 68, wherein the mammal is a
pig.
73. The non-human vertebrate of claim 66, which is selected from
the group consisting of wild and domesticated animals.
74. A germ cell obtained from a vertebrate of claims 24 or 61
comprising a native germ cell carrying in its genome at least one
xenogeneic polynucleotide.
75. Vertebrate semen comprising the germ cell of claim 74.
76. A gene therapy method, comprising the method of claim 42,
wherein the polynucleotide encoding a desired trait or product is
derived from the same species of vertebrate as the recipient
vertebrate.
77. A non-human transgenic vertebrate produced by the method of
claim 42, wherein the polynucleotide encoding a desired trait or
product is derived from any genome.
78. An in vitro method of incorporating at least one polynucleotide
encoding a desired trait into a maturing male germ cell, comprising
obtaining a maturing male germ cell from a vertebrate; transfecting
the germ cell in vitro with at least one polynucleotide encoding a
desired trait in the presence of a gene delivery mixture comprising
at least one transfecting agent, and optionally a polynucleotide
encoding a genetic selection marker, at about or below the
vertebrate's body temperature and for a transfection-effective
period of time; and allowing the polynucleotide encoding a desired
trait to be taken up by, and released into the germ cell.
79. The method of claim 78, further comprising allowing the
incorporation of the polynucleotide into the genome of the germ
cell.
80. The method of claim 78, wherein the encoding a desired trait is
incorporated into the vertebrate germ cell's genome.
81. The method of claim 78, wherein the maturing male germ cell
comprises a spermatogonia or other undifferentiated male germ
cell.
82. The method of claim 78, wherein the transfection is conducted
under conditions of temperature of about 25.degree. C. to about
38.degree. C.
83. The method of claim 78, wherein the transfecting agent is
selected from the group consisting of liposomes, viral vectors,
transferrin-polylysine enhanced viral vectors, retroviral vectors,
lentiviral vectors, and other uptake enhancing DNA segments, or
comprises a mixture of any members of said group.
84. The method of claim 83, wherein the transfecting agent
comprises a viral vector selected from the group consisting of
retroviral vectors, adenoviral vectors, transferrin-polylysine
enhanced adenoviral vectors, human immunodeficiency virus vectors,
lentiviral vectors, Moloney murine leukemia virus-derived vectors,
mumps vectors, and virus-derived DNAs that enhance polynucleotide
uptake by and release into the cytoplasm of germ cells, or said
transfecting agent comprises an operative fragment of- or mixture
of any members of said group.
85. The method of claim 84, wherein the transfecting agent
comprises an adenovirus vector having endosomal lytic activity, and
the polynucleotide encoding a desired trait is operatively linked
to the vector.
86. The method of claim 78, wherein the polynucleotide encoding a
desired trait is in the form of a complex with a viral vector.
87. The method of claim 78, wherein the transfecting agent
comprises a lipid transfecting agent.
88. The method of claim 78, wherein the transfecting agent further
comprises an agent selected from the group consisting of a
male-germ-cell-targeting molecule and at least one genetic
selection marker; and the method further comprises isolating or
selecting a maturing male germ cell carrying at least one
polynucleotide encoding a desired trait or product and at least one
polynucleotide encoding a genetic selection marker, from a donor
male vertebrate with the aid of the genetic selection marker.
89. The method of claim 88, wherein the male-germ-cell-targeting
molecule is specifically targeted to spermatogonia and comprises a
c-kit ligand, and the genetic selection marker comprises a gene
expressing a detectable product, driven by a spermatogonia-specific
promoter selected from the group consisting of c-kit promoter,
b-Myb promoter, c-raf-1 promoter, ATM (axataia-telangiectasia)
promoter, RBM (ribosome binding motif) promoter, DAZ (deleted in
azoospermia) promoter, XRCC-1 promoter, HSP 90 (heat shock gene)
promoter, and FRMI (from fragile X site) promoter.
90. The method of claim 78, wherein the vertebrate is a mammal.
91. The method of claim 90, wherein the mammal is a human.
92. The method of claim 90, wherein the mammal is selected from the
group consisting of human and non-human primates and farm and
marine mammals.
93. The method of claim 78, wherein the polynucleotide encoding a
desired trait is derived from the same vertebrate species as the
maturing germ cell.
94. The method of claim 78, wherein the vertebrate is selected from
the group consisting of wild and domesticated vertebrates.
95. The method of claim 78, wherein the polynucleotide encoding a
desired trait is derived from a mammal selected from the group
consisting of human and non-human primates, canines, felines,
swines, farm mammals, pachyderms, marine mammals, equines, murine,
ovine and bovine, or from a bird selected from the group consisting
of ducks, geese, turkeys and chickens.
96. The method of claim 95, wherein the polynucleotide is derived
from a human.
97. A non-human transgenic vertebrate, or its progeny, comprising a
native germ cell carrying in its genome at least one xenogeneic
polynucleotide, said polynucleotide having been incorporated into
the genome of said germ cell through the method of claim 78.
98. The non-human transgenic vertebrate of claim 97, wherein the
polynucleotide comprises at least one biologically functional
gene.
99. The non-human transgenic vertebrate of claim 98, being a
male.
100. The non-human transgenic vertebrate of claim 99, harboring
native male germ cells transfected with a xenogeneic
polynucleotide.
101. The progeny resulting from breeding the non-human transgenic
vertebrate of claim 99 or progeny thereof, with a female of the
same species.
102. A non-human vertebrate, carrying in its germ cells at least
one xenogeneic polynucleotide sequence, said vertebrate obtained by
breeding the vertebrate of claim 98 or progeny thereof, with a
member of the opposite sex of the same species, and selecting the
bred progeny for the presence of the transfected xenogeneic
polynucleotide.
103. The non-human vertebrate of claim 102, which is selected from
the group consisting of mammals and birds.
104. The non-human vertebrate of claim 103, which is a mammal
selected from the group consisting of humans and non-human
primates, canines, felines, swine, farm and marine mammals,
pachyderms, equines, murine, ovines and bovine, or a bird selected
from the group consisting of ducks, geese, turkeys and
chickens.
105. The non-human vertebrate of claim 104, which is a bird
selected from the group consisting of ducks, geese, turkeys and
chickens.
106. The non-human vertebrate of claim 104, wherein the mammal is a
farm or marine mammal.
107. The non-human vertebrate of claim 104, wherein the mammal is a
bull.
108. The non-human vertebrate of claim 104, wherein the mammal is a
pig.
109. The non-human vertebrate of claim 102, which is selected from
the group consisting of wild and domesticated animals.
110. A germ cell obtained from the vertebrate of claim 97, or its
progeny.
111. Vertebrate semen comprising a plurality of the germ cells
obtained from the vertebrate of claim 98.
112. A gene therapy method, comprising the method of claim 78;
further comprising the step of introducing said transfected male
germ cell into the testis of a recipient vertebrate, wherein the
polynucleotide encoding a desired trait is derived from the same
vertebrate species as the recipient vertebrate.
113. A non-human transgenic vertebrate produced by the method of
claim 78, wherein the polynucleotide encoding a desired trait is
derived from any genome.
114. A kit for the transfection and storage of a male vertebrate's
germ cells, comprising a transfection mixture, said tranfection
mixture comprising at least one transfecting agent, and optionally
a genetic selection marker, whereby said kit may be used to
transfect and store said germ cells in a viable condition.
115. The kit of claim 114, wherein the transfecting agent is
selected from the group consisting of liposomes, viral vectors,
transferrin-polylysine enhanced viral vectors, retroviral vectors,
lentiviral vectors, and uptake enhancing DNA segments, or comprises
a mixture of any members of said group.
116. The kit of claim 114, wherein the transfecting agent comprises
a viral vector selected from the group consisting of retroviral
vectors, adenoviral vectors, transferrin-polylysine enhanced
adenoviral vectors, human immunodeficiency virus vectors,
lentiviral vectors, Moloney murine leukemia virus-derived vectors,
mumps vectors, DNAs that facilitate polynucleotide uptake by and
release into the cytoplasm of germ cells, or comprises an operative
fragment of- or mixture of any members of said group.
117. The kit of claim 114,wherein the transfecting agent comprises
an adenovirus vector having endosomal lytic activity, and the
polynucleotide is operatively linked to the vector.
118. The kit of claim 114, wherein the transfecting agent comprises
a lipid transfecting agent.
119. The kit of claim 114, wherein the transfecting agent further
comprises a male-germ-cell-targeting molecule.
120. The kit of claim 119, wherein the male-germ-cell-targeting
molecule is specific for targeting spermatogonia and comprises a
c-kit ligand.
121. The kit of claim 114, where the transfection mixture further
comprises an immunosuppressing agent.
122. The kit of claim 121, wherein the immunosuppressing agent is
selected from the group consisting of cyclosporin and
corticosteroids.
123. The kit of claim 119, wherein the male-germ-cell-targeting
molecule is specifically targeted to spermatogonia and comprises a
c-kit ligand; and the genetic selection marker comprises a gene
expressing a detectable product driven by a spermatogonia-specific
promoter.
124. The kit of claim 119, wherein the male-germ-cell-targeting
molecule is specifically targeted to spermatogonia and comprises a
c-kit ligand; and the genetic selection marker comprises a gene
expressing a detectable product, driven by a spermatogonia-specific
promoter, said promoter selected from the group consisting of c-kit
promoter, b-Myb promoter, c-raf-1 promoter, ATM
(axataia-telangiectasia) promoter, RBM (ribosome binding motif)
promoter, DAZ (deleted in azoospermia) promoter, XRCC-1 promoter,
HSP 90 (heat shock gene) promoter, and FRMI (from fragile X site)
promoter.
125. The kit of claim 114, wherein at least one polynucleotide
comprises at least one polynucleotide sequence encoding a genetic
selection marker.
126. The kit of claim 114, further comprising a cryoprotectant.
127. The germ cell as in any of claims 35, 36, 37, 74, or 110,
wherein said germ cell has been cryopreserved in a viable and
functional condition.
128. A transgenic male germ cell produced by the method of any of
claims 17, 18, 19, 20, 21, or 22, wherein the transgenic male germ
cell has been cryopreserved in a viable and functional
condition.
129. A transgenic male germ cell produced by the method of any of
claims 54, 55, 56, or 58, wherein the transgenic male germ cell has
been cryopreserved in a viable and functional condition.
130. A transgenic male germ cell produced by the method of any of
claims 90, 91, 92, or 94, wherein the transgenic male germ cell has
been cryopreserved in a viable and functional condition.
131. The method of any of claims 1 or 78, wherein the
polynucleotide encoding a desired trait or product is operatively
linked to a germ cell-specific promoter.
132. The method of any of claims 41, 78, or 88, wherein the
polynucleotide encoding a genetic selection marker is operatively
linked to a germ cell-specific promoter.
133. The method of claim 42, wherein support cells are
co-administered to a testis along with isolated or selected germ
cells.
134. The method of claim 42, wherein transfected support cells are
isolated or selected, and co-administered to a testis of a
recipient male vertebrate along with said isolated or selected germ
cells.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to the field of transgenics
and gene therapy. More specifically, this invention relates to in
vitro and in vivo methods for transfecting germ cells and, in some
instances, incorporating a nucleic acid segment encoding a specific
trait into the male germ cells of an animal. When the nucleic acid
becomes incorporated into the germ cell genome, upon mating, or in
vitro fertilization and the like, the trait may be transmitted to
the progeny. The present technology is suitable for breeding
progeny with or without a desired trait by modifying their genome.
This technology is also suitable for use in introducing a
therapeutic gene into the germ or support cells (e.g., Leydig and
Sertoli cells) of the testis and is, therefore, suitable for use in
gene therapy for males with fertility problems associated with
genetic defects.
[0003] 2. Description of the Background
[0004] The field of transgenics was initially developed to
understand the action of a single gene in the context of the whole
animal and phenomena of gene activation, expression, and
interaction. This technology has been used to produce models for
various diseases in humans and other animals. Transgenic technology
is amongst the most powerful tools available for the study of
genetics, and the understanding of genetic mechanisms and function.
It is also used to study the relationship between genes and
diseases. About 5,000 diseases are caused by a single genetic
defect. More commonly, other diseases are the result of complex
interactions between one or more genes and environmental agents,
such as viruses or carcinogens. The understanding of such
interactions is of prime importance for the development of
therapies, such gene therapy and drug therapies, and also
treatments such as organ transplantation. Such treatments
compensate for functional deficiencies and/or may eliminate
undesirable functions expressed in an organism. Transgenesis has
also been used for the improvement of livestock, and for the large
scale production of biologically active pharmaceuticals.
[0005] Historically, transgenic animals have been produced almost
exclusively by micro injection of the fertilized egg. The pronuclei
of fertilized eggs are micro injected in vitro with foreign, i.e.
xenogeneic or allogeneic DNA or hybrid DNA molecules. The micro
injected fertilized eggs are then transferred to the genital tract
of a pseudopregnant female. The generation of transgenic animals by
this technique is generally reproducible, and for this reason
little has been done to improve on it. This technique, however,
requires large numbers of fertilized eggs. This is partly because
there is a high rate of egg loss due to lysis during micro
injection. Moreover manipulated embryos are less likely to implant
and survive in utero. These factors contribute to the technique's
extremely low efficiency. For example, 300-500 fertilized eggs may
need to be micro injected to produce perhaps three transgenic
animals. Partly because of the need to micro inject large numbers
of embryos, transgenic technology has largely been exploited in
mice because of their high fecundity. Whilst small animals such as
mice have proved to be suitable models for certain diseases, their
value in this respect is limited. Larger animals would be much more
suitable to study the effects and treatment of most human diseases
because of their greater similarity to humans in many aspects, and
also the size of their organs. Now that transgenic animals with the
potential for human xenotransplantation are being developed, larger
animals, of a size comparable to man will be required. Transgenic
technology will allow that such donor animals will be
immunocompatible with the human recipient. Historical transgenic
techniques, however, require that there be an ample supply of
fertilized female germ cells or eggs. Most large mammals, such as
primates, cows, horses and pigs produce only 10-20 or less eggs per
animal per cycle even after hormonal stimulation. Consequently,
generating large animals with these techniques is prohibitively
expensive.
[0006] This invention relies on the fact that vast numbers of male
germ cells are more readily available. Most male mammals generally
produce at least 10.sup.8 spermatozoa (male germ cells) in each
ejaculate. This is in contrast to only 10-20 eggs in a mouse even
after treatment with superovulatory drugs. A similar situation is
true for ovulation in nearly all larger animals. For this reason
alone, male germ cells will be a better target for introducing
foreign DNA into the germ line, leading to the generation of
transgenic animals with increased efficiency and after simple,
natural mating.
[0007] Initially, attempts were made to produce transgenic animals
by adding DNA to spermatozoa which were then used to fertilize
mouse eggs in vitro. The fertilized eggs were then transferred to
pseudopregnant foster females, and of the pups born, 30% were
reported to be transgenic and express the transgene. Despite
repeated efforts by others, however, this experiment could not be
reproduced and no transgenic pups were obtained. Indeed, there
remains little doubt that the transgenic animals reputed to have
been obtained by this method were not transgenic at all and the DNA
incorporation reported was mere experimental artifact. Data
collected from laboratories around the world engaged in testing
this method showed that no transgenics were obtained from a total
of 890 pups generated.
[0008] In summary, it is currently possible to produce live
transgenic progeny but the available methods are costly and
extremely inefficient. Spermatogenic transfection in accordance
with this invention, either in vitro or in vivo, provides a simple,
less costly and less invasive method of producing transgenic
animals and one that is potentially highly effective in
transferring allogeneic as well as xenogeneic genes into the
animal's germ cells. The present technology is also of great value
in producing transgenic animals in large species as well as for
repairing genetic defects which lead to male infertility. The
present technology is also suitable for germ line gene therapy in
humans and other animal species. Male germ cells that have stably
integrated the DNA could be selected.
SUMMARY OF THE INVENTION
[0009] The present invention relates to the in vivo and ex vivo (in
vitro) transfection of eukaryotic animal germ cells with a desired
genetic material. Briefly, the in vivo method involves injection of
genetic material together with a suitable vector directly into the
testicle of the animal. In this method, all or some of the male
germ cells within the testicle are transfected in situ, under
effective conditions. The ex vivo method involves extracting germ
cells from the gonad of a suitable donor or from the animal's own
gonad, using a novel isolation method, transfecting them in vitro,
and then returning them to the testis under suitable conditions
where they will spontaneously repopulate it. The ex vivo method has
the advantage that the transfected germ cells may be screened by
various means before being returned to the testis to ensure that
the transgene is incorporated into the genome in a stable state.
Moreover, after screening and cell sorting only enriched
populations of germ cells may be returned. This approach provides a
greater chance of transgenic progeny after mating.
[0010] This invention also relates to a novel method for the
isolation of spermatogonia, comprising obtaining spermatogonia from
a mixed population of testicular cells by extruding the cells from
the seminiferous tubules and gentle enzymatic disaggregation. The
spermatogonia or stem cells which are to be genetically modified,
may be isolated from a mixed cell population by a novel method
including the utilization of a promoter sequence, which is only
active in cycling spermatogonia stem cell populations, for example,
b-Myb or a spermotogonia specific promoter, such as the c-kit
promoter region, c-raf-1 promoter, ATM (axataia-telangiectasia)
promoter, RBM (ribosome binding motif) promoter, DAZ (deleted in
azoospermia) promoter, XRCC-1 promoter, HSP 90 (heat shock gene)
promoter, or FRMI (from fragile X site) promoter, optionally linked
to a reporter construct, for example, the Green Fluorescent Protein
Gene (EGFP). These unique promoter sequences drive the expression
of the reporter construct only in the cycling spermatogonia. The
spermatogonia, thus, are the only cells in the mixed population
which will express the reporter construct and they, thus, may be
isolated on this basis. In the case of the green fluorescent
reporter construct, the cells may be sorted with the aid of, for
example, a FACs scanner set at the appropriate wavelength or they
may be selected by chemical methods.
[0011] This invention also relates to the repopulation of a testis
with germ cells that have been isolated from a fresh or frozen
testicular biopsy. These germ cells may or may not be genetically
manipulated prior to reimplantation.
[0012] For transfection, the method of the invention comprises
administering to the animal, or to germ cells in vitro, a
composition comprising amounts of nucleic acid comprising
polynucleotides encoding a desired trait. In addition, the
composition comprises, for example, a relevant controlling promoter
region made up of nucleotide sequences. This is combined with, for
example, a gene delivery system comprising a cell transfection
promotion agent such as retro viral vectors, adenoviral and
adenoviral related vectors, or liposomal reagents or other agents
used for gene therapy. These introduced under conditions effective
to deliver the nucleic acid segments to the animal's germ cells
optionally with the polynucleotide inserted into the genome of the
germ cells. Following incorporation of the DNA, the treated animal
is either allowed to breed naturally, or reproduced with the aid of
assisted reproductive technologies, and the progeny selected for
the desired trait.
[0013] This technology is applicable to the production of
transgenic animals for use as animal models, and to the
modification of the genome of an animal, including a human, by
addition, modification, or subtraction of genetic material, often
resulting in phenotypic changes. The present methods are also
applicable to altering the carrier status of an animal, including a
human, where that individual is carrying a gene for a recessive or
dominant gene disorder, or where the individual is prone to pass a
multigenic disorder to his offspring.
[0014] A preparation suitable for use with the present methods
comprises a polynucleotide segment encoding a desired trait and a
transfection promotion agent, and optionally an uptake promotion
agent which is sometime equipped with agents protective against DNA
breakdown. The different components of the transfection composition
are also provided in the form of a kit, with the components
described above in measured form in two or more separate
containers. The kit generally contains the different components in
separate containers. Other components may also be provided in the
kit as well as a carrier.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0015] The present invention arose from a desire by the present
inventors to improve on existing methods for the genetic
modification of an animal's germ cells and for producing transgenic
animals. The pre-existing art methods rely on direct injection of
DNA into zygotes produced in vitro or in vivo, or by the production
of chimeric embryos using embryonal stem cells incorporated into a
recipient blastocyst. Following this, such treated embryos are
transferred to the primed uterus or oviduct. The available methods
are extremely slow and costly, rely on several invasive steps, and
only produce transgenic progeny sporadically and unpredictably.
[0016] In their search for a less costly, faster, and more
efficient approach for producing transgenics, the present inventors
devised the present method which relies on the in vivo or ex vivo
(in vitro) transfection of male animal germ cells with a nucleic
acid segment encoding a desired trait. The present method relies on
at least one of the following strategies. A first method delivers
the nucleic acid segment using known gene delivery systems in situ
to the gonad of the animal (in vivo transfection), allows the
transfected germ cells to differentiate in their own milieu, and
then selects for animals exhibiting the nucleic acid's integration
into its germ cells (transgenic animals). The thus selected animals
may be mated, or their sperm utilized for insemination or in vitro
fertilization to produce transgenic progeny. The selection may take
place after biopsy of one or both gonads, or after examination of
the animal's ejaculate amplified by the polymerase chain reaction
to confirm the incorporation of the desired nucleic acid sequence.
In order to simplify the confirmation of the actual incorporation
of the desired nucleic acid, the initial transfection may include a
co-transfected reporter gene, such as a gene encoding for Green
Fluorescent Protein, which fluoresces under suitable wave-lengths
of ultra-violet light.
[0017] Alternatively, male germ cells may be isolated from a donor
animal and transfected, or genetically altered in vitro to impart
the desired trait. Following this genetic manipulation, germ cells
which exhibit any evidence that the DNA has been modified in the
desired manner are selected, and transferred to the testis of a
suitable recipient animal. Further selection may be attempted after
biopsy of one or both gonads, or after examination of the animal's
ejaculate amplified by the polymerase chain reaction to confirm
whether the desired nucleic acid sequence was actually
incorporated. As described above, the initial transfection may have
included a co-transfected reporter gene, such as a gene encoding
the Green Fluorescent Protein. Before transfer of the germ cells,
the recipient testis are generally treated in one, or a
combination, of a number of ways to inactivate or destroy
endogenous germ cells, including by gamma irradiation, by chemical
treatment, by means of infectious agents such as viruses, or by
autoimmune depletion or by combinations thereof. This treatment
facilitates the colonization of the recipient testis by the altered
donor cells.
[0018] Animals that were shown to carry suitably modified sperm
cells then may be either allowed to mate naturally, or
alternatively their spermatozoa are used for insemination or in
vitro fertilization. The thus obtained transgenic progeny may be
bred, whether by natural mating or artificial insemination, to
obtain further transgenic progeny. The method of this invention has
a lesser number of invasive procedures than other available
methods, and a high rate of success in producing incorporation into
the progeny's genome of the nucleic acid sequence encoding a
desired trait.
[0019] Primordial germ cells are thought to arise from the
embryonic ectoderm, and are first seen in the epithelium of the
endodermal yolk sac at the E8 stage. From there they migrate
through the hindgut endoderm to the genital ridges. The primitive
spermatogonial stem cells, known as A0/As, differentiate into type
B spermatogonia The latter further differentiate to form primary
spermatocytes, and enter a prolonged meiotic prophase during which
homologous chromosomes pair and recombine. Several morphological
stages of meiosis are distinguishable: preleptotene, leptotene,
zygotene, pachytene, secondary spermatocytes, and the haploid
spermatids. The latter undergo further morphological changes during
spermatogenesis, including the reshaping of their nucleus, the
formation of acrosome, and assembly of the tail. The final changes
in the spermatozoon take place in the genital tract of the female,
prior to fertilization. The uptake of the nucleic acid segment
administered by the present in vivo method to the gonads will reach
germ cells that are at one or more of these stages, and be taken up
by those that are at a more receptive stage. In the ex vivo (in
vitro) method of genetic modification, generally only diploid
spermatogonia are used for nucleic acid modification. The cells may
be modified in vivo using gene therapy techniques, or in vitro
using a number of different transfection strategies.
[0020] The inventors are, thus, providing in this patent a novel
and unobvious method for; isolation of male germ cells, and for the
in vivo and ex vivo (in vitro) transfection of allogeneic as well
as xenogeneic DNA into an animal's germ cells. This comprises the
administration to an animal of a composition comprising a gene
delivery system and at least one nucleic acid segment, in amounts
and under conditions effective to modify the animal's germ cells,
and allowing the nucleic acid segment to enter, and be released
into, the germ cells, and to integrate into their genome.
[0021] The in vivo introduction of the gene delivery mixture to the
germ cells may be accomplished by direct delivery into the animal's
testis (es), where it is distributed to male germ cells at various
stages of development. The in vivo method utilizes novel
technology, such as injecting the gene delivery mixture either into
the vasa efferentia, directly into the seminiferous tubules, or
into the rete testis using, for example, a micropipette. To ensure
a steady infusion of the gene delivery mixture, under pressures
which will not damage the delicate tubule system in the testis, the
injection may be made through the micropipette with the aid of a
picopump delivering a precise measured volume under controlled
amounts of pressure. The micropipette may be made of a suitable
material, such as metal or glass, and is usually made from glass
tubing which has been drawn to a fine bore at its working tip, e.g.
using a pipette puller. The tip may be angulated in a convenient
manner to facilitate its entry into the testicular tubule system.
The micropipette may be also provided with a beveled working end to
allow a better and less damaging penetration of the fine tubules at
the injection site. This bevel may be produced by means of a
specially manufactured grinding apparatus. The diameter of the tip
of the pipette for the in vivo method of injection may be about 15
to 45 microns, although other sizes may be utilized as needed,
depending on the animal's size. The tip of the pipette may be
introduced into the rete testis or the tubule system of the
testicle, with the aid of a binocular microscope with coaxial
illumination. with care taken not to damage the wall of the tubule
opposite the injection point, and keeping trauma to a minimum. On
average, a magnification of about .times.25 to .times.80 is
suitable, and bench mounted micromanipulators are not severally
required as the procedure may be carried out by a skilled artisan
without additional aids. A small amount of a suitable, non-toxic
dye, may be added to the gene delivery fluid to confirm delivery
and dissemination to the tubules of the testis. It may include a
dilute solution of a suitable, non-toxic dye, which may be
visualized and tracked under the microscope.
[0022] In this manner, the gene delivery mixture is brought into
intimate contact with the germ cells. The gene delivery mixture
typically comprises the modified nucleic acid encoding the desired
trait, together with a suitable promoter sequence, and optionally
agents which increase the uptake of the nucleic acid sequence, such
as liposomes, retroviral vectors, adenoviral vectors, adenovirus
enhanced gene delivery systems, or combinations thereof. A reporter
construct such as the gene encoding for Green Fluorescent Protein
may further be added to the gene delivery mixture. Targeting
molecules such as c-kit ligand may be added to the gene delivery
mixture to enhance the transfer of the male germ cell.
[0023] For the ex vivo (in vitro) method of genetic alteration, the
introduction of the modified germ cells into the recipient testis
may be accomplished by direct injection using a suitable
micropipette. Support cells, such as Leydig or Sertoli cells that
provide hormonal stimulus to spermatogonial differentiation, may be
transferred to a recipient testis along with the modified germ
cells. These transferred support cells may be unmodified, or,
alternatively, may themselves have been transfected, together with-
or separately from the germ cells. These transferred support cells
may be autologous or heterologous to either the donor or recipient
testis. A preferred concentration of cells in the transfer fluid
may easily be established by simple experimentation, but will
likely be within the range of about 1.times.10.sup.5
.times.10.times.10.sup.5 cells per 10 .mu.l of fluid. This
micropipette may be introduced into the vasa efferentia, the rete
testis or the seminiferous tubules, optionally with the aid of a
picopump to control pressure and/or volume, or this delivery may be
done manually. The micropipette employed is in most respects
similar to that used for the in vivo injection, except that its tip
diameter generally will be about 70 microns. The microsurgical
method of introduction is similar in all respects to that used for
the in vivo method described above. A suitable dyestuff may also be
incorporated into the carrier fluid for easy identification of
satisfactory delivery of the transfected germ cells.
[0024] Once in contact with germ cells, whether they are in situ in
the animal or vitro, the gene delivery mixture facilitates the
uptake and transport of the xenogeneic genetic material into the
appropriate cell location for integration into the genome and
expression. A number of known gene delivery methods may be used for
the uptake of nucleic acid sequences into the cell.
[0025] "Gene delivery (or tansfection) mixture", in the context of
this patent, means selected genetic material together with an
appropriate vector mixed, for example, with an effective amount of
lipid transfection agent. The amount of each component of the
mixture is chosen so that the transfection of a specific species of
germ cell is optimized. Such optimization requires no more than
routine experimentation. The ratio of DNA to lipid is broad,
preferably about 1:1, although other proportions may also be
utilized depending on the type of lipid agent and the DNA utilized.
This proportion is not crucial.
[0026] "Transfecting agent", as utilized herein, means a
composition of matter added to the genetic material for enhancing
the uptake of exogenous DNA segment(s) into a eukaryotic cell,
preferably a mammalian cell, and more preferably a mammalian germ
cell. The enhancement is measured relative to the uptake in the
absence of the transfecting agent. Examples of transfecting agents
include adenovirus-transferrin-polylysine- -DNA complexes. These
complexes generally augment the uptake of DNA into the cell and
reduce its breakdown during its passage through the cytoplasm to
the nucleus of the cell. These complexes may be targeted to the
male germ cells using specific ligands which are recognized by
receptors on the cell surface of the germ cell, such as the c-kit
ligand or modifications thereof.
[0027] "Virus", as used herein, means any virus, or transfecting
fragment thereof, which may facilitate the delivery of the genetic
material into male germ cells. Examples of viruses which are
suitable for use herein are adenoviruses, adeno-associated viruses,
retroviruses such as human immune-deficiency virus, lentiviruses,
such as Moloney murine leukemia virus and the retrovirus vector
derived from Moloney virus called
vesicular-stomatitis-virus-glycoprotein (VSV-G)-Moloney murine
leukemia virus, mumps virus, and transfecting fragments of any of
these viruses, and other viral DNA segments that facilitate the
uptake of the desired DNA segment by, and release into, the
cytoplasm of germ cells and mixtures thereof. The mumps virus is
particularly suited because of its affinity for immature sperm
cells including spermatogonia. All of the above viruses may require
modification to render them non-pathogenic or less antigenic. Other
known vector systems, however, may also be utilized within the
confines of the invention.
[0028] "Genetic material", as used herein, means DNA sequences
capable of imparting novel genetic modification(s), or biologically
functional characteristic(s) to the recipient animal. The novel
genetic modification(s) or characteristic(s) may be encoded by one
or more genes or gene segments, or may be caused by removal or
mutation of one or more genes, and may additionally contain
regulatory sequences. The transfected genetic material is
preferably functional, that is it expresses a desired trait by
means of a product or by suppressing the production of another.
Examples of other mechanisms by which a gene's function may be
expressed are genomic imprinting, i.e. inactivation of one of a
pair of genes (alleles) during very early embryonic development, or
inactivation of genetic material by mutation or deletion of gene
sequences, or by expression of a dominant negative gene product,
among others.
[0029] In addition, novel genetic modification(s) may be
artificially induced mutations or variations, or natural allelic
mutations or variations of a gene(s). Mutations or variations may
be induced artificially by a number of techniques, all of which are
well known in the art, including chemical treatment, gamma
irradiation treatment, ultraviolet radiation treatment, ultraviolet
radiation, and the like. Chemicals useful for the induction of
mutations or variations include carcinogens such as ethidium
bromide and others known in the art.
[0030] DNA segments of specific sequences may also be constructed
to thereby incorporate any desired mutation or variation or to
disrupt a gene or to alter genomic DNA. Those skilled in the art
will readily appreciate that the genetic material is inheritable
and is, therefore, present in almost every cell of future
generations of the progeny, including the germ cells.
[0031] Among novel characteristics are the expression of a
previously unexpressed trait. augmentation or reduction of an
expressed trait, over expression or under expression of a trait,
ectopic expression, that is expression of a trait in tissues where
it normally would not be expressed, or the attenuation or
elimination of a previously expressed trait. Other novel
characteristics include the qualitative change of an expressed
trait, for example, to palliate or alleviate, or otherwise prevent
expression of an inheritable disorder with a multigenic basis.
[0032] The method of the invention is suitable for application to a
variety of vertebrate animals, all of which are capable of
producing gametes, i.e. sperm or ova. Thus, in accordance with the
invention novel genetic modification(s) and/or characteristic(s)
may be imparted to animals, including mammals, such as humans,
non-human primates, for example simians, marmosets, domestic
agricultural animals such as sheep, cows, pigs, horses,
particularly race horses, marine mammals, feral animals, rodents
such as mice and rats, and the like. Other animals include fowl
such as chickens, turkeys, ducks, ostriches, geese, rare and
ornamental birds, and the like. Of particular interest are
endangered species of wild animal, such rhinoceros, tigers,
cheetahs, certain species of condor, and the like.
[0033] Broadly speaking, a "transgenic" animal is one that has had
foreign DNA permanently introduced into its cells. The foreign
gene(s) which (have) been introduced into the animal's cells is
(are) called a "transgene(s)". The present invention is applicable
to the production of transgenic animals containing xenogeneic,
i.e., exogenous, transgenic genetic material, or material from a
different species, including biologically functional genetic
material, in its native, undisturbed form in which it is present in
the animal's germ cells. In other instances, the genetic material
is "allogeneic" genetic material, obtained from different strains
of the same species, for example, from animals having a "normal"
form of a gene, or a desirable allele thereof Also the gene may be
a hybrid construct consisting of promoter DNA sequences and DNA
coding sequences linked together. These sequences may be obtained
from different species or DNA sequences from the same species that
are not normally juxtaposed. The DNA construct may also contain DNA
sequences from prokaryotic organisms, such as bacteria, or
viruses.
[0034] In one preferred embodiment, the transfected germ cells of
the transgenic animal have the non-endogenous (exogenous) genetic
material integrated into their chromosomes. This is what is
referred to as a "stable transfection". This is applicable to all
vertebrate animals, including humans. Those skilled in the art will
readily appreciate that any desired traits generated as a result of
changes to the genetic material of any transgenic animal produced
by this invention are inheritable. Although the genetic material
was originally inserted solely into the germ cells of a parent
animal, it will ultimately be present in the germ cells of future
progeny and subsequent generations thereof. The genetic material is
also present in the differentiated cells, i.e. somatic cells, of
the progeny. This invention also encompasses progeny resulting from
breeding of the present transgenic animals. The transgenic animals
bred with other transgenic or non-transgenic animals of the same
species will produce some transgenic progeny, which should be
fertile. This invention, thus, provides animal line(s) which result
from breeding of the transgenic animal(s) provided herein, as well
as from breeding their fertile progeny.
[0035] "Breeding", in the context of this patent, means the union
of male and female gametes so that fertilization occurs. Such a
union may be brought about by natural mating, i.e. copulation, or
by in vitro or in vivo artificial means. Artificial means include,
but are not limited to, artificial insemination, in vitro
fertilization, cloning and embryo transfer, intracytoplasmic
spermatozoal microinjection, cloning and embryo splitting, and the
like. However, others may also be employed.
[0036] The transfection of mature male germ cells may be also
attained utilizing the present technology upon isolation of the
cells from a vertebrate, as is known in the art, and exemplified in
Example 10. The thus isolated cells may then be transfected ex vivo
(in vitro), or cryopreserved as is known in the art and exemplified
in Example 11. The actual transsection of the isolated testicular
cells may be accomplished, for example, by isolation of a
vertebrate's testes, decapsulation and teasing apart and mincing of
the seminiferous tubules. The separated cells may then be incubated
in an enzyme mixture comprising enzymes known for gently breaking
up the tissue matrix and releasing undamaged cells such as, for
example, pancreatic trypsin, collagenase type I, pancreatic DNAse
type I, as well as bovine serum albumin and a modified DMEM medium.
The cells may be incubated in the enzyme mixture for a period of
about 5 min to about 30 min, more preferably about 15 to about 20
min, at a temperature of about 33.degree. C. to about 37.degree.
C., more preferably about 36 to 37.degree. C. After washing the
cells free of the enzyme mixture, they may be placed in an
incubation medium such as DMEM, and the like, and plated on a
culture dish. Any of a number of commercially available
transfection mixtures may be admixed with the polynucleotide
encoding a desire trait or product for transfection of the cells.
The transfection mixture may then be admixed with the cells and
allowed to interact for a period of about 2 hrs to about 16 hrs,
preferably about 3 to 4 hrs, at a temperature of about 33 .degree.
C. to about 37.degree. C., preferably about 36.degree. C. to
37.degree. C., and more preferably in a constant and/or controlled
atmosphere. After this period, the cells are preferably placed at a
lower temperature of about 33 .degree. C. to about 34.degree. C.,
preferably about 30-35 .degree. C for a period of about 4 hrs to
about 20 hrs, preferably about 16 to 18 hrs. Other conditions which
do not deviate radically from the ones described may also be
utilized as an artisan would know.
[0037] The present method is applicable to the field of gene
therapy, since it permits the introduction of genetic material
encoding and regulating specific genetic traits. Thus, in the
human, for example, by treating parents it is possible to correct
many single gene disorders which otherwise might affect their
children. It is similarly possible to alter the expression of fully
inheritable disorders or those disorders having at least a
partially inherited basis, which are caused by interaction of more
than one gene, or those which are more prevalent because of the
contribution of multiple genes. This technology may also be applied
in a similar way to correct disorders in animals other than human
primates. In some instances, it may be necessary to introduce one
or more "gene(s)" into the germ cells of the animal to attain a
desired therapeutic effect, as in the case where multiple genes are
involved in the expression or suppression of a defined trait. In
the human, examples of multigenic disorders include diabetes
mellitus caused by deficient production of, or response to,
insulin, inflammatory bowel disease, certain forms of atheromatus
cardiovascular disease and hypertension, schizophrenia and some
forms of chronic depressive disorders, among others. In some cases,
one gene may encode an expressible product, whereas another gene
encodes a regulatory function, as is known in the art. Other
examples are those where homologous recombinant methods are applied
to repair point mutations or deletions in the genome, inactivation
of a gene causing pathogenesis or disease, or insertion of a gene
that is expressed in a dominant negative manner, or alterations of
regulating elements such as gene promoters, enhancers, the
untranslated tail region of a gene, or regulation of expansion of
repeated sequences of DNA which cause such diseases as Huntingdon's
chorea, Fragile-X syndrome and the like.
[0038] A specific reproductive application of the present method is
to the treatment of animals, particularly humans, with disorders of
spermatogenesis. Defective spermatogenesis or spermiogenesis
frequently has a genetic basis, that is, one or mutations in the
genome may result in failure of production of normal sperm cells.
This may happen at various stages of the development of germ cells,
and may result in male infertility or sterility. The present
invention is applicable, for example, to the insertion or
incorporation of nucleic acid sequences into a recipient's genome
and, thereby, establish spermatogenesis in the correction of
oligozoospermia or azoospermia in the treatment of infertility.
Similarly, the present methods are also applicable to males whose
subfertility or sterility is due to a motility disorder with a
genetic basis.
[0039] The present method is additionally applicable to the
generation of transgenic animals expressing agents which are of
therapeutic benefit for use in human and veterinary medicine or
well being. Examples include the production of pharmaceuticals in
domestic cows' milk, such as factors which enhance blood clotting
for patients with types of haemophilia, or hormonal agents such as
insulin and other peptide hormones.
[0040] The present method is further applicable to the generation
of transgenic animals of a suitable anatomical and physiological
phenotype for human xenograft transplantation. Transgenic
technology permits the generation of animals which are
immune-compatible with a human recipient. Appropriate organs, for
example, may be removed from such animals to allow the
transplantation of, for example, the heart, lung and kidney.
[0041] In addition, germ cells transfected in accordance with this
invention may be extracted from the transgenic animal, and stored
under conditions effective for later use, as is known in the art.
Storage conditions include the use of cryopreservation using
programmed freezing methods and/or the use of cryoprotectants, and
the use of storage in substances such as liquid nitrogen. The germ
cells may be obtained in the form of a male animal's semen, or
separated spermatozoa, or immature spermatocytes, or whole biopsies
of testicular tissue containing the primitive germ cells. Such
storage techniques are particularly beneficial to young adult
humans or children, undergoing oncological treatments for such
diseases such as leukemia or Hodgkin's lymphoma. These treatments
frequently irreversibly damage the testicle and, thus, render it
unable to recommence spermatogenesis after therapy by, for example,
irradiation or chemotherapy. The storage of germ cells and
subsequent testicular transfer allows the restoration of fertility.
In such circumstances, the transfer and manipulation of germ cells
as taught in this invention are accomplished, but transfection is
generally not relevant or needed.
[0042] In species other than humans, the present techniques are
valuable for transport of gametes as frozen germ cells. Such
transport will facilitate the establishment of various valued
livestock or fowl, at a remote distance from the donor animal. This
approach is also applicable to the preservation of endangered
species across the globe.
[0043] The invention will now be described in greater detail by
reference to the following non-limiting examples. The pertinent
portions of the contents of all references, and published patent
applications cited throughout this patent necessary for enablement
purposes are hereby incorporated by reference.
EXAMPLES
Transfection of Male Germ Cells in Vivo
[0044] In Vivo Adenovirus-Enhanced Transferrin-Polylysine-Mediated
Delivery of Green Lantern Reporter Gene Delivery System to
Testicular Cells
[0045] The adenovirus enhanced transferrin-polylysine-mediated gene
delivery system has been described and patented by Curiel al.
(Curiel D. T., et al. Adenovirus enhancement of
transferrin-polylysine-mediated gene delivery, PNAS USA 88:
8850-8854 (1991). The delivery of DNA depends upon endocytosis
mediated by the transferrin receptor (Wagner et al.,
Transferrin-polycation conjugates as carriers for DNA uptake into
cells, PNAS (USA) 87: 3410-3414 (1990). In addition this method
relies on the capacity of adenoviruses to disrupt cell vesicles,
such as endosomes and release the contents entrapped therein. This
system can enhance the gene delivery to mammalian cells by as much
as 2,000 fold over other methods.
[0046] The gene delivery system employed for the in vivo
experiments was prepared as shown in examples below.
Example 1
[0047] Preparation of Transferrin-Poly-L-Lysine Complexes
[0048] Human transferrin was conjugated to poly (L-lysine) using
EDC (1-ethyl-3-(3-dimethyl aminopropyl carbodiimide hydrochloride)
(Pierce), according to the method of Gabarek and Gergely (Gabarek
& Gergely, Zero-length cross-linking procedure with the use of
active esters, Analyt. Biochem 185 : 131 (1990)). In this reaction,
EDC reacts with a carboxyl group of human transferrin to form an
amine-reactive intermediate. The activated protein was allowed to
react with the poly (L-lysine) moiety for 2 hrs at room
temperature, and the reaction was quenched by adding hydroxylamine
to a final concentration of 10 mM. The conjugate was purified by
gel filtration, and stored at -20.degree. C.
Example 2
[0049] Preparation of DNA for In Vivo Trasfection
[0050] The Green Lantern-1 vector (Life Technologies, Gibco BRL,
Gaithersberg, Md.) is a reporter construct used for monitoring gene
transfection in mammalian cells. It consists of the gene encoding
the Green Fluorescent Protein (GFP) driven by the cytomegalovirus
(CMV) immediate early promoter. Downstream of the gene is a SV40
polyadenylation signal. Cells transfected with Green Lantern-1
fluoresce with a bright green light when illuminated with blue
light. The excitation peak is 490 nm.
Example 3
[0051] Preparation of Adenoviral Particles
[0052] Adenovirus dI312, a replication-incompetent strain deleted
in the Ela region, was propagated in the Ela trans-complementing
cell line 293 as described by Jones and Shenk (Jones and Shenk,
PNAS USA (1979) 79: 3665-3669). A large scale preparation of the
virus was made using the method of Mittereder and Trapnell
(Mittereder et al., "Evaluation of the concentration and
bioactivity of adenovirus vectors for gene therapy", J. Urology,
70: 7498-7509 (1996)). The virion concentration was determined by
UV spectroscopy, 1 absorbance unit being equivalent to 10 viral
particles/ml. The purified virus was stored at -70.degree. C.
Example 4
[0053] Formation of Transferrin-Poly-L Lysine-DNA-Viral
Complexes
[0054] 6 .mu.g transferrin-polylysine complex from Example 1 were
mixed in 7.3.times.10.sup.7 adenovirus d1312 particles prepared as
in Example 3, and then mixed with 5 ug of the Green Lantern DNA
construct of Example 2, and allowed to stand at room temperature
for 1 hour. About 100 ul of the mixture were drawn up into a
micropipette, drawn on a pipette puller, and slightly bent on a
microforge. The filled micropipette was then attached to a picopump
(Eppendorf), and the DNA complexes were delivered under continuous
pressure, in vivo to mice as described in Example 6.
[0055] Controls were run following the same procedure, but omitting
the transferrin-poly-lysine-DNA-viral complexes from the
administered mixture.
Example 5
[0056] Comparison of Adenovirus-Enhanced Transferrin-Polylysine
& Lipofectin Mediated Transfection Efficiency
[0057] The conjugated adenovirus particle complexed with DNA were
tested on CHO cells in vitro prior to in vivo testing. For these
experiments a luciferase reporter gene was used due to the ease of
quantifying luciferase activity. The expression construct consists
of a reporter gene encoding luciferase, is driven by the CMV
promoter (Invitrogen, Carlsbad, Calif. 92008). CHO cells were grown
in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf
serum. For gene transfer experiments CHO cells were seeded into 6
cm tissue culture plates and grown to about 50% confluency
(5.times.10.sup.5 cells). Prior to transfection the medium was
aspirated and replaced with serum free DMEM. Cells were either
transfected with transferrin-polylysine-DNA complexes or with
lipofectin DNA aggregates. For the transferrin-polylysine mediated
DNA transfer, the DNA-adenovirus complexes were added to the cells
at a concentration of 0.05-3.2.times.10.sup.4 adenovirus particles
per cell. Plates were returned to the 5% CO.sub.2 incubator for 1
hour at 37.degree. C. After 1 hour 3 ml of complete media was added
to the wells and the cells were allowed to incubate for 48 hours
before harvesting. The cells were removed from the plate, counted
and then lysed for measurement of luciferase activity.
[0058] For cells transfected by lipofectin, 1 .mu.g of
CMV-luciferase DNA was incubated with 7 .mu.l of Lipofectin (Life
Technologies). The DNA-lipofectin aggregates were added to the CHO
cells and allowed to incubate at 37.degree. C. at 5% CO.sub.2 for 4
hours. Three mls of complete medium was added then to the cells and
they were allowed to incubate for 48 hours. The cells were
harvested, counted and lysed for luciferase activity. The
luciferase activity was measured by a luminometer. The results
obtained are shown in Table 1.
[0059] The data included in Table 1 below show that the
adenovirus-enhanced transferrin-polylysine gene delivery system is
1,808 fold more efficient than lipofection for transfection of CHO
cells.
1TABLE 1 Comparison of Lipofection & Adenovirus Enhanced
Transferrin-polylysine Transfection of CHO Cells Luciferase Sample
Treatment Activity (RLU) 1 1 .times. 10.sup.7 particles + 6 ug
CMV-Luc 486 2 2.5 .times. 10.sup.7 particles + 6 ug CMV-Luc 1,201 3
5.0 .times. 10.sup.7 particles + 6 ug CMV-luc 11,119 4 1 .times.
10.sup.7 particles + 6 ug CMV-Luc 2,003,503 5 Lipofection 1,108 6
Unmanipulated cells 155
Example 6
[0060] In Vivo Delivery of DNA to Animal's Germ Cells via
Tranferrin-L-lysine-DNA-Viral Complexes
[0061] The GFP DNA-transferrin-polylysine viral complexes, prepared
as described in Example 4 above, were delivered into the
seminiferous tubules of three (3)-week-old B6D2F1 male mice. The
DNA delivery by transferrin receptor-mediated endocytosis is
described by Schmit et al. and Wagner et al. (Schmit et al., Cell
4: 41-51 (1986); Wagner, E., et al. PNAS (1990), (USA) 81:
3410-3414 (1990)). In addition, this delivery system relies on the
capacity of adenoviruses to disrupt cell vesicles, such as
endosomes and release the contents entrapped therein. The
transfection efficiency of this system is almost 2,000 fold higher
than lipofection.
[0062] The male mice were anesthetized with 2% Avertin (100%
Avertin comprises 10 g 2,2,2-tribromoethanol (Aldrich) and 10 ml
t-amyl alcohol (Sigma), and a small incision made in their skin and
body wall, on the ventral side of the body at the level of the hind
leg. The animal's testis was pulled out through the opening by
grasping at the testis fat pad with forceps, and the vas efferens
tubules exposed and supported by a glass syringe. The GFP
DNA-transferrin-polylysine viral complexes were injected into a
single vasa efferentia using a glass micropipette attached to a
hand held glass syringe or a pressurized automatic pipettor
(Eppendorf), and Trypan blue added to visualize the entry of the
mixture into the seminiferous tubules. The testes were then placed
back in the body cavity, the body wall was sutured, the skin closed
with wound clips, and the animal allowed to recover on a warm
pad.
Example 7
[0063] Detection of DNA and Transcribed Message
[0064] Nine (9) days after delivery of the genetic material to the
animals' testis, two of the animals were sacrificed, their testes
removed, cut in half, and frozen in liquid nitrogen. The DNA from
one half of the tissues, and the RNA from the other half of the
tissues were extracted and analyzed.
[0065] (a) Detection of DNA
[0066] The presence of GFP DNA in the extracts was tested 9 days
after administration of the transfection mixture using the
polymerase chain reaction, and GFP specific oligonucleotides. GFP
DNA was present in the testes of the animals that had received the
DNA complexes, but was absent from sham operated animals.
[0067] (b) Detection of RNA
[0068] The presence of GFP mRNA was assayed in the testes of
experimental animals as follows. RNA was extracted from injected,
and non-injected testes, and the presence of the GFP messages was
detected using reverse transcriptase PCR (RTPCR) with GFP specific
primers. The GFP message was present in the injected testes, but
not in the control testes. Thus, the DNA detected above by PCR
analysis is, in fact, episomal GFP DNA, or GFP DNA which has
integrated into the chromosomes of the animal. The transfected gene
was being expressed.
Example 8
[0069] Expression of Non-Endogenous DNA
[0070] Two males, one having received an injection with the GFP
transfection mixture and a control to whom only surgery was
administered, were sacrificed 4 days after injection, and their
testes excised, and fixed in 4% paraformaldehyde for 18 hours at
4.degree. C. The fixed testis was then placed in 30% sucrose in PBS
with 2 mM MgCl.sub.2 for 18 hours at 4.degree. C., embedded in OCT
frozen on dry ice, and sectioned. When the testes of both animals
were examined with a confocal microscope with fluorescent light at
a wavelength of 488 nM. bright fluorescence was detected in the
tubules of the GFP-injected mice, but not in the testes of the
controls. Many cells within the seminferous tubules of the
GFP-injected mouse showed bright fluorescence, which evidences that
they were expressing Fluorescent Green Protein.
Example 9
[0071] Generation of Offspring from Normal Matings
[0072] GFP transfected males were mated with normal females. The
females were allowed to complete gestation, and the pups to be
born. The pups (F1 offspring or progeny) were screened for the
presence of the novel genetic material(s).
Example 10
[0073] In Vitro Transfection of Testicular Cells in Vitro
[0074] Cells were isolated from the testes of three 10-day-old
mice. The testes were decapsulated and the seminiferous tubules
were teased apart and minced with sterile needles. The cells were
incubated in enzyme mixture for 20 minutes at 37.degree. C. The
enzyme mixture was made up of 10 mg bovine serum albumin (embryo
tested), 50 mg bovine pancreatic trypsin type III, Clostridium
collagenase type I, 1 mg bovine pancreatic DNAse type I in 10 mls
of modified HTF medium (Irvine Scientific, Irvine, Calif.). The
enzymes were obtained from Sigma Company (St. Louis, Mo. 63178).
After digestion, the cells were washed twice by centrifugation at
500.times.g with HTF medium and resuspended in 250 .mu.l HTF
medium. The cells were counted, and 0.5.times.10.sup.6 cells were
plated in a 60 mm culture dish in a total volume of 5 ml DMEM
(Gibco-BRL, Life Technologies, Gaithesburg, Md. 20884). A
transfection mixture was prepared by mixing 5 .mu.g Green Lantern
DNA (Gibco-BRL, Life Technologies, Gaithesburg, Md. 20884) with 20
.mu.l Superfect (Quagen, Santa Clarita, Calif. 91355) and 150,
.mu.l DMEM. The transfection mix was added to the cells and they
were allowed to incubate for 3 hours at 37.degree. C., 5% CO.sub.2
The cells were transferred to a 33.degree. C. incubator and
incubated overnight.
[0075] The following morning the cells were assessed for
transfection efficiency by counting the number of fluorescent
cells. In this experiment the transfection efficiency was 90%
(Figure not shown). The testicular cells transfected with Green
Lantern viewed with Nomaski optics .times.20 show the same cells
viewed with FITC. Nearly all the cells were fluorescent, which is
confirmation of their successful transfection.
[0076] The cells were injected into the testis via the vasa
efferentia using a micropipette. 3 .times.10.sup.5 cells in a total
volume of 50 .mu.l were used for the injection. The cells were
mixed with Trypan blue prior to the injection. Three adult mice
were injected with transfected cells. The Balb/cByJ recipient mice
had been irradiated 6 weeks prior to the injection with 800 Rads of
gamma irradiation. One mouse became sick and was sacrificed 48
hours after the injection. The testes from this mouse were
dissected, fixed and processed for histology.
[0077] The two remaining males were bred with normal females as
shown. After 4 months pups were born. Litters are currently being
screened for the integration of the transgene.
Example 11
[0078] Preparation of a Cell Suspension from Testicular Tissue for
Cryopreservation
[0079] A cell suspension was prepared from mice of different ages
as described below.
2 Group I: 7-10 day olds Group II: 15-17 day olds Group III: 24-26
day olds
[0080] The mice's testes were dissected, placed in phosphate
buffered saline (PBS) decapsulated, and the seminiferous tubules
were teased apart. Seminiferous tubules from groups I and II were
transferred to HEPES buffered culture medium (D-MEM) (Gibco-BRL,
Life Technologies, Gaithesburg, Md. 20884) containing 1mg/ml Bovine
serum albumin (BSA) (Sigma, St. Louis, Mo. 63178) and Collagenase
Type I (Sigma) for the removal of interstitial cells. After a 10
minute incubation at 33.degree. C., the tubules were lifted into
fresh culture medium. This enzymatic digestion was not carried out
on the testes from group I because of their fragility.
[0081] The tubules from group II and III mice or the whole tissue
from group I mice were transferred to a Petri dish with culture
medium and were cut into 0.1-1 mm pieces using a sterile scalpel
and needle. The minced tissue was centrifuged at 500.times.g for 5
minutes and the pellet was resuspended in 1ml of enzyme mix. The
enzyme mix was made up in D-DMEM with HEPES (GibcoBRL) and
consisted of 1mg/ml bovine serum albumin (BSA) (Sigma, embryo
tested), 1 mg/ml collagenase I (Sigma) and 5 mg/ml bovine
pancreatic trypsin (Sigma) and 0.1 mg/ml deoxyribonuclease I
(DN-EP, Sigma). The tubules were incubated in enzyme mix for 30
minutes at 33.degree. C. After the incubation, 1 ml of medium was
added to the mix and the cells were centrifuged at 500.times.g for
5 min. The cells were washed twice in medium by centrifugation and
resuspension. After the final wash the cell pellet was resuspended
in 250 .mu.l of culture medium and counted.
Example 12
[0082] Cryopreservation of Methods for Testicular Cells
[0083] (a) Propanediol (PROH)-sucrose Method Testicular cells from
a total of 31 mice (age 8-12 weeks) were cryopreserved using 6
different freezing and thawing protocols. In addition to freezing
cell supsensions, pieces of testicular tissue were frozen (see
freezing method above). The cell suspension was prepared as
described above.
[0084] The cell suspension was incubated in a buffer stock solution
consisting of 80% phosphate buffered saline (PBS) and 20% human
serum (SPR, Helsinki, Finland) for 5 minutes. The cells were then
incubated in 1.5M PROH for 10 minutes, pelleted by centrifugation
and resuspended in 1.5M PROH with 0.1M sucrose. The cell suspension
was loaded into straws (0.25 .mu.m, Paillette, L'Aigle, France) or
1 ml cryogenic vials (Nunc cryotube). Samples were frozen in a
controlled temperature freezing machine (Planer Kryo, Series III,
Planer Biomed, Sunbury on Thames, UK). The samples were cooled at a
rate of 2.degree. C./min to -8.degree. C., and seeded manually
using forceps cooled in liquid nitrogen. After 10 min the samples
were cooled at 0.3.degree. C./min to -30.degree. C. after which
they were cooled at a rate of -50.degree. C./min to -150.degree. C.
Samples were then stored in liquid nitrogen at -196.degree. C.
[0085] The samples were removed from liquid nitrogen and kept at
room temperature for 2 min. The samples were incubated in 1M PROH
+0.1M sucrose for 5 min, followed by an incubation in 0.5M
PROH+0.1M sucrose for 5 min and then in 0.1M sucrose for 10 min.
The cell suspension was placed in buffer stock.
[0086] (b) Glycerol Yolk Buffer Method
[0087] The cell suspension was pipetted into a vial and the yolk
buffer freezing medium (Irvine Scientific, Santa Ana, Calif.) was
added drop by drop to make up approximately 50% of the total
volume. The samples were cooled in a controlled freezer at an
initial cooling rate of 0.5.degree. C./min to a temperature of
1.5.degree. C. The samples were then cooled at 10.degree.C./min
until they reached a temperature of -80.degree. C. On reaching this
temperature the samples were placed in liquid nitrogen for
storage.
[0088] Samples were removed from liquid nitrogen and thawed at room
temperature. The suspension was centrifuged and the pelleted cells
were resuspended in PBS.
[0089] (c) DMSO Method
[0090] Cells were pipetted into a cryogenic vial containing 60%
medium 199 with Earle's salts (Gibco, Gaithesburg,Md.), 20% human
AB serum. 20% DMSO was added to the cells drop by drop to make up
50% of the total volume. The cells were cooled at a rate of
4.degree.C./min to 0C. and then at 1.degree. C./min to -80.degree.
C., then at 10.degree.C./min to -100.degree.C. and finally at
20.degree.C./min to-160.degree. C. The samples were then stored in
liquid nitrogen.
[0091] Samples were removed from liquid nitrogen and thawed at room
temperature. The suspension was centrifuged and the pelleted cells
were resuspended in PBS.
[0092] (d) DMSO-Heparin Method
[0093] Cells were pipetted into a cryogenic vial. A solution
containing 45% 5000 U/ml heparin (Tovens medicinske fabrik,
Ballerup, Denmark), 15% DMSO and 40% albumin (SPR) in PBS was added
drop by drop to make up 50% of the total volume. The freezing and
thawing programme was the same as that used for the glycerol yolk
buffer method.
[0094] (e) Quick DMSO method
[0095] Cells were pipetted into a cryogenic vial and a freezing
solution containing 90% fetal calf serum and 10% DMSO was added at
room temperature to make up 90% of the total volume. The samples
were placed in a -70.degree. C. freezer (Revco Scientific Corp.,
Asheville, N.C.) for 24 hours. The samples were then stored in
liquid nitrogen. The thawing procedure was that same as that used
for the Glycerol yolk method.
[0096] (f) Quick Glycerol Method
[0097] The cells were pipetted into a cryogenic vial and a freezing
solution containing 70% DMEM, 20% fetal calf serum and 10% filtered
glycerol was added to the cells to make up 90% of the total volume.
The resuspension was incubated at 37.degree. C. for 10 min. The
samples were placed in a -70.degree. C. freezer for 24 hours after
which they were stored in liquid nitrogen.
[0098] The thawing procedure was the same as that described for the
Glycerol yolk method.
[0099] (g) Freezing Testicular Tissue
[0100] The method used for freezing whole testicular tissue was the
same as the method we described previously for freezing ovarian
tissue (Hovatta, et al., Human Reprod. 11:1268-1272 (1996). The
testicles of 6 mice were decapsulated in culture medium (D-MEM) and
cut into 0.3-1.0 mm pieces. The tissue pieces were placed in medium
containing 1.5M PROH in PBS with 20% serum for 10 min. at room
temperature. They were transferred to cyrogenic vials and cooled at
2.degree. C./min to -8.degree. C. The vials were seeded manually
with forceps dipped in liquid nitrogen. After 10 min the cooling
was continued at a rate of 0.3.degree. C./min to -30.degree. C. and
then at a rate of 50.degree. C./min to -150.degree. C. When the
samples reached this temperature they were transferred to liquid
nitrogen.
[0101] The vials were removed from the liquid nitrogen and allowed
to come to room temperature for 2 min. They were then placed in a
water bath at 30.degree. C. until they had thawed. The tissue
pieces were transferred to a Petri dish containing 1.0M PROH, 0.1M
sucrose and 20% serum in PBS for 5 min. They were then transferred
to a solution containing 0.5M PROH, 0.1M sucrose and 20% serum in
PBS for 5 min and then to a solution containing 0.1M sucrose with
20% serum in PBS for 10 min. The cells were kept in culture
medium.
[0102] The results obtained from the above experimental procedures
are summarized in Table 2 below.
3TABLE 2 Comparison of Results by Different Methods Method Cell
Viability after Freeze/Thaw Propanediol-Sucrose 63% Glyerol-Yolk
Buffer 56% DMSO 50% Quick DMSO 33% DMSO-Heparin 23% Quick-Glycerol
13%
[0103] From Table 2 above, it may be seen that the testicular cells
that had been frozen using the propanediol-sucrose method had the
highest percentage of viable cells upon thawing than cells frozen
using the other methods. The propanediol-sucrose freezing method
was significantly less damaging to testicular cells than the DMSO
method used by Avarbock et al., 1996 for freezing testicular cells
prior to transfer. The propanediol-sucrose method was also shown to
be good for freezing human ovarian tissue as described by Hovatta
et al. (Hovatta et al., Human Reprod. 11: 1268-1272 (1996a), the
relevant part of which is incorporated herein by reference, and
pieces of testicular tissue.
[0104] The testicular spermatozoa from a human biopsy were
frozen-thawed using the glycerol-yolk buffer method, and then used
for intracytoplasmic injection of eggs (ICSI). A successful
pregnancy resulted (Hovatta, O. et al., Pregnancy resulting from
intracytoplasmic injection of spermatozoa from a frozen thawed
testicular biopsy, Human Reprod. 11: 2472-2473 (1996b).
* * * * *