U.S. patent application number 10/155904 was filed with the patent office on 2003-04-10 for method of cloning animals.
This patent application is currently assigned to Infigen, Inc.. Invention is credited to Betthauser, Jeffrey M., Bishop, Michael D., Jurgella, Gail L., Pace, Marvin M., Strelchenko, Nikolai S..
Application Number | 20030070186 10/155904 |
Document ID | / |
Family ID | 26754032 |
Filed Date | 2003-04-10 |
United States Patent
Application |
20030070186 |
Kind Code |
A1 |
Strelchenko, Nikolai S. ; et
al. |
April 10, 2003 |
Method of cloning animals
Abstract
The present invention relates to cloning technologies. The
invention relates in part to immortalized and totipotent cells
useful for cloning animals, the embryos produced from these cells
using nuclear transfer techniques, animals that arise from these
cells and embryos, and materials, methods, and processes for
establishing such cells, embryos, and animals.
Inventors: |
Strelchenko, Nikolai S.;
(DeForest, WI) ; Betthauser, Jeffrey M.; (Windsor,
WI) ; Jurgella, Gail L.; (Madison, WI) ; Pace,
Marvin M.; (DeForest, WI) ; Bishop, Michael D.;
(Rio, WI) |
Correspondence
Address: |
Michael A. Whittaker
FOLEY & LARDNER
P.O. Box 80278
San Diego
CA
92138-0278
US
|
Assignee: |
Infigen, Inc.
1825 Infinity Drive
DeForest
WI
53532
|
Family ID: |
26754032 |
Appl. No.: |
10/155904 |
Filed: |
May 22, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10155904 |
May 22, 2002 |
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09354276 |
Jul 15, 1999 |
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6395958 |
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09354276 |
Jul 15, 1999 |
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09239922 |
Jan 28, 1999 |
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6011197 |
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09354276 |
Jul 15, 1999 |
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08812851 |
Mar 6, 1997 |
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08812851 |
Mar 6, 1997 |
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PCT/US98/04345 |
Mar 5, 1998 |
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PCT/US98/04345 |
Mar 5, 1998 |
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08812031 |
Mar 6, 1997 |
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60073019 |
Jan 29, 1998 |
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Current U.S.
Class: |
800/21 ; 435/325;
800/15 |
Current CPC
Class: |
C12N 5/0603 20130101;
C12N 2510/04 20130101; C12N 2502/13 20130101; C12N 15/8771
20130101; C12N 5/0611 20130101 |
Class at
Publication: |
800/21 ; 435/325;
800/15 |
International
Class: |
A01K 067/027; C12N
005/06 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 5, 1998 |
WO |
PCT/US98/04345 |
Claims
What is claimed is:
1. A totipotent non-ovine mammalian cell, wherein said cell is
cultured.
2. A totipotent nonovine mammalian cell, wherein said cell is
cultured, prepared by a process comprising: (a) isolating one or
more precursor cells; and (b) introducing said one or more
precursor cells to a stimulus that converts said one or more
precursor cells into said totipotent mammalian cell.
3. The totipotent nonovine mammalian cell of claims 1 and 2,
wherein said totipotent nonovine mammalian cell is a non-embryonic
cell.
4. The totipotent nonovine mammalian cell of claims 1 and 2,
wherein said totipotent non-ovine mammalian cell is a bovine
cell.
5. The totipotent nonovine mammalian cell of claim 2, wherein said
stimulus comprises a receptor ligand cocktail.
6. The nonembryonic cell of claims 1 and 2, wherein said
nonembryonic cell arises from the group consisting of a primordial
germ cell, an amniotic cell, a fetal fibroblast cell, an ovarian
follicular cell, a cumulus cell, and a hepatic cell.
7. The totipotent nonovine mammalian cell of claims 1 and 2,
wherein said totipotent nonovine mammalian cell comprises modified
nuclear DNA.
8. The totipotent nonovine mammalian cell of claims 1 and 2,
wherein said totipotent nonovine mammalian cell is subject to
manipulation.
9. The totipotent nonovine mammalian cell of claim 2, comprising
the step of co-culturing said precursor cells with feeder
cells.
10. A method for preparing a totipotent nonovine mammalian cell,
wherein said cell is cultured, the method comprising: (a) isolating
one or more precursor cells; and (b) introducing said one or more
precursor cells to a stimulus that converts said one or more
precursor cells into said totipotent nonovine mammalian cell.
11. A totipotent mammalian cell, wherein said cell is cultured and
wherein said cell is not serum starved.
12. A totipotent mammalian cell, wherein said cell is cultured and
wherein said cell is not serum starved, the cell prepared by a
process comprising: (a) isolating one or more precursor cells; and
(b) introducing said one or more precursor cells to a stimulus that
converts said one or more precursor cells into said totipotent
mammalian cell.
13. The totipotent mammalian cell of claims 11 and 12, wherein said
totipotent mammalian cell is a non-embryonic cell.
14. The totipotent mammalian cell of claims 11 and 12, wherein said
totipotent mammalian cell is a bovine cell.
15. The totipotent mammalian cell of claim 12, wherein said
stimulus comprises a receptor ligand cocktail.
16. The nonembryonic cell of claims 11 and 12, wherein said
totipotent mammalian cell arises from the group consisting of a
primordial germ cell, an amniotic cell, a fetal fibroblast cell, an
ovarian follicular cell, a cumulus cell, and a hepatic cell.
17. The totipotent mammalian cell of claims 11 and 12, wherein said
totipotent mammalian cell comprises modified nuclear DNA.
18. The totipotent mammalian cell of claims 11 and 12, wherein said
totipotent mammalian cell is subject to manipulation.
19. The totipotent mammalian cell of claim 12, comprising the step
of co-culturing said precursor cells with feeder cells.
20. A method for preparing a totipotent mammalian cell, wherein
said cell is cultured and wherein said cell is not serum starved,
the method comprising: (a) isolating one or more precursor cells;
and (b) introducing said one or more precursor cells to a stimulus
that converts said one or more precursor cells into said totipotent
mammalian cell.
21. A cloned non-ovine mammalian embryo, wherein said embryo is
totipotent, and wherein said embryo arises from a totipotent
non-ovine mammalian cell, wherein said cell is cultured.
22. A cloned non-ovine mammalian embryo, wherein said embryo is
totipotent, prepared by a process comprising the step of nuclear
transfer between (a) a totipotent non-ovine mammalian cell, wherein
said cell is cultured; and (b) an oocyte, wherein said oocyte is at
a stage allowing formation of said embryo.
23. The cloned nonovine mammalian embryo of any one of claims 21
and 22, wherein said embryo is a bovine embryo.
24. The cloned nonovine mammalian embryo of any one of claims 21
and 22, wherein one or more cells of said embryo comprise modified
nuclear DNA.
25. The cloned nonovine mammalian embryo of claim 22, Wherein said
totipotent nonovine mammalian cell and said oocyte originate from
different species.
26. The cloned nonovine mammalian embryo of claim 22, wherein said
nuclear transfer comprises the step of activation of said
totipotent non-ovine mammalian cell and said oocyte.
27. The cloned nonovine mammalian embryo of any one of claims 21
and 22, wherein said embryo is subject to manipulation.
28. The cloned nonovine mammalian embryo of claim 27, wherein said
manipulation comprises the step of implanting said embryo into the
uterus of a suitable maternal host.
29. The cloned nonovine mammalian embryo of claim 27, wherein said
manipulation comprises the steps of: (a) separating said embryo
into one or more individual cells; and (b) performing at least one
subsequent nuclear transfer between (i) an individual cell of (a);
and (ii) an oocyte.
30. A method for preparing a cloned non-ovine mammalian embryo,
comprising the step of a nuclear transfer between: (a) a totipotent
non-ovine mammalian cell, wherein said cell is cultured; and (b) an
oocyte, wherein said oocyte is at a stage allowing formation of
said embryo.
31. A cloned non-ovine mammalian animal arising from an embryo of
anyone of claims 21, 22, 23, 24, 25, 26, 27, 28, and 29.
32. A cloned non-ovine mammalian animal prepared by a process
comprising: (a) preparation of a cloned nonovine mammalian embryo
of any one of claims 21, 22, 23, 24, 25, 26, 27, 28, and 29; and
(b) manipulation of said cloned nonovine mammalian embryo such that
it develops into an animal.
33. The cloned non-ovine mammalian animal of claim 32, wherein said
non-ovine mammalian animal is a bovine animal.
34. The cloned non-ovine mammalian animal of any one of claims 31
and 32, wherein one or more cells of said animal comprise modified
nuclear DNA.
35. A method of using a cloned non-ovine mammalian animal,
comprising the step of isolating at least one component from said
non-ovine mammalian animal, wherein said component is selected from
the group consisting of fluid, cell, tissue, and organ.
36. The method of claim 35, wherein said fluid is semen.
37. A method for preparing a cloned non-ovine mammalian animal,
comprising the steps of: (a) preparation of a cloned mammalian
embryo by the method of claim 30; and (b) manipulation of said
cloned mammalian embryo such that it develops into an animal.
38. A cloned mammalian embryo, wherein said embryo is totipotent,
and wherein said embryo arises from a totipotent mammalian cell,
wherein said cell is cultured and wherein said cell is not serum
starved.
39. A cloned mammalian embryo, wherein said embryo is totipotent,
prepared by a process comprising the step of nuclear transfer
between (a) a totipotent mammalian cell, wherein said cell is
cultured and wherein said cell is not serum starved; and (b) an
oocyte, wherein said oocyte is at a stage allowing formation of
said embryo.
40. The cloned mammalian embryo of any one of claims 38 and 39,
wherein said mammalian embryo is an ungulate embryo.
41. The cloned mammalian embryo of claim 40, wherein said ungulate
embryo is a bovine embryo.
42. The cloned mammalian embryo of any one of claims 38 and 39,
wherein one or more cells of said embryo comprise modified nuclear
DNA.
43. The cloned mammalian embryo of claim 39, wherein said
totipotent mammalian cell originates from one specie of ungulate
and wherein said oocyte originates from another specie of
ungulate.
44. The cloned mammalian embryo of claim 39, wherein said nuclear
transfer comprises the step of activation of said totipotent
mammalian cell and said oocyte.
45. The cloned mammalian embryo of any one of claims 38 and 39,
wherein said embryo is subject to manipulation.
46. The cloned mammalian embryo of claim 45, wherein said
manipulation comprises the step of implanting said embryo into the
uterus of a suitable maternal host.
47. The cloned mammalian embryo of claim 45, wherein said
manipulation comprises: (a) separating said embryo into one or more
individual cells; and (b) performing at least one subsequent
nuclear transfer between (i) an individual cell of (a); and (ii) an
oocyte.
48. A method for preparing a cloned mammalian embryo, comprising
the step of a nuclear transfer between: (a) a totipotent mammalian
cell, wherein said cell is cultured and wherein said cell is not
serum starved; and (b) an oocyte, wherein said oocyte is at a stage
allowing formation of said embryo.
49. A cloned mammalian animal arising from an embryo of anyone of
claims 38, 39, 40, 41, 42, 43, 44, 45, 46, and 47.
50. A cloned mammalian animal prepared by a process comprising the
steps of: (a) preparation of a cloned mammalian embryo of any one
of claims 38, 39, 40, 41, 42, 43, 44, 45, 46, and 47; and (b)
manipulation of said cloned mammalian embryo such that it develops
into an animal.
51. The cloned mammalian animal of any one of claims 49 and 50,
wherein said mammalian animal is an ungulate animal.
52. The cloned mammalian animal of claim 51, wherein said ungulate
animal is a bovine animal.
53. The cloned mammalian animal of any one of claims 49 and 50,
wherein one or more cells of said animal comprise modified nuclear
DNA.
54. A method of using a cloned mammalian animal, comprising the
step of isolating at least one component from said mammalian
animal, wherein said component is selected from the group
consisting of fluid, cell, tissue, and organ.
55. The method of claim 54, wherein said fluid is semen.
56. A method for preparing a cloned mammalian animal, comprising
the steps of: (a) preparation of a cloned mammalian embryo by the
method of claim 48; and (b) manipulation of said cloned mammalian
embryo such that it developes into an animal.
Description
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 60/073,019, filed Jan. 29, 1998, entitled
"Cloning of Biological Organisms from Immortalized Totipotent
Cells" (pending); U.S. application Ser. No. 08/812,851, filed Mar.
6, 1997, entitled "Method of Cloning Animals" (pending); P.C.T
application Ser. No. PCT/US 98/04345, filed Mar. 5, 1998, entitled
"Method of Cloning Animals" (pending); and U.S. application Ser.
No. 08/812,031, filed Mar. 6, 1997, entitled "Method of Cloning
Bovines" (pending), each of which is hereby incorporated by
reference in its entirety including any drawings, and from each of
which priority is claimed.
FIELD OF THE INVENTION
[0002] The invention relates to the cloning of animals.
BACKGROUND OF THE INVENTION
[0003] The following discussion of the background of the invention
is merely provided to aid the reader in understanding the invention
and is not admitted to describe or constitute prior art to the
present invention.
[0004] Researchers have been developing methods for cloning
mammalian animals over the past two decades. These reported methods
typically include the steps of (1) isolating a cell, most often an
embryonic cell; (2) inserting the cell or nucleus isolated from the
cell into an enucleated oocyte (e.g., the oocyte's nucleus was
previously extracted), and (3) allowing the embryo to mature in
vivo.
[0005] The first successful nuclear transfer experiment using
mammalian cells was reported in 1983, where the pronuclei isolated
from a murine (mouse) zygote were inserted into an enucleated
oocyte and resulted in like offspring(s). McGrath & Solter,
1983, Science 220:1300-1302. Subsequently, others described the
production of chimeric murine embryos (e.g., embryos that contain a
subset of cells having significantly different nuclear DNA from
other cells in the embryo) using murine primordial germ cells
(PGC). These cells are and can give rise to pluripotent cells
(e.g., cells that can differentiate into other types of cells but
do not differentiate into a grown animal). Matsui et al., 1992,
Cell 70:841-847 and Resnick et al., 1992, Nature 359:550; Kato et
al., 1994, Journal of Reproduction and Fertility Abstract Series,
Society For the Study of Fertility, Annual Conference, Southampton,
13:38.
[0006] Some publications related to murine pluripotent cells stress
the importance of steel factor for converting precursor cells into
pluripotent cells. U.S. Pat. Nos. 5,453,357 and 5,670,372, entitled
"Pluripotent Embryonic Stem Cells and Methods of Making Same,"
issued to Hogan. These same publications indicate that murine
pluripotent cells exhibit strong, uniform alkaline phosphatase
staining.
[0007] Although murine animals were never clearly cloned from
nuclear transfer techniques using embryonic cells, some progress
was reported in the field of cloning ovine (sheep) animals. One of
the first successful nuclear transfer experiments utilizing ovine
embryonic cells as nuclear donors was reported in 1986. Willadsen,
1986, Nature 320:63-65. A decade later, others reported that
additional lambs were cloned from ovine embryonic cells. Campbell
et al., 1996, Nature 380.64-66 and PCT Publication WO 95/20042.
Recently, another lamb was reported to be cloned from ovine somatic
mammary tissue. Wilmut et al., 1997, Nature 385:810-813. Some
methods for cloning ovine animals focused upon utilizing serum
deprived somatic ovine cells and cells isolated from ovine
embryonic discs as nuclear donors. PCT Publications WO 96/07732 and
WO 97/07669. Other methods for cloning ovine animals involved
manipulating the activation state of an in vivo matured oocyte
after nuclear transfer. PCT Publication WO 97/07668.
[0008] While few lambs were produced, publications that disclose
cloned lambs report a cloning efficiency that is, at best,
approximately 0.4%. Cloning efficiency, as calculated for the
previous estimate, is a ratio equal to the number of cloned lambs
divided by the number of nuclear transfers used to produce that
number of cloned lambs.
[0009] Despite the slower progress endemic to the field of cloning
bovine animals, a bovine animal was cloned using embryonic cells
derived from 2-64 cell embryos. This bovine animal was cloned by
utilizing the nuclear transfer techniques set forth in U.S. Pat.
Nos. 4,994,384 and 5,057,420. Others reported that cloned bovine
embryos were formed by nuclear transfer techniques utilizing the
inner cell mass cells of a blastocyst stage embryo. Sims &
First, 1993, Theriogenology 39:313 and Keefer et al., 1994, Mol.
Reprod. Dev. 38:264-268. In addition, another publication reported
that cloned bovine embryos were prepared by nuclear transfer
techniques that utilized PGCs isolated from fetal tissue. Delhaise
et al., 1995, Reprod. Fert. Develop. 7:1217-1219; Lavoir 1994,J.
Reprod. Dev. 37:413-424; and PCT application WO 95/10599 entitled
"Embryonic Stem Cell-Like Cells." However, the reports demonstrated
that cloned PGC-derived bovine embryos never clearly developed past
the first trimester during gestation. Similarly, embryonic stem
cell (e.g., cell line derived from embryos which are
undifferentiated, pluripotent, and can establish a permanent cell
line which exhibits a stable karyotype), ESC, derived bovine
embryos never developed past fifty-five days, presumably due to
incomplete placental development. Stice et al., 1996, Biol. Reprod.
54: 100-110.
[0010] Despite the progress of cloning ovine and bovine animals,
there remains a great need in the art for methods and materials
that increase cloning efficiency. In addition there remains a great
need in the art to expand the variety of cells that can be utilized
as nuclear donors, especially expanding nuclear donors to
non-embryonic cells. Furthermore, there remains a long felt need in
the art for karyotypically stable permanent cell lines that can be
used for genome manipulation and production of transgenic cloned
animals.
SUMMARY
[0011] The present invention relates to cloning technologies. The
invention relates in part to immortalized, totipotent cells useful
for cloning animals, the embryos produced from these cells using
nuclear transfer techniques, animals that arise from these cells
and embryos, and the methods and processes for creating such cells,
embryos, and animals.
[0012] The present invention provides multiple advantages over the
tools and methods currently utilized in the field of mammalian
cloning. Such features and advantages include:
[0013] (1) Production of cloned animals from virtually any type of
cell. The invention provides materials and methods for
reprogramming non-totipotent cells into totipotent cells. These
non-totipotent cells may be of non-embryonic origin. This feature
of the invention allows for the ability to assess the phenotype of
an existing animal and then readily establish a permanent cell line
for cloning that animal.
[0014] (2) Creation of permanent cell lines from virtually any type
of cell. Permanent cell lines provide a nearly unlimited source of
genetic material for nuclear transfer cloning techniques. In one
aspect of the invention, non-totipotent precursor cells can be
reprogrammed into totipotent and permanent cells. These
non-totipotent precursor cells may be non-embryonic cells.
Permanent cell lines provide the advantage of enhancing cloning
efficiency due to the lower cellular heterogeneity within the cell
lines (e.g., permanent cells that have lower rates of
differentiation than primary culture cell lines currently used for
cloning). In addition, the permanent cell lines can be manipulated
in vitro to produce cells, embryos, and animals whose genomes have
been manipulated (e.g., transgenic). Furthermore, permanent cell
lines can be more easily stored, transported, and re-established in
culture than other types of cell lines.
[0015] (3) Enhancement of the efficiency for cloning embryos as a
result of utilizing asynchronous, permanent, and karyotypically
stable cell lines in a complete in vitro embryo production
system.
[0016] Cloning efficiency can be expressed by the ratio between the
number of embryos resulting from nuclear transfer and the number of
nuclear transfers performed to give rise to the embryos.
Alternatively, cloning efficiency can be expressed as the ratio
between the number of live born animals and the number of nuclear
transfers performed to give rise to these animals.
[0017] Immortalized and Totipotent Cells of the Invention
[0018] In a first aspect, the invention features a totipotent
mammalian cell. Preferably, the totipotent mammalian cell is (1) a
cultured cell; (2) a cell cultured in a cell line; and (3) an
immortalized cell. In addition, the mammalian cell is preferably an
ungulate cell and more preferably a bovine cell.
[0019] The term "mammalian" or "mammal" as used herein refers to
any animal of the class Mammalia. A mammalian animal of the
invention is preferably an endangered animal, or, more preferably,
a farm animal. Most preferably, animal is an ungulate.
[0020] The term "non-ovine" as used herein refers to any animal
other than an animal of the family Ovidae. Members of the family
Oviadae include sheep. A non-ovine mammal is any member of the
class Mammalia other than an animal of the family Ovidae.
Preferable non-ovine animals are ungulate animals and most
preferably are bovine and porcine animals.
[0021] The term "ungulate" as used herein refers to a four-legged
animal having hooves. In other preferred embodiments, the ungulate
is selected from the group consisting of domestic or wild
representatives of bovids, ovids, cervids, suids, equids and
camelids. Examples of such representatives are cows or bulls,
bison, buffalo, sheep, big-horn sheep, horses, ponies, donkeys,
mule, deer, elk, caribou, goat, water buffalo, camels, llama,
alpaca, and pigs. Especially preferred in the bovine species are
Bos taurus, Bos indicus, and Bos buffaloes cows or bulls.
[0022] The term "bovine" as used herein refers to a family of
ruminants belonging to the genus Bos or any closely related genera
of the family Bovidae. The family Bovidae includes true antelopes,
oxen, sheep, and goats, for example. Preferred bovine animals are
the cow and ox. Especially preferred bovine species are Bos taurus,
Bos indicus, and Bos buffaloes. Other preferred bovine species are
Bos primigenius and Bos longifrons.
[0023] The term "totipotent" as used herein refers to a cell that
gives rise to all of the cells in a developing cell mass, such as
an embryo, fetus, and animal. In preferred embodiments, the term
"totipotent" also refers to a cell that gives rise to all of the
cells in an animal. A totipotent cell can give rise to all of the
cells of a developing cell mass when it is utilized in a procedure
for creating an embryo from one or more nuclear transfer steps. An
animal may be an animal that functions ex utero. An animal can
exist, for example, as a live born animal. Totipotent cells may
also be used to generate incomplete animals such as those useful
for organ harvesting, e.g., having genetic modifications to
eliminate growth of a head such as by manipulation of a homeotic
gene.
[0024] The terms "developing cell mass" as used herein refers to a
group of cells in which all cells or a portion of the cells are
undergoing cell division. The developing cell mass may be an
embryo, a fetus, and/or an animal, for example. The developing cell
mass may be dividing in vitro (e.g., in culture) or in vivo (e.g.,
in utero). The developing cell mass may be a product of one or more
nuclear transfer processes or may be the product of oocyte
activation (e.g., sperm mediated fertilization).
[0025] The term "live born" as used herein preferably refers to an
animal that exists ex utero. A "live born" animal may be an animal
that is alive for at least one second from the time it exits the
maternal host. A "live born" animal may not require the circulatory
system of an in utero environment for survival. A "live born"
animal may be an ambulatory animal. Such animals can include
pre-and post-pubescent animals. In addition, a "live born animal"
may also be deceased for a certain period of time. As discussed
previously, a "live born" animal may lack a portion of what exists
in a normal animal of its kind. For example, a "live born" animal
may lack a head as a result of the deletion or manipulation of one
or more homeotic genes.
[0026] The term "totipotent" as used herein is to be distinguished
from the term "pluripotent." The latter term refers to a cell that
differentiates into a sub-population of cells within a developing
cell mass, but is a cell that may not give rise to all of the cells
in that developing cell mass. Thus, the term "pluripotent" can
refer to a cell that cannot give rise to all of the cells in a live
born animal.
[0027] The term "totipotent" as used herein is also to be
distinguished from the term "chimer" or "chimera." The latter term
refers to a developing cell mass that comprises a sub-group of
cells harboring nuclear DNA with a significantly different
nucleotide base sequence than the nuclear DNA of other cells in
that cell mass. The developing cell mass can, for example, exist as
an embryo, fetus, and/or animal.
[0028] The term "immortalized" or "permanent" as used herein in
reference to cells refers to cells that have exceeded the Hayflick
limit. The Hayflick limit can be defined as the number of cell
divisions that occur before a cell line becomes senescent. Hayflick
set this limit to approximately 60 divisions for most
non-immortalized cells. See, e.g., Hayflick and Moorhead, 1961,
Exp. Cell. Res. 25: 585-621; and Hayflick, 1965, Exp. Cell Research
37: 614-636, incorporated herein by reference in their entireties
including all figures, tables, and drawings. Therefore, an
immortalized cell line can be distinguished from non-immortalized
cell lines if the cells in the cell line are able to undergo more
than 60 divisions. If the cells of a cell line are able to undergo
more than 60 cell divisions, the cell line is an immortalized or
permanent cell line. The immortalized cells of the invention are
preferably able to undergo more than 70 divisions, are more
preferably able to undergo more than 80 divisions, and are most
preferably able to undergo more than 90 cell divisions.
[0029] Typically, immortalized or permanent cells can be
distinguished from non-immortalized and non-permanent cells on the
basis that immortalized and permanent cells can be passaged at
densities lower than those of non-immortalized cells. Specifically,
immortalized cells can be grown to confluence (e.g., when a cell
monolayer spreads across an entire plate) when plating conditions
do not allow physical contact between the cells. Hence,
immortalized cells can be distinguished from non-immortalized cells
when cells are plated at cell densities where the cells do not
physically contact one another.
[0030] The term "plated" or "plating" as used herein in reference
to cells refers to establishing cell cultures in vitro. For
example, cells can be diluted in cell culture media and then added
to a cell culture plate or cell culture dish. Cell culture plates
are commonly known to a person of ordinary skill in the art. Cells
may be plated at a variety of concentrations and/or cell
densities.
[0031] The meaning of the term "cell plating" can also extend to
the term "cell passaging." Immortalized cells of the invention can
be passaged using cell culture techniques well known to those
skilled in the art. The term "cell passaging" can refer to such
techniques which typically involve the steps of (1) releasing cells
from a solid support and disassociation of these cells, and (2)
diluting the cells in fresh media suitable for cell proliferation.
Immortalized cells can be successfully grown by plating the cells
in conditions where they lack cell to cell contact. Cell passaging
may also refer to removing a portion of liquid medium bathing
cultured cells and adding liquid medium from another source to the
cell culture.
[0032] The term "proliferation" as used herein in reference to
immortalized or permanent cells refers to a group of cells that can
increase in size and/or can increase in numbers over a period of
time.
[0033] The term "confluence" as used herein refers to a group of
cells where a large percentage of the cells are physically
contacted with at least one other cell in that group. Confluence
may also be defined as a group of cells that grow to a maximum cell
density in the conditions provided. For example, if a group of
cells can proliferate in a monolayer and they are placed in a
culture vessel in a suitable growth medium, they are confluent when
the monolayer has spread across a significant surface area of the
culture vessel. The surface area covered by the cells preferably
represents about 50% of the total surface area, more preferably
represents about 70% of the total surface area, and most preferably
represents about 90% of the total surface area.
[0034] The term "culture" as used herein in reference to cells
refers to one or more cells that are undergoing cell division or
not undergoing cell division in an in vitro environment. An in
vitro environment can be any medium known in the art that is,
suitable for maintaining cells in vitro, such as suitable liquid
media or agar. Specific examples of suitable in vitro environments
for cell cultures are described in Culture of animal Cells: a
manual of basic techniques (3.sup.rd edition), 1994, R. I. Freshney
(ed.), Wiley-Liss, Inc.; Cells: a laboratory manual (vol. 1), 1998,
D. L. Spector, R. D. Goldman, L. A. Leinwand (eds.), Cold Spring
Harbor Laboratory Press; and Animal Cells: culture and media, 1994,
D. C. Darling, S. J. Morgan, John Wiley and Sons, Ltd., each of
which is incorporated herein by reference in its entirety including
all figures, tables, and drawings. Preferred media are
AminoMax.TM.-C 100 Basal Medium (Gibco 1701-082), AminoMax.TM.
C-100 Supplement Medium (Gibco 17002-080), and Knockout.TM. D-MEM
Medium (Gibco 10829-108).
[0035] Nearly any type of cell can be placed in cell culture
conditions. Cells may be cultured in suspension and/or in
monolayers with one or more substantially similar cells. Cells may
be cultured in suspension and/or in monolayers with a heterogeneous
population cells. The term "heterogeneous" as utilized in the
previous sentence can relate to any cell characteristics, such as
cell type and cell cycle stage, for example. Cells may be cultured
in suspension and/or in monolayers with feeder cells. The term
"feeder cells" is defined hereafter. In preferred embodiments,
cells may be successfully cultured by plating the cells in
conditions where they lack cell to cell contact. Cell cultures can
also be utilized to establish a cell line.
[0036] In preferred embodiments, (1) cultured cells undergo cell
division; (2) cells are cultured for greater than 5 hours; (3)
cells are cultured for greater than 7 hours; (4) cells are cultured
for greater than 10 hours; (5) cells are cultured for greater than
12 hours; (6) cells are cultured for greater than 24 hours; (7)
cells are cultured for and greater than 48 hours; (8) cells are
cultured greater than 3 days; (9) cells are cultured for greater
than 5 days; (10) cells are cultured for greater than 10 days; and
(11) cells are cultured for greater than 30 days.
[0037] The term "suspension" as used herein refers to cell culture
conditions in which the cells are not attached to a solid support.
Cells proliferating in suspension can be stirred while
proliferating using apparatus well known to those skilled in the
art.
[0038] The term "monolayer" as used herein refers to cells that are
attached to a solid support while proliferating in suitable culture
conditions. A small portion of the cells proliferating in the
monolayer under suitable growth conditions may be attached to cells
in the monolayer but not to the solid support. Preferably less than
15% of these cells are not attached to the solid support, more
preferably less than 10% of these cells are not attached to the
solid support, and most preferably less than 5% of these cells are
not attached to the solid support. Cells can also grow in culture
in multilayers. The term "multilayer" as used herein refers to
cells proliferating in suitable culture conditions where at least
15% of the cells are indirectly attached to the solid support
through an attachment to other cells. Preferably, at least 25% of
the cells are indirectly attached to the solid support, more
preferably at least 50% of the cells are indirectly attached to the
solid support, and most preferably at least 75% of the cells are
indirectly attached to the solid support.
[0039] The term "substantially similar" as used herein in reference
to immortalized bovine cells refers to cells from the same organism
and the same tissue. In preferred embodiments, substantially
similar also refers to cell populations that have not significantly
differentiated. For example, preferably less than 15% of the cells
in a population of cells have differentiated, more preferably less
than 10% of the cell population have differentiated, and most
preferably less than 5% of the cell population have
differentiated.
[0040] The term "cell line" as used herein refers to cultured cells
that can be passaged more than once. The invention relates to cell
lines that can be passaged more than 2, 5, 6, 7, 8, 9, 10, 15, 20,
30, 50, 80, 100, and 200 times, or preferably more than any integer
between 2 and 200, each number not having been explicitly set forth
in the interest of conciseness. The concept of cell passaging is
defined previously.
[0041] In preferred embodiments, (1) the totipotent cells are not
alkaline phosphatase positive; (2) the totipotent cells arise from
at least one precursor cell; (3) the precursor cell is isolated
from and/or arises from any region of an animal; (4) the precursor
cell is isolated from and/or arises from any cell in culture; (5)
the precursor cell is selected from the group consisting of a
non-embryonic cell, a non-fetal cell, a differentiated cell, a
somatic cell, an embryonic cell, a fetal cell, an embryonic stem
cell, a primordial germ cell, a genital ridge cell, an amniotic
cell, a fetal fibroblast cell, an ovarian follicular cell, a
cumulus cell, an hepatic cell, an endocrine cell, an endothelial
cell, an epidermal cell, an epithelial cell, a fibroblast cell, a
hematopoietic cell, a keratinocyte, a renal cell, a lymphocyte, a
melanocyte, a muscle cell, a myeloid cell, a neuronal cell, an
osetoblast, a mesenchymal cell, a mesodermal cell, an adherent
cell, a cell isolated from an asynchronous population of cells, and
a cell isolated from a synchronized population of cells where the
synchronous population is not arrested in the Go stage of the cell
cycle; and (6) the precursor cell is preferably isolated and/or
arises from a mammalian animal, more preferably an ungulate animal,
and most preferably a bovine animal.
[0042] The term "alkaline phosphatase positive" as used herein
refers to a detectable presence of cellular alkaline phosphatase.
Cells that are not alkaline phosphatase positive do not stain
appreciably using a procedure for visualizing cellular alkaline
phosphatase. Procedures for detecting the presence of cellular
alkaline phosphatase are well-known to a person of ordinary skill
in the art. See, e.g., Matsui et al., 1991, "Effect of Steel Factor
and Leukemia Inhibitory Factor on Murine Primordial Germ Cells in
Culture," Nature 353: 750-752. Examples of cells that stain
appreciably for alkaline phosphatase can be found in the art. See,
e.g., U.S. Pat. No. 5,453,357, Entitled "Pluripotent Embryonic Stem
Cells and Methods of Making Same," issued to Hogan on Sept. 26,
1995, which is incorporated by reference herein in its entirety,
including all figures, tables, and drawings.
[0043] The term "precursor cell" or "precursor cells" as used
herein refers to a cell or cells used to create a cell line of
totipotent cells. The cell line is preferably permanent. Precursor
cells can be isolated from any mammal, preferably from an ungulate
and more preferably from a bovine animal. The precursor cell or
cells may be isolated from nearly any cellular entity. For example,
a precursor cell or cells may be isolated from blastocysts,
embryos, fetuses, and cell lines (e.g., cell lines established from
embryonic cells), preferably isolated from fetuses and/or cell
lines established from fetal cells, and more preferably isolated
from ex utero animals and/or cell cultures and/or cell lines
established from such ex utero animals. An ex utero animal may
exist as a newborn animal, adolescent animal, yearling animal, and
adult animal. The ex utero animals may be alive or post mortem. The
precursor cell or cells may be immortalized or non-immortalized.
These examples are not meant to be limiting and a further
description of these exemplary precursor cells is provided
hereafter.
[0044] The term "arises from" as used herein refers to the
conversion of one or more cells into one or more other cells. For
example, a non-totipotent precursor cell can be converted into a
totipotent cell by utilizing features of the invention described
hereafter. This conversion process can be referred to as a
reprogramming step. In another example, a precursor cell can give
rise to a feeder layer of cells, as defined hereafter. In addition,
the term "arises from" can refer to the creation of totipotent
embryos from immortalized, totipotent cells of the invention, as
described hereafter.
[0045] The term "reprogramming" or "reprogrammed" as used herein
refers to materials and methods that can convert a non-totipotent
cell into an totipotent cell. Distinguishing features between
totipotent and non-totipotent cells are described previously. An
example of materials and methods for converting non-totipotent
cells into totipotent cells is to incubate precursor cells with a
receptor ligand cocktail. Receptor ligand cocktails are described
hereafter.
[0046] The term "isolated" as used herein refers to a cell that is
mechanically separated from another group of cells. Examples of a
group of cells are a developing cell mass, a cell culture, a cell
line, and an animal. These examples are not meant to be limiting
and the invention relates to any group of cells.
[0047] The term "non-embryonic cell" as used herein refers to a
cell that is not isolated from an embryo. Non-embryonic cells can
be differentiated or non-differentiated. Non-embryonic cells can
refer to nearly any somatic cell, such as cells isolated from an ex
utero animal. These examples are not meant to be limiting.
[0048] For the purposes of the present invention, the term "embryo"
or "embryonic" as used herein refers to a developing cell mass that
has not implanted into the uterine membrane of a maternal host.
Hence, the term "embryo" as used herein can refer to a fertilized
oocyte, a cybrid (defined herein), a pre-blastocyst stage
developing cell mass, and/or any other developing cell mass that is
at a stage of development prior to implantation into the uterine
membrane of a maternal host. Embryos of the invention may not
display a genital ridge. Hence, an "embryonic cell" is isolated
from and/or has arisen from an embryo.
[0049] An embryo can represent multiple stages of cell development.
For example, a one cell embryo can be referred to as a zygote, a
solid spherical mass of cells resulting from a cleaved embryo can
be referred to as a morula, and an embryo having a blastocoel can
be referred to as a blastocyst.
[0050] The term "fetus" as used herein refers to a developing cell
mass that has implanted into the uterine membrane of a maternal
host. A fetus can include such defining features as a genital
ridge, for example. A genital ridge is a feature easily identified
by a person of ordinary skill in the art, and is a recognizable
feature in fetuses of most animal species. The term "fetal cell" as
used herein can refer to any cell isolated from and/or has arisen
from a fetus or derived from a fetus. The term "non-fetal cell" is
a cell that is not derived or isolated from a fetus.
[0051] The term "primordial germ cell" as used herein refers to a
diploid somatic cell capable of becoming a germ cell. Primordial
germ cells can be isolated from the genital ridge of a developing
cell mass. The genital ridge is a section of a developing cell mass
that is well-known to a person of ordinary skill in the art. See,
e.g., Strelchenko, 1996, Theriogenology 45: 130-141 and Lavoir
1994, J. Reprod. Dev. 37: 413-424.
[0052] The terms "embryonic germ cell" and "EG cell" as used herein
refers to a cultured cell that has a distinct flattened morphology
and can grow within monolayers in culture. An EG cell may be
distinct from a fibroblast cell. This EG cell morphology is to be
contrasted with cells that have a spherical morphology and form
multicellular clumps on feeder layers. Embryonic germ cells may not
require the presence of feeder layers or presence of growth factors
in cell culture conditions. Embryonic germ cells may also grow with
decreased doubling rates when these cells approach confluence on
culture plates. Embryonic germ cells of the invention may be
totipotent. Embryonic germ cells of the invention may not
appreciably stain for alkaline phosphatase. Preferably, embryonic
germ cells are established in culture media that contains a
significant concentration of glucose.
[0053] Embryonic germ cells may be established from a cell culture
of nearly any type of precursor cell. Examples of precursor cells
are discussed herein, and a preferred precursor cell for
establishing an embryonic germ cell culture is a genital ridge cell
from a fetus. Genital ridge cells are preferably isolated from
porcine fetuses where the fetus is between 20 days and parturition,
between 30 days and 100 days, more preferably between 35 days and
70 days and between 40 days and 60 days, and most preferably about
a 55 day fetus. An age of a fetus can be determined as described
above. The term "about" with respect to fetuses can refer to plus
or minus five days. As described herein, EG cells may be physically
isolated from a primary culture of cells, and these isolated EG
cells may be utilized to establish a cell culture that eventually
forms a homogenous or nearly homogenous line of EG cells.
[0054] The term "embryonic stem cell" as used herein refers to
pluripotent cells isolated from an embryo that are maintained in in
vitro cell culture. Embryonic stem cells may be cultured with or
without feeder cells. Embryonic stem cells can be established from
embryonic cells isolated from embryos at any stage of development,
including blastocyst stage embryos and pre-blastocyst stage
embryos. Embryonic stem cells are well known to a person of
ordinary skill in the art. See, e.g., WO 97/37009, entitled
"Cultured Inner Cell Mass Cell-Lines Derived from Ungulate
Embryos," Stice and Golueke, published Oct. 9, 1997, and Yang &
Anderson, 1992, Theriogenology 38: 315-335, both of which are
incorporated herein by reference in their entireties, including all
figures, tables, and drawings.
[0055] The term "amniotic cell" as used herein refers to any
cultured or non-cultured cell isolated from amniotic fluid.
Examples of methods for isolating and culturing amniotic cells are
discussed in Bellow et al., 1996, Theriogenology 45: 225; Garcia
& Salaheddine, 1997, Theriogenology 47: 1003-1008; Leibo &
Rail, 1990, Theriogenology 33: 531-552; and Vos et al., 1990, Vet.
Rec. 127: 502-504, each of which is incorporated herein by
reference in its entirety, including all figures tables and
drawings. Particularly preferred are cultured amniotic cells that
are spherical (e.g., cultured amniotic cells that do not display a
fibroblast-like morphology). Also preferred amniotic cells are
fetal fibroblast cells. The terms "fibroblast," fibroblast-like,"
"fetal," and "fetal fibroblast" are defined hereafter.
[0056] The terms "fibroblast-like" and "fibroblast" as used herein
refer to cultured cells that have a distinct flattened morphology
and that are able to grow within monolayers in culture.
[0057] The term "fetal fibroblast cell" as used herein refers to
any differentiated fetal cell having a fibroblast appearance. While
fibroblasts characteristically have a flattened appearance when
cultured on culture media plates, fetal fibroblast cells can also
have a spindle-like morphology. Fetal fibroblasts may require
density limitation for growth, may generate type I collagen, and
may have a finite life span in culture of approximately fifty
generations. Preferably, fetal fibroblast cells rigidly maintain a
diploid chromosomal content. For a description of fibroblast cells,
see, e.g., Culture of Animal Cells: a manual of basic techniques
(3.sup.rd edition), 1994, R. I. Freshney (ed), Wiley-Liss, Inc.,
incorporated herein by reference in its entirety, including all
figures, tables, and drawings.
[0058] The terms "morphology" and "cell morphology" as used herein
refer to form, structure, and physical characteristics of cells.
For example, one cell morphology is significant levels of alkaline
phosphatase, and this cell morphology can be identified by
determining whether a cell stains appreciably for alkaline
phosphatase. Another example of a cell morphology is whether a cell
is flat or round in appearance when cultured on a surface or in the
presence of a layer of feeder cells. Many other cell morphologies
are known to a person of ordinary skill in the art and are cell
morphologies are readily identifiable using materials and methods
well known to those skilled in the art. See, e.g., Culture of
Animal Cells: a manual of basic techniques (3.sup.rd edition),
1994, R. I. Freshney (ed.), Wiley-Liss, Inc.
[0059] The term "ovarian follicular cell" as used herein refers to
a cultured or non-cultured cell obtained from an ovarian follicle,
other than an oocyte. Follicular cells may be isolated from ovarian
follicles at any stage of development, including primordial
follicles, primary follicles, secondary follicles, growing
follicles, vesicular follicles, maturing follicles, mature
follicles, and graafian follicles. Furthermore, follicular cells
may be isolated when an oocyte in an ovarian follicle is immature
(i.e., an oocyte that has not progressed to metaphase II) or when
an oocyte in an ovarian follicle is mature (i.e., an oocyte that
has progressed to metaphase II or a later stage of development).
Preferred follicular cells include, but are not limited to,
pregranulosa cells, granulosa cells, theca cells, columnar cells,
stroma cells, theca interna cells, theca externa cells, mural
granulosa cells, luteal cells, and corona radiata cells.
Particularly preferred follicular cells are cumulus cells. Various
types of follicular cells are known and can be readily
distinguished by those skilled in the art. See, e.g., Laboratory
Production of Cattle Embryos, 1994, Ian Gordon, CAB International;
Anatomy and Physiology of Farm Animals (5.sup.th ed.), 1992, R. D.
Frandson and T. L. Spurgeon, Lea & Febiger, each of which is
incorporated herein by reference in its entirety including all
figures, drawings, and-tables. Individual types of follicular cells
may be cultured separately, or a mixture of types may be cultured
together.
[0060] The term "cumulus cell" as used herein refers to any
cultured or non-cultured cell isolated from cells and/or tissue
surrounding an oocyte. Persons skilled in the art can readily
identify cumulus cells. Examples of methods for isolating and/or
culturing cumulus cells are discussed in Damiani et al., 1996, Mol.
Reprod. Dev. 45: 521-534; Long et al., 1994, J. Reprod. Fert. 102:
361-369; and Wakayama et al., 1998, Nature 394: 369-373, each of
which is incorporated herein by reference in its entireties,
including all figures, tables, and drawings. Cumulus cells may be
isolated from ovarian follicles at any stage of development,
including primordial follicles, primary follicles, secondary
follicles, growing follicles, vesicular follicles, maturing
follicles, mature follicles, and graafian follicles. Cumulus cells
may be isolated from oocytes in a number of manners well known to a
person of ordinary skill in the art. For example, cumulus cells can
be separated from oocytes by pipeting the cumulus cell/oocyte
complex through a small bore pipette, by exposure to hyaluronidase,
or by mechanically disrupting (e.g. vortexing) the cumulus
cell/oocyte complex. Additionally, exposure to
Ca.sup.++/Mg.sup.++free media can remove cumulus from immature
oocytes. Also, cumulus cell cultures can be established by placing
matured oocytes in cell culture media. Once cumulus cells are
removed from media containing increased LH/FSH concentrations, they
can to attach to the culture plate.
[0061] The term "hepatic cell" as used herein refers to any
cultured or non-cultured cell isolated from a liver. Particularly
preferred hepatic cells include, but are not limited to, a hepatic
parenchymal cell, a Kupffer cell, an Ito cell, a hepatocyte, a
fat-storing cell, a pit cell, and a hepatic endothelial cell.
Persons skilled in the art can readily identify the various types
of hepatic cells. See, e.g., Regulation of Hepatic Metabolism,
1986, Thurman et al. (eds.), Plenum Press, which is incorporated
herein by reference in its entirety including all figures,
drawings, and tables.
[0062] The term "differentiated cell" as used herein refers to a
precursor cell that has developed from an unspecialized phenotype
to that of a specialized phenotype. For example, embryonic cells
can differentiate into an epithelial cell lining the intestine. It
is highly unlikely that differentiated cells revert into their
precursor cells in vivo or in vitro. However, materials and methods
of the invention can reprogram differentiated cells into
immortalized, totipotent cells. Differentiated cells can be
isolated from a fetus or a live born animal, for example.
[0063] In contrast to the totipotent and/or immortalized cells of
the invention that arise from non-embryonic cells, an example of
embryonic cells is discussed in WO 96/07732, entitled "Totipotent
Cells for Nuclear Transfer," hereby incorporated herein by
reference in its entirety including all figures, drawings, and
tables. The WO 96/07732 publication relates primarily to ovine
animals. A unique feature of the present invention is that
immortalized, totipotent cells are reprogrammed from SD-101848.1
non-embryonic cells by utilizing the materials and methods
described herein in descriptions of the preferred embodiments and
exemplary embodiments.
[0064] The term "asynchronous population" as used herein refers to
cells that are not arrested at any one stage of the cell cycle.
Many cells can progress through the cell cycle and do not arrest at
any one stage, while some cells can become arrested at one stage of
the cell cycle for a period of time. Some known stages of the cell
cycle are G.sub.0, G.sub.1, S, G.sub.2, and M. An asynchronous
population of cells is not manipulated to synchronize into any one
or predominantly into any one of these phases. Cells can be
arrested in the G.sub.0 stage of the cell cycle, for example, by
utilizing multiple techniques known in the art, such as by serum
deprivation. Examples of methods for arresting non-immortalized
cells in one part of the cell cycle are discussed in WO 97/07669,
entitled "Quiescent Cell Populations for Nuclear Transfer," hereby
incorporated herein by reference in its entirety, including all
figures, tables, and drawings.
[0065] The terms "synchronous population" and "synchronizing" as
used herein refer to a fraction of cells in a population that are
arrested (i.e., the cells are not dividing) in a discreet stage of
the cell cycle. Synchronizing a population of cells, by techniques
such as serum deprivation, may render the cells quiescent. The term
"quiescent" is defined below. Preferably, about 50% of the cells in
a population of cells are arrested in one stage of the cell cycle,
more preferably about 70% of the cells in a population of cells are
arrested in one stage of the cell cycle, and most preferably about
90% of the cells in a population of cells are arrested in one stage
of the cell cycle. Cell cycle stage can be distinguished by
relative cell size as well as by a variety of cell markers well
known to a person of ordinary skill in the art. For example, cells
can be distinguished by such markers by using flow cytometry
techniques well known to a person of ordinary skill in the art.
Alternatively, cells can be distinguished by size utilizing
techniques well known to a person of ordinary skill in the art,
such as by the utilization of a light microscope and a micrometer,
for example.
[0066] The terms "serum deprivation," "serum starved," and "serum
starvation" as used herein refer to culturing cells in a medium
comprising a serum concentration sufficiently low as to render
cultured cells quiescent. The term "quiescent" is defined
hereafter. A number of sera are used by those skilled in the art to
supplement cell culture media. Particularly preferred is fetal
bovine serum. Preferred serum starvation conditions are culturing
cells in a medium comprising less than 1% fetal bovine serum.
Particularly preferred conditions are culturing cells in a medium
comprising not more than 0.5% fetal bovine serum. A length of time
cultured cells are serum starved to be rendered quiescent can vary
depending upon cell type. Cultured cells can be serum starved for
at least 1 hour, at least 5 hours, at least 12 hours, and at least
24 hours. Preferably, cultured cells are serum starved for more
than 1 day. Most preferably, cultured cells are serum starved for
more than 3 days. These conditions are not meant to be limiting,
and other serum starvation conditions can easily be identified by
those skilled in the art without undue experimentation.
[0067] The term "quiescent" as used herein in reference to cells
refers to cells which are not dividing. A "quiescent cell culture"
refers to a culture in which a majority of cells in the culture are
not dividing. More preferably, in a quiescent cell culture all
cells in the culture are not dividing. As discussed herein, a cell
culture may be rendered quiescent by serum starvation, but other
methods which render cell cultures quiescent are known to those of
ordinary skill in the art. Cells may be made permanently quiescent,
and more preferably, quiescent cells may be made to resume dividing
at a later time.
[0068] In preferred embodiments, (1) the totipotent cells of the
invention comprise modified nuclear DNA; (2) the modified nuclear
DNA includes a DNA sequence that encodes a recombinant product; (3)
the recombinant product is a polypeptide; (4) the recombinant
product is a ribozyme; (4) the recombinant product is expressed in
a biological fluid or tissue; (5) the recombinant product confers
or partially confers resistance to one or more diseases; (6) the
recombinant product confers resistance or partially confers
resistance to one or more parasites; (7) the modified nuclear DNA
comprises at least one other DNA sequence that can function as a
regulatory element; (8) the regulatory element is selected from the
group consisting of promotor, enhancer, insulator, and repressor;
and (9) the regulatory element is selected from the group
consisting of milk protein promoter, urine protein promoter, blood
protein promoter, tear duct protein promoter, synovial protein
promoter, mandibular gland protein promoter, casein promoter,
.beta.-casein promoter, melanocortin promoter, milk serum protein
promoter, .alpha.-lactalbumin promoter, whey acid protein promoter,
uroplakin promoter, .alpha.-actin promoter.
[0069] The term "modified nuclear DNA" as used herein refers to the
nuclear deoxyribonucleic acid sequence of a cell, embryo, fetus, or
animal of the invention that has been manipulated by one or more
recombinant DNA techniques. Examples of these recombinant DNA
techniques are well known to a person of ordinary skill in the art,
which can include (1) inserting a DNA sequence from another
organism (e.g., a human organism) into target nuclear DNA, (2)
deleting one or more DNA sequences from target nuclear DNA, and (3)
introducing one or more base mutations (e.g., site-directed
mutations) into target nuclear DNA. Cells with modified nuclear DNA
can be referred to as "transgenic cells" for the purposes of the
invention. Transgenic cells can be useful as materials for nuclear
transfer cloning techniques provided herein.
[0070] Methods and tools for insertion, deletion, and mutation of
nuclear DNA of mammalian cells are well-known to a person of
ordinary skill in the art. See, Molecular Cloning, a Laboratory
Manual, 2nd Ed., 1989, Sambrook, Fritsch, and Maniatis, Cold Spring
Harbor Laboratory Press; U.S. Pat. No. 5 ,633,067 , "Method of
Producing a Transgenic Bovine or Transgenic Bovine Embryo," DeBoer
et al., issued May 27, 1997; U.S. Pat. No. 5,612,205, "Homologous
Recombination in Mammalian Cells," Kay et al., issued Mar. 18,
1997; and PCT publication WO 93/22432, "Method for Identifying
Transgenic Pre-Implantation Embryos," all of which are incorporated
by reference herein in their entirety, including all figures,
drawings, and tables. These methods include techniques for
transfecting cells with foreign DNA fragments and the proper design
of the foreign DNA fragments such that they effect insertion,
deletion, and/or mutation of the target DNA genome.
[0071] Transgenic cells may be obtained in a variety of manners.
For example, transgenic cells can be isolated from a transgenic
animal. Examples of transgenic animals are well known in the art,
as described herein with regard to transgenic bovine and ovine
animals. Cells isolated from a transgenic animal can be converted
into totipotent and/or immortalized cells by using the materials
and methods provided herein. In another example, transgenic cells
can be created from totipotent and/or immortalized cells of the
invention. Materials and methods for converting non-transgenic
cells into transgenic cells are well known in the art, as described
previously.
[0072] Any of the cell types defined herein can be altered to
harbor modified nuclear DNA. For example, embryonic stem cells,
fetal cells, and any totipotent and immortalized cell defined
herein can be altered to harbor modified nuclear DNA.
[0073] Examples of methods for modifying a target DNA genome by
insertion, deletion, and/or mutation are retroviral insertion,
artificial chromosome techniques, gene insertion, random insertion
with tissue specific promoters, homologous recombination, gene
targeting, transposable elements, and/or any other method for
introducing foreign DNA. Other modification techniques well known
to a person of ordinary skill in the art include deleting DNA
sequences from a genome, and/or altering nuclear DNA sequences.
Examples of techniques for altering nuclear DNA sequences are
site-directed mutagenesis and polymerase chain reaction procedures.
Therefore, the invention provides for bovine cells that are
simultaneously totipotent, immortalized, and transgenic. These
transgenic, totipotent, immortalized cells can serve as nearly
unlimited sources of donor cells for production of cloned
transgenic animals.
[0074] The term "recombinant product" as used herein refers to the
product produced from a DNA sequence that comprises at least a
portion of the modified nuclear DNA. This product can be a peptide,
a polypeptide, a protein, an enzyme, an antibody, an antibody
fragment, a polypeptide that binds to a regulatory element (a term
described hereafter), a structural protein, an RNA molecule, and/or
a ribozyme, for example. These products are well defined in the
art. This list of products is for illustrative purposes only and
the invention relates to other types of products.
[0075] The term "ribozyme" as used herein refers to ribonucleic
acid molecules that can cleave other RNA molecules in
specific-regions. Ribozymes can bind to discrete regions on a RNA
molecule, and then specifically cleave a region within that binding
region or adjacent to the binding region. Ribozyme techniques can
thereby decrease the amount of polypeptide translated from formerly
intact message RNA molecules. For specific descriptions of
ribozymes, see U.S. Pat. No. 5,354,855, entitled "RNA Ribozyme
which Cleaves Substrate RNA without Formation of a Covalent Bond,"
Cech et al., issued on Oct. 11, 1994, and U.S. Pat. 5,591,610,
entitled "RNA Ribozyme Polymerases, Dephosphorylases, Restriction
Endoribonucleases and Methods," Cech et al., issued on Jan. 7,
1997, both of which are incorporated by reference in their
entireties including all figures, tables, and drawings.
[0076] The term "biological fluid or tissue" as used herein refers
to any fluid or tissue in a biological organism. The fluids may
include, but are not limited to, tears, saliva, milk, urine,
amniotic fluid, semen, plasma, oviductal fluid, and synovial fluid.
The tissues may include, but are not limited to, lung, heart,
blood, liver, muscle, brain, pancreas, skin, and others.
[0077] The term "confers resistance" as used herein refers to the
ability of a recombinant product to completely abrogate or
partially alleviate the symptoms of a disease or parasitic
condition. Hence, if the disease is related to inflammation, for
example, a recombinant product can confer resistance to that
inflammation if the inflammation decreases upon expression of the
recombinant product. A recombinant product may confer resistance or
partially confer resistance to a disease or parasitic condition,
for example, if the recombinant product is an anti-sense RNA
molecule that specifically binds to an mRNA molecule encoding a
polypeptide responsible for the inflammation. Other examples of
conferring resistance to diseases or parasites are described
hereafter. In addition, examples of diseases are described
hereafter.
[0078] Examples of parasites and strategies for conferring
resistance to these parasites are described hereafter. These
examples include, but are not limited to, worms, insects,
invertebrate, bacterial, viral, and eukaryotic parasites. These
parasites can lead to diseased states that can be controlled by the
materials and methods of the invention.
[0079] The term "regulatory element" as used herein refers to a DNA
sequence that can increase or decrease the amount of product
produced from another DNA sequence. The regulatory element can
cause the constitutive production of the product (e.g., the product
can be expressed constantly). Alternatively, the regulatory element
can enhance or diminish the production of a recombinant product in
an inducible fashion (e.g., the product can be expressed in
response to a specific signal). The regulatory element can be
controlled, for example, by nutrition, by light, or by adding a
substance to the transgenic organism's system. Examples of
regulatory elements well-known to those of ordinary skill in the
art are promoters, enhancers, insulators, and repressors. See,
e.g., Transgenic Animals, Generation and Use, 1997, Edited by L. M.
Houdebine, Hardwood Academic Publishers, Australia, hereby
incorporated herein by reference in its entirety including all
figures, tables, and drawings.
[0080] The term "promoters" or "promoter" as used herein refers to
a DNA sequence that is located adjacent to a DNA sequence that
encodes a recombinant product. A promoter is preferably operatively
linked to the adjacent DNA sequence. A promoter typically increases
the amount of recombinant product expressed from a DNA sequence as
compared to the amount of the expressed recombinant product when no
promoter exists. A promoter from one organism can be utilized to
enhance recombinant product expression from a DNA sequence that
originates from another organism. In addition, one promoter element
can increase an amount of recombinant products expressed for
multiple DNA sequences attached in tandem. Hence, one promoter
element can enhance the expression of one or more recombinant
products. Multiple promoter elements are well-known to persons of
ordinary skill in the art. Examples of promoter elements are
described hereafter.
[0081] The term "enhancers" or "enhancer" as used herein refers to
a DNA sequence that is located adjacent to the DNA sequence that
encodes a recombinant product. Enhancer elements are typically
located upstream of a promoter element or can be located downstream
of the coding DNA sequence (e.g., the DNA sequence transcribed or
translated into a recombinant product or products). Hence, an
enhancer element can be located 100 base pairs, 200 base pairs, or
300 or more base pairs upstream of the DNA sequence that encodes
the recombinant product. Enhancer elements can increase the amount
of recombinant product expressed from a DNA sequence above the
increased expression afforded by a promoter element. Multiple
enhancer elements are readily available to persons of ordinary
skill in the art.
[0082] The term "insulators" or "insulator" as used herein refers
to DNA sequences that flank the DNA sequence encoding the
recombinant product. Insulator elements can direct the recombinant
product expression to specific tissues in an organism. Multiple
insulator elements are well known to persons of ordinary skill in
the art. See, e.g., Geyer, 1997, Curr. Opin. Genet. Dev. 7:
242-248, hereby incorporated herein by reference in its entirety,
including all figures, tables, and drawings.
[0083] The term "repressor" or "repressor element" as used herein
refers to a DNA sequence located in proximity to the DNA sequence
that encodes the recombinant product, where the repressor sequence
can decrease the amount of recombinant product expressed from that
DNA sequence. Repressor elements can be controlled by the binding
of a specific molecule or specific molecules to the repressor
element DNA sequence. These molecules can either activate or
deactivate the repressor element. Multiple repressor elements are
available to a person of ordinary skill in the art.
[0084] The terms "milk protein promoter," "urine protein promoter,"
"blood protein promoter," "tear duct protein promoter," "synovial
protein promoter," and "mandibular gland protein promoter" refer to
promoter elements that regulate the specific expression of proteins
within the specified fluid or gland or cell type in an animal. For
example, a milk protein promoter is a regulatory element that can
control the expression of a protein that is expressed in the milk
of an animal. Other promoters, such as casein promoter,
.alpha.-lactalbumin promoter, whey acid protein promoter, uroplakin
promoter, and .alpha.-actin promoter, for example, are well known
to a person of ordinary skill in the art.
[0085] In preferred embodiments, (1) the totipotent cell is subject
to manipulation; (2) the manipulation comprises the step of
utilizing a totipotent cell in a nuclear transfer procedure; (3)
the manipulation comprises the step of cryopreserving the
totipotent cells; (4) the manipulation comprises the step of
thawing the totipotent cells; (5) the manipulation comprises the
step of passaging totipotent cells; (6) the manipulation comprises
the step of synchronizing totipotent cells; (7) the manipulation
comprises the step of transfecting totipotent cells with foreign
DNA; and (8) the manipulation comprises the step of dissociating a
cell from another cell or group of cells.
[0086] The term "manipulation" as used herein refers to the common
usage of the term, which is the management or handling directed
towards some object. Examples of manipulations are described
herein.
[0087] The term "nuclear transfer" as used herein refers to
introducing a full complement of nuclear DNA from one cell to an
enucleated cell. Nuclear transfer methods are well known to a
person of ordinary skill in the art. See, U.S. Pat. No. 4,994,384,
entitled "Multiplying Bovine Embryos," Prather et al., issued on
Feb. 19, 1991 and U.S. Pat. No. 5,057,420, entitled "Bovine Nuclear
Transplantation," Massey, issued on Oct. 15, 1991, both of which
are hereby incorporated by reference in their entirety including
all figures, tables and drawings. Nuclear transfer may be
accomplished by using oocytes that are not surrounded by a zona
pellucida.
[0088] Although the basic principals of nuclear transfer have been
described previously, the technique can be sensitive to the
introduction of any new parameters. Therefore, significant
modifications to the techniques described in the area of nuclear
transfer may require some experimentation to determine the
practical effect of these modifications upon the efficiency of
nuclear transfer. An example of a variable that can affect nuclear
transfer efficiency is the age of the oocyte utilized for
enucleation and nuclear transfer.
[0089] The term "cryopreserving" as used herein refers to freezing
a cell, embryo, or animal of the invention. The cells, embryos, or
portions of animals of the invention are frozen at temperatures
preferably lower than 0.degree. C., more preferably lower than
-80.degree. C., and most preferably at temperatures lower than
-196.degree. C. Cells and embryos in the invention can be
cryopreserved for an indefinite amount of time. It is known that
biological materials can be cryopreserved for more than fifty
years. For example, semen that is cryopreserved for more than fifty
years can be utilized to artificially inseminate a female bovine
animal. Methods and tools for cryopreservation are well-known to
those skilled in the art. See, e.g., U.S. Pat. No. 5,160,312,
entitled "Cryopreservation Process for Direct Transfer of Embryos,"
issued to Voelkel on Nov. 3, 1992.
[0090] The term "thawing" as used herein refers to the process of
increasing the temperature of a cryopreserved cell, embryo, or
portions of animals. Methods of thawing cryopreserved materials
such that they are active after the thawing process are well-known
to those of ordinary skill in the art.
[0091] The terms "transfected," "transformation," and
"transfection" as used herein refer to methods of inserting foreign
DNA into a cellular organism. These methods involve a variety of
techniques, such as treating the cells with high concentrations of
salt, an electric field, liposomes, polycationic micelles, or
detergent, to render the host cell outer membrane or wall permeable
to nucleic acid molecules of interest. Transfection techniques are
well known to a person of ordinary skill in the art and materials
and methods for carrying out transfection of DNA constructs into
cells are commercially available. Materials typically used to
transfect cells with DNA constructs are lipophilic compounds such
as Lipofectin.TM.. Particular lipophilic compounds can be induced
to form liposomes for mediating transfection of the DNA construct
into the cells. These specified methods are not limiting and the
invention relates to any transformation technique well known to a
person of ordinary skill in the art. See, e.g., Molecular Cloning,
a Laboratory Manual, 2nd Ed., 1989, Sambrook, Fritsch, and
Maniatis, Cold Spring Harbor Laboratory Press and Transgenic
Animals, Generation and Use, 1997, Edited by L. M. Houdebine,
Hardwood Academic Publishers, Australia, both of which were
previously incorporated by reference.
[0092] The term "foreign DNA" as used herein refers to DNA that can
be transfected into a target cell, where the foreign DNA harbors at
least one base pair modification as compared to the nuclear DNA of
the target organism. Foreign DNA and transfection can be further
understood and defined in conjunction with the term "modified
nuclear DNA," described previously.
[0093] The term "dissociating" as used herein refers to the
materials and methods useful for pulling a cell away from another
cell. For example, a blastomere (i.e., a cellular member of a
blastocyst stage embryo) can be pulled away from the rest of the
developing cell mass by techniques and apparatus well known to a
person of ordinary skill in the art. See, e.g., U.S. Pat. No.
4,994,384, entitled "Multiplying Bovine Embryos," issued on Feb.
19, 1991, hereby incorporated herein by reference in its entirety,
including all figures, tables, and drawings. Alternatively, cells
proliferating in culture can be separated from one another to
facilitate such processes as cell passaging, which is described
previously. In addition, dissociation of a cultured cell from a
group of cultured cells can be useful as a first step in the
process of nuclear transfer, as described hereafter. When a cell is
dissociated from an embryo, the dissociation manipulation can be
useful for such processes as re-cloning, a process described
herein, as well as a step for multiplying the number of
embryos.
[0094] In another aspect, the invention features a totipotent
mammalian cell, where the cell is immortalized, prepared by a
process comprising the steps of: (a) isolating at least one
precursor cell; and (b) introducing a stimulus to the precursor
cell that converts the precursor cell into the totipotent mammalian
cell.
[0095] The term "converts" as used herein refers to the phenomenon
in which precursor cells become immortalized and/or totipotent. The
term "convert" is synonymous with the term "reprogram" as used
herein when the precursor cell is non-immortalized and/or
non-totipotent. Precursor cells can be converted into totipotent,
immortalized cells in varying proportions. For example, it is
possible that only a small portion of precursor cells are converted
into totipotent, immortalized cells. In the art, some researchers
have discussed techniques for converting precursor cells into
pluripotent cells. Matsui et al., 1992, Cell 70: 841-847.
[0096] The term "stimulus" as used herein refers to materials
and/or methods useful for converting precursor cells into
immortalized and/or totipotent cells. The stimulus can be
electrical, mechanical, temperature-related, and/or chemical, for
example The stimulus may be a combination of one or more different
types of stimuli. As described herein in exemplary embodiments,
placing precursor cells in culture can be a sufficient stimulus to
convert precursor cells into immortalized and/or totipotent cells.
A stimulus can be introduced to precursor cells for any period of
time that accomplishes the conversion of precursor cells into
immortalized and/or totipotent cells.
[0097] The term "introduce" as used herein in reference to a
stimulus refers to a step or steps in which precursor cells are
contacted with a stimulus. If the stimulus is chemical in nature,
for example, the stimulus may be introduced to the precursor cells
by mixing the stimulus with cell culture medium.
[0098] In preferred embodiments (1) the precursor cells are
co-cultured with feeder cells; (2) the precursor cells are not
co-cultured with feeder cells; (3) the feeder cells are established
from fetal cells; (4) the fetal cells arise from a fetus where no
cell types have been removed from the fetus; (5) the fetal cells
arise from a fetus where one or more cell types have been removed
from the fetus; (6) the stimulus is introduced to precursor cells
by feeder cells; (7) the feeder cells are the only source of the
stimulus; (8) the stimulus is introduced to the precursor cells in
a mechanical fashion; (9) the only stimulus is introduced to the
precursor cells in a mechanical fashion; (10) the stimulus is
introduced to the precursor cells by feeder cells and in a
mechanical fashion; ( 11) the stimulus comprises the step of
incubating the precursor cells with a receptor ligand cocktail;
(12) the precursor cells are isolated from an ungulate animal and
preferably a bovine animal; (13) the precursor cells are selected
from the group consisting of non-embryonic cells, primordial germ
cells, genital ridge cells, amniotic cells, fetal fibroblast cells,
ovarian follicular cells, cumulus cells, hepatic cells,
differentiated cells, cells that originate from an animal,
embryonic stem cells, fetal cells, and embryonic cells; (14) the
receptor ligand cocktail comprises at least one component selected
from the group consisting of cytokine, growth factor, trophic
factor, and neurotrophic factor, LIF, and FGF; (15) the LIF has an
amino acid sequence substantially similar to the amino acid
sequence of human LIF; and (16) the FGF has an amino acid sequence
substantially similar to the amino acid sequence of bovine
bFGF.
[0099] The terms "mechanical fashion" and "mechanical stimulus" as
used herein refers to introducing a stimulus to cells where the
stimulus is not introduced by feeder cells. For example, purified
LIF and bFGF (defined hereafter) can be introduced as a stimulus to
precursor cells by adding these purified products to a cell culture
medium in which the precursor cells are growing.
[0100] The term "feeder cells" as used herein refers to cells grown
in co-culture with target cells. Target cells can be precursor
cells and totipotent cells, for example. Feeder cells can provide,
for example, peptides, polypeptides, electrical signals, organic
molecules (e.g., steroids), nucleic acid molecules, growth factors
(e.g., bFGF), other factors (e.g., cytokines such as LIF and steel
factor), and metabolic nutrients to target cells. Certain cells,
such as immortalized, totipotent cells may not require feeder cells
for healthy growth. Feeder cells preferably grow in a
mono-layer.
[0101] Feeder cells can be established from multiple cell types.
Examples of these cell types are fetal cells, mouse cells, Buffalo
rat liver cells, and oviductal cells. These examples are not meant
to be limiting. Tissue samples can be broken down to establish a
feeder cell line by methods well known in the art (e.g., by using a
blender). Feeder cells may originate from the same or different
animal species as the precursor cells. In an example of feeder
cells established from fetal cells, ungulate fetuses and preferably
bovine fetuses may be utilized to establish a feeder cell line
where one or more cell types have been removed from the fetus
(e.g., primordial germs cells, cells in the head region, and cells
in the body cavity region). When an entire fetus is utilized to
establish a fetal feeder cell line, feeder cells (e.g., fibroblast
cells) and precursor cells (e.g., primordial germ cells) can arise
from the same source (e.g., one fetus).
[0102] The term "receptor ligand cocktail" as used herein refers to
a mixture of one or more receptor ligands. A receptor ligand refers
to any molecule that binds to a receptor protein located on the
outside or the inside of a cell. Receptor ligands can be selected
from molecules of the cytokine family of ligands, neurotrophin
family of ligands, growth factor family of ligands, and mitogen
family of ligands, all of which are well known to a person of
ordinary skill in the art. Examples of receptor/ligand pairs are:
epidermal growth factor receptor/epidermal growth factor,
insulin/insulin receptor, cAMP-dependent protein kinase/cAMP,
growth hormone receptor/growth hormone, and steroid
receptor/steroid. It has been shown that certain receptors exhibit
cross-reactivity. For example, heterologous receptors, such as
insulin-like growth factor receptor 1 (IGFR1) and insulin-like
growth factor receptor 2 (IGFR2) can both bind IGF1. When a
receptor ligand cocktail comprises the stimulus, the receptor
ligand cocktail can be introduced to the precursor cell in a
variety of manners known to a person of ordinary skill in the
art.
[0103] The term "cytokine" as used herein refers to a large family
of receptor ligands well-known to a person of ordinary skill in the
art. The cytokine family of receptor ligands includes such members
as leukemia inhibitor factor (LIF), cardiotrophin 1 (CT-1), ciliary
neurotrophic factor (CNTF), stem cell factor (SCF), oncostatin M
(OSM), and any member of the interleukin (IL) family, including
IL-6, IL-11, and IL-12. The teachings of the invention do not
require the mechanical addition of steel factor (also known as stem
cell factor in the art) for the conversion of precursor cells into
totipotent cells.
[0104] The term "growth factor" as used herein refers to any
receptor ligand that causes a cell growth and/or cell proliferation
effect. Examples of growth factors are well known in the art.
Fibroblast growth factor (FGF) is one example of a growth factor.
The term "bFGF" can refer to basic FGF.
[0105] The term "substantially similar" as used herein in reference
to amino acid sequences refers to two amino acid sequences having
preferably 50% or more amino acid identity, more preferably 70% or
more amino acid identity or most preferably 90% or more amino acid
identity. Amino acid identity is a property of amino acid sequence
that measures their similarity or relationship. Identity is
measured by dividing the number of identical residues in the two
sequences by the total number of residues and multiplying the
product by 100. Thus, two copies of exactly the same sequence have
100% identity, while sequences that are less highly conserved and
have deletions, additions, or replacements have a lower degree of
identity. Those of ordinary skill in the art will recognize that
several computer programs are available for performing sequence
comparisons and determining sequence identity.
[0106] In another aspect, the invention features a method for
preparing a totipotent mammalian cell, where the cell is
immortalized, comprising the following steps: (a) isolating one or
more precursor cells; and (b) introducing the precursor cell to a
stimulus that converts the precursor cell into the totipotent cell.
Any of the embodiments defined previously herein in reference to
totipotent mammalian cells relate to the method for preparing a
totipotent mammalian cell.
[0107] Cloned Totipotent Embryos of the Invention
[0108] The invention relates in part to cloned totipotent embryos.
Hence, aspects of the invention feature cloned mammalian embryos
where (1) the embryo is totipotent; (2) the embryo arises from an
immortalized and/or totipotent cell; and (3) the embryo arises from
a non-embryonic cell; and (4) any combination of the foregoing.
[0109] The term "totipotent" as used herein in reference to embryos
refers to embryos that can develop into a live born animal. The
term "live born" is defined previously.
[0110] The term "cloned" as used herein refers to a cell, embryonic
cell, fetal cell, and/or animal cell having a nuclear DNA sequence
that is substantially similar or identical to the nuclear DNA
sequence of another cell, embryonic cell, fetal cell, and/or animal
cell. The terms "substantially similar" and "identical" are
described herein. The cloned embryo can arise from one nuclear
transfer, or alternatively, the cloned embryo can arise from a
cloning process that includes at least one re-cloning step. If the
cloned embryo arises from a cloning procedure that includes at
least one re-cloning step, then the cloned embryo can indirectly
arise from an immortalized, totipotent cell since the re-cloning
step can utilize embryonic cells isolated from an embryo that arose
from an immortalized, totipotent cell.
[0111] In preferred embodiments, (1) the cloned mammalian embryo is
preferably an ungulate embryo and more preferably a bovine embryo;
(2) the cloned bovine embryo can be one member of a plurality of
embryos, where the plurality of embryos share a substantially
similar nuclear DNA sequence; (3) the cloned mammalian embryo can
be one member of a plurality of embryos, and the plurality of
embryos can have an identical nuclear DNA sequence; (4) the cloned
mammalian embryo has a nuclear DNA sequence that is substantially
similar to a nuclear DNA sequence of a live born mammalian animal;
(5) one or more cells of the cloned mammalian embryo have modified
nuclear DNA; (6) the cloned mammalian embryo is subject to
manipulation; (7) the manipulation comprises the step of culturing
the embryo in a suitable medium; (8) the suitable medium for
culturing the embryo is CR-2 medium; (9) the medium can comprise
feeder cells; (10) the manipulation of an embryo comprises the step
of implanting the embryo into the uterus of a female; (11) the
female animal is preferably an ungulate animal and more preferably
a bovine animal; (12) the estrus cycle of the female is
synchronized with the development cycle of the embryo; and (13) the
manipulation comprises the step of incubating the embryo in an
artificial environment.
[0112] All preferred embodiments related to modified nuclear DNA
for totipotent cells of the invention extend to cloned embryos of
the invention. In addition, any of the manipulations described in
conjunction with totipotent cells of the invention apply to cloned
embryos of the invention.
[0113] The term "substantially similar" as used herein in reference
to nuclear DNA sequences refers to two nuclear DNA sequences that
are nearly identical. The two sequences may differ by copy error
differences that normally occur during the replication of a nuclear
DNA. Substantially similar DNA sequences are preferably greater
than 97% identical, more preferably greater than 98% identical, and
most preferably greater than 99% identical. The term "identity" as
used herein in reference to nuclear DNA sequences can refer to the
same usage of the term in reference to amino acid sequences, which
is described previously herein.
[0114] The term "plurality" as used herein in reference to embryos
refers to a set comprising at least two embryos having a
substantially similar nuclear DNA sequence. In preferred
embodiments, the plurality consists of five or more embryos, ten or
more embryos, one-hundred or more embryos, or one-thousand or more
embryos. Because the occurrence of more than three embryos
progressing to term only occurs with a probability of approximately
1/100,000, a plurality of at least five embryos or animals relates
to cloned embryos or cloned animals rather than naturally occurring
embryos or animals.
[0115] The term "culturing" as used herein with respect to embryos
refers to laboratory procedures that involve placing an embryo in a
culture medium. The embryo can be placed in the culture medium for
an appropriate amount of time to allow the embryo to remain static
but functional in the medium, or to allow the embryo to grow in the
medium. Culture media suitable for culturing embryos are well-known
to those skilled in the art. See, e.g., U.S. Pat. No. 5,213,979,
entitled "In vitro Culture of Bovine Embryos," First et al., issued
May 25, 1993, and U.S. Pat. No. 5,096,822, entitled "Bovine Embryo
Medium," Rosenkrans, Jr. et al., issued Mar. 17, 1992, incorporated
herein by reference in their entireties including all figures,
tables, and drawings.
[0116] The term "suitable medium" as used herein refers to any
medium that allows cell proliferation. The suitable medium need not
promote maximum proliferation, only measurable cell proliferation.
A suitable medium for embryo development is discussed
previously.
[0117] The term "CR-2 medium" as used herein refers to a medium
suitable for culturing embryos. CR-2 medium can comprise one or
more of the following components: sodium chloride; potassium
chloride; sodium bicarbonate; hemicalcium L-lactate; and fatty-acid
free BSA. These components may exist in the medium in
concentrations of about 115 mM for sodium chloride; about 3 mM for
potassium chloride; about 25 mM for sodium bicarbonate; about 5 mM
for hemicalcium L-lactate; and about 3 mg/mL for fatty-acid free
BSA. Alternatively, the concentrations of these components may
exist in the medium in concentrations of 0-1M sodium chloride;
0-100 mM potassium chloride; 0-500 mM sodium bicarbonate; 0-100 mM
hemicalcium L-lactate; and 0-100 mg/mL fatty-acid free BSA.
[0118] The term "feeder cells" is defined previously herein.
Embryos of the invention can be cultured in media with or without
feeder cells. In other preferred embodiments, the feeder cells can
be cumulus cells.
[0119] The term "implanting" as used herein in reference to embryos
refers to impregnating a female animal with an embryo described
herein. This technique is well known to a person of ordinary skill
in the art. See, e.g., Seidel and Elsden, 1997, Embryo Transfer in
Dairy Cattle, W. D. Hoard & Sons, Co., Hoards Dairyman. The
embryo may be allowed to develop in utero, or alternatively, the
fetus may be removed from the uterine environment before
parturition.
[0120] The term "synchronized" as used herein in reference to
estrus cycle, refers to assisted reproductive techniques well known
to a person of ordinary skill in the art. These techniques are
fully described in the reference cited in the previous paragraph.
Typically, estrogen and progesterone hormones are utilized to
synchronize the estrus cycle of the female animal with the
developmental cycle of the embryo. The term "developmental cycle"
as used herein refers to embryos of the invention and the time
period that exists between each cell division within the embryo.
This time period is predictable for embryos from ungulates, and can
be synchronized with the estrus cycle of a recipient animal.
[0121] The term "artificial environment" refers to one that
promotes the development of an embryo or other developing cell
mass. An artificial environment can be a uterine environment or an
oviductal environment of a species different from that of the
developing cell mass. For example, a developing bovine embryo can
be placed into the uterus or oviduct of an ovine animal. Stice
& Keefer, 1993, "Multiple generational bovine embryo cloning,"
Biology of Reproduction 48: 715-719. Alternatively, an artificial
development environment can be assembled in vitro. This type of
artificial uterine environment can be synthesized using biological
and chemical components known in the art.
[0122] In another aspect the invention features a cloned mammalian
embryo, where the embryo is totipotent, prepared by a process
comprising the step of nuclear transfer. Preferably, nuclear
transfer occurs between (a) a totipotent mammalian cell, where the
cell is immortalized, and (b) an oocyte, where the oocyte is at a
stage allowing formation of the embryo.
[0123] In preferred embodiments, (1) the oocyte is an enucleated
oocyte; (2) the totipotent mammalian cell and the oocyte preferably
originate from an ungulate animal and more preferably originate
from a bovine animal; (3) the totipotent mammalian cell can
originate from one specie of ungulate and the oocyte can originate
from another specie of ungulate; (4) the oocyte is a young oocyte;
(5) the totipotent mammalian cell is placed in the perivitelline
space of the oocyte; (6) the totipotent cell utilized for nuclear
transfer can arise from any of the cells described previously
(e.g., a non-embryonic cell, a primordial germ cell, a genital
ridge cell, a differentiated cell, a fetal cell, a non-fetal cell,
a non-primordial germ cell, an amniotic cell, a fetal fibroblast
cell, an ovarian follicular cell, a cumulus cell, an hepatic cell,
a cell isolated from an asynchronous population of cells, a cell
isolated from a synchronous population of cells, a cell isolated
from an existing animal, and an embryonic stem cell); (7) the
nuclear transfer comprises the step of translocation of the
totipotent mammalian cell into the recipient oocyte; (8) the
translocation can comprise the step of injection of the totipotent
mammalian cell nuclear donor into the recipient oocyte; (9) the
translocation can comprise the step of fusion of the totipotent
mammalian cell and the oocyte; (10) the fusion can comprise the
step of delivering one or more electrical pulses to the totipotent
mammalian cell and the oocyte; (11) the fusion can comprise the
step of delivering a suitable concentration of at least one fusion
agent to the totipotent mammalian cell and the oocyte; (12) the
nuclear transfer may comprise the step of activation of the
totipotent mammalian cell and the oocyte; and (13) the activation
is accomplished by introducing DMAP and/or ionomycin to an oocyte
and/or a cybrid.
[0124] The term "enucleated oocyte" as used herein refers to an
oocyte which has had part of its contents removed. Typically a
needle can be placed into an oocyte and the nucleus can be
aspirated into the inner space of the needle. The needle can be
removed from the oocyte without rupturing the plasma membrane. This
enucleation technique is well known to a person of ordinary skill
in the art. See, U.S. Pat. No. 4,994,384; U.S. Pat. No. 5,057,420;
and Willadsen, 1986, Nature 320:63-65. An enucleated oocyte can be
prepared from a young or an aged oocyte.
[0125] Definitions of "young oocyte" and "aged oocyte" are provided
herein. Nuclear transfer may be accomplished by combining one
nuclear donor and more than one enucleated oocyte. In addition,
nuclear transfer may be accomplished by combining one nuclear
donor, one or more enucleated oocytes, and the cytoplasm of one or
more enucleated oocytes.
[0126] The term "cybrid" as used herein refers to a construction
where an entire nuclear donor is translocated into the cytoplasm of
a recipient oocyte. See, e.g. In Vitro Cell. Dev. Biol. 26: 97-101
(1990).
[0127] The invention specifically relates to cloned mammalian
embryos created by nuclear transfer, where the nucleus of the
oocyte is not physically extracted from the nucleus. It is possible
to create a cloned embryo where the nuclear DNA from the donor cell
is the material replicated during cellular divisions. See, e.g.,
Wagoner et a!., 1996, "Functional enucleation of bovine oocytes:
effects of centrifugation and ultraviolet light," Theriogenology
46: 279-284.
[0128] The term "another ungulate" as used herein refers to a
situation where the nuclear donor originates from an ungulate of a
different species, genera or family than the ungulate from which
the recipient oocyte originates. For example, the totipotent
mammalian cell used as a nuclear donor can arise from a water
buffalo, while the oocyte recipient can arise from a domestic cow.
This example is not meant to be limiting and any ungulate
species/family combination of nuclear donors and recipient oocytes
are foreseen by the invention.
[0129] The term "young oocyte" as used herein refers to an oocyte
that has been matured in vitro and/or ovulated in vivo for less
than 28 hours since the onset of maturation. Oocytes can be
isolated from live animals using methods well known to a person of
ordinary skill in the art. See, e.g., Pieterse et al., 1988,
"Aspiration of bovine oocytes during transvaginal ultrasound
scanning of the ovaries," Theriogenology 30: 751-762. Oocytes can
be isolated from ovaries or oviducts or deceased or live born
animals. Suitable media for in vitro culture of oocytes are well
known to a person of ordinary skill in the art. See, e.g., U.S.
Pat. No. 5,057,420, which is incorporated by reference herein.
[0130] The term "maturation" as used herein refers to process in
which an oocyte is incubated in a medium in vitro. Oocytes can be
incubated with multiple media well known to a person of ordinary
skill in the art. See, e.g., Saito et al., 1992, Roux's Arch. Dev.
Biol. 201: 134-141 for bovine organisms and Wells et al., 1997,
Biol. Repr. 57: 385-393 for ovine organisms, both of which are
incorporated herein by reference in their entireties including all
figures, tables, and drawings. Maturation media can comprise
multiple types of components, including microtubule inhibitors
(e.g., cytochalasin B). Other examples of components that can be
incorporated into maturation media are discussed in WO 97/07668,
entitled "Unactivated Oocytes as Cytoplast Recipients for Nuclear
Transfer," Campbell & Wilmut, published on Mar. 6, 1997, hereby
incorporated herein by reference in its entirety, including all
figures, tables, and drawings. The time of maturation can be
determined from the time that an oocyte is placed in a maturation
medium and the time that the oocyte is then utilized in a nuclear
transfer procedure.
[0131] Young oocytes can be identified by the appearance of their
ooplasm. Because certain cellular material (e.g., lipids) have not
yet dispersed within the ooplasm. Young oocytes can have a pycnotic
appearance. A pycnotic appearance can be characterized as clumping
of cytoplasmic material. A "pycnotic" appearance is to be
contrasted with the appearance of oocytes that are older than 28
hours, which have a more homogenous appearing ooplasm.
[0132] The term "translocation" as used herein in reference to
nuclear transfer refers to the combining of a totipotent mammalian
cell nuclear donor and a recipient oocyte. The translocation may be
performed by such techniques as fusion and/or direct injection, for
example.
[0133] The term "injection" as used herein in reference to embryos,
refers to the perforation of the oocyte with a needle, and
insertion of the nuclear donor in the needle into the oocyte. In
preferred embodiments, the nuclear donor may be injected into the
cytoplasm of the oocyte or in the perivitelline space of the
oocyte. This direct injection approach is well known to a person of
ordinary skill in the art, as indicated by the publications already
incorporated herein in reference to nuclear transfer. For the
direct injection approach to nuclear transfer, the whole totipotent
mammalian cell may be injected into the oocyte, or alternatively, a
nucleus isolated from the totipotent mammalian cell may be injected
into the oocyte. Such an isolated nucleus may be surrounded by
nuclear membrane only, or the isolated nucleus may be surrounded by
nuclear membrane and plasma membrane in any proportion. The oocyte
may be pre-treated to enhance the strength of its plasma membrane,
such as by incubating the oocyte in sucrose prior to injection of
the nuclear donor.
[0134] Techniques for placing a nuclear donor (e.g., an
immortalized and totipotent cell of the invention) into the
perivitelline space of an enucleated oocyte are well known to a
person of ordinary skill in the art, and are fully described in the
patents and references cited previously herein in reference to
nuclear transfer.
[0135] The term "fusion" as used herein refers to the combination
of portions of lipid membranes corresponding to the totipotent
mammalian cell nuclear donor and the recipient oocyte. Lipid
membranes can correspond to the plasma membranes of cells or
nuclear membranes, for example. The fusion can occur between the
nuclear donor and recipient oocyte when they are placed adjacent to
one another, or when the nuclear donor is placed in the
perivitelline space of the recipient oocyte, for example. Specific
examples for translocation of the totipotent mammalian cell into
the oocyte are described hereafter in other preferred embodiments.
These techniques for translocation are fully described in the
references cited previously herein in reference to nuclear
transfer.
[0136] The term "electrical pulses" as used herein refers to
subjecting the nuclear donor and recipient oocyte to electric
current. For nuclear transfer, the nuclear donor and recipient
oocyte can be aligned between electrodes and subjected to
electrical current. The electrical current can be alternating
current or direct current. The electrical current can be delivered
to cells for a variety of different times as one pulse or as
multiple pulses. The cells are typically cultured in a suitable
medium for the delivery of electrical pulses. Examples of
electrical pulse conditions utilized for nuclear transfer are
described in the references and patents previously cited herein in
reference to nuclear transfer.
[0137] The term "fusion agent" as used herein refers to any
compound or biological organism that can increase the probability
that portions of plasma membranes from different cells will fuse
when a totipotent mammalian cell nuclear donor is placed adjacent
to the recipient oocyte. In preferred embodiments fusion agents are
selected from the group consisting of polyethylene glycol (PEG),
trypsin, dimethylsulfoxide (DMSO), lectins, agglutinin, viruses,
and Sendai virus. These examples are not meant to be limiting and
other fusion agents known in the art are applicable and included
herein.
[0138] The term "suitable concentration" as used herein in
reference to fusion agents, refers to any concentration of a fusion
agent that affords a measurable amount of fusion. Fusion can be
measured by multiple techniques well known to a person of ordinary
skill in the art, such as by utilizing a light microscope, dyes,
and fluorescent lipids, for example.
[0139] The term "activation" refers to any materials and methods
useful for stimulating a cell to divide before, during, and after a
nuclear transfer step. Cybrids may require stimulation in order to
divide after a nuclear transfer has occurred. The invention
pertains to any activation materials and methods known to a person
of ordinary skill in the art. Although electrical pulses are
sometimes sufficient for stimulating activation of cybrids, other
means are sometimes useful or necessary for proper activation of
the cybrid. Chemical materials and methods useful for activating
embryos are described below in other preferred embodiments of the
invention.
[0140] Examples of non-electrical means for activation include
agents such as ethanol; inositol trisphosphate (IP.sub.3);
Ca.sup.++ ionophores (e.g., ionomycin) and protein kinase
inhibitors (e.g., 6-dimethylaminopurine (DMAP)); temperature
change; protein synthesis inhibitors (e.g., cyclohexamide); phorbol
esters such as phorbol 12-myristate 13-acetate (PMA); mechanical
techniques; and thapsigargin. The invention includes any activation
techniques known in the art. See, e.g., U.S. Pat. No. 5,496,720,
entitled "Parthenogenic Oocyte Activation," issued on Mar. 5, 1996,
Susko-Parrish et al., incorporated by reference herein in its
entirety, including all figures, tables, and drawings.
[0141] In other preferred embodiments, (1) one or more cells of the
cloned embryo comprise modified nuclear DNA; (2) the cloned embryo
is subject to manipulation; (3) the manipulation comprises the step
of disaggregating at least one individual cell from a cloned
embryo; (4) the manipulation comprises the step of utilizing the
individual cell as a nuclear donor in a nuclear transfer procedure;
(5) the individual cell is disaggregated from the inner cell mass
of a blastocyst stage embryo; (6) the individual cell is
disaggregated from a pre-blastocyst stage embryo; (7) the
manipulation comprises the process of re-cloning; (8) the recloning
process comprises the steps of: (a) separating the embryo into one
or more individual cells, and (b) performing at least one
subsequent nuclear transfer between (i) an individual cell of (a),
and (ii) an oocyte; (9) the oocyte utilized for the subsequent
nuclear transfer is an aged oocyte; (10) the individual cell is
placed in the perivitelline space of the enucleated oocyte for the
subsequent nuclear transfer; (11) the subsequent nuclear transfer
comprises at least one of the steps of translocation, injection,
fusion, and activation of the individual cell and/or the enucleated
oocyte; (12) one or more cells of the cloned mammalian embryo
arising from the subsequent nuclear transfer comprises modified
nuclear DNA; and (13) the cloned mammalian embryo arising from the
subsequent nuclear transfer may be subject to a subsequent
manipulation, where the subsequent manipulation is any of the
manipulation steps defined previously herein in relation to
immortalized cells and/or cloned embryos.
[0142] The term "individual cells" as used herein refers to cells
that have been isolated from a cloned mammalian embryo of the
invention. An individual single cell can be isolated from the rest
of the embryonic mass by techniques well known to those skilled in
the art. See, U.S. Pat. Nos. 4,994,384 and 5,957,420, previously
incorporated herein by reference in their entireties.
[0143] The term "subsequent nuclear transfer" as described herein
is also referred to as a "re-cloning" step. Preferably, a
re-cloning step can be utilized to enhance nuclear reprogramming
during nuclear transfer, such that the product of nuclear transfer
is a live born animal. The re-cloning step is distinct, since
previous efforts towards re-cloning have been directed to
multiplying embryo number and not for enhancement of nuclear
reprogramming. The number of subsequent nuclear transfer steps is
discussed in greater detail hereafter.
[0144] Any of the preferred embodiments related to the
translocation, injection, fusion, and activation steps described
previously herein relate to the subsequent nuclear transfer
step.
[0145] The term "inner cell mass" as used herein refers to the
cells that gives rise to the embryo proper. The cells that line the
outside of a blastocyst are referred to as the trophoblast of the
embryo. The methods for isolating inner cell mass cells from an
embryo are well known to a person of ordinary skill in the art.
See, Sims and First, 1993, Theriogenology 39:313; and Keefer et
al., 1994, Mol. Reprod. Dev. 38:264-268, hereby incorporated by
reference herein in their entireties, including all figures,
tables, and drawings. The term "pre-blastocyst" is well known in
the art and is referred to previously.
[0146] The term "aged oocyte" as used herein refers to an oocyte
that has been matured in vitro or ovulated in vivo for more than 28
hours since the onset of maturation or ovulation. An aged oocyte
can be identified by its characteristically homogenous ooplasm.
This appearance is to be contrasted with the pycnotic appearance of
young oocytes as described previously herein. The age of the oocyte
can be defined by the time that has elapsed between the time that
the oocyte is placed in a suitable maturation medium and the time
that the oocyte is activated. The age of the oocyte can
dramatically enhance the efficiency of nuclear transfer.
[0147] The term "ovulated in vivo" as used herein refers to an
oocyte that is isolated from an animal a certain number of hours
after the animal exhibits characteristics that it is in estrus. The
characteristics of an animal in estrus are well known to a person
of ordinary skill in the art, as described in references disclosed
herein.
[0148] In another aspect the invention relates to a method for
preparing a cloned mammalian embryo. The method comprises the step
of a nuclear transfer between: (a) a totipotent mammalian cell,
where the cell is immortalized; and (b) an oocyte, where the oocyte
is at a stage allowing formation of the embryo. In preferred
embodiments, any of the embodiments of the invention concerning
cloned mammalian embryos apply to methods for preparing cloned
mammalian embryos.
[0149] Cloned Fetuses of the Invention
[0150] In another aspect, the invention features cloned mammalian
fetuses arising from totipotent embryos of the invention.
Preferably, the mammalian fetuses are ungulate fetuses, and more
preferably, the ungulate fetuses are bovine fetuses. A fetus may be
isolated from the uterus of a pregnant female animal.
[0151] In preferred embodiments, (1) one or more cells of the
fetuses harbor modified nuclear DNA (defined previously herein);
and (2) the fetuses may be subject to any of the manipulations
defined herein. For example, one manipulation may comprise the
steps of isolating a fetus from the uterus of a pregnant female
animal, isolating a cell from the fetus (eg., a primordial germ
cell), and utilizing the isolated cell as a nuclear donor for
nuclear transfer.
[0152] Other aspects of the invention feature (1) a cloned
mammalian fetus prepared by a process comprising the steps of (a)
preparation of a cloned mammalian embryo defined previously, and
(b) manipulation of the cloned mammalian embryo such that it
develops into a fetus; (2) a method for preparing a cloned
mammalian fetus comprising the steps of (a) preparation of a cloned
mammalian embryo defined previously, and (b) manipulation of the
cloned mammalian embryo such that it develops into a fetus; (3) a
method of using a cloned fetus of the invention comprising the step
of isolating at least one cell type from a fetus (eg., for creating
a feeder cell layer); and (4) a method of using a cloned fetus of
the invention comprising the step of separating at least one part
of a fetus into individual cells (e.g., for establishing a feeder
cell layer).
[0153] Cloned Animals of the Invention
[0154] In another aspect the invention features a cloned mammalian
animal arising from a cloned embryo of the invention. The embryo is
totipotent and can arise from any of the processes or methods
described previously herein.
[0155] In preferred embodiments, the cloned mammalian animal (1) is
preferably a cloned ungulate animal and more preferably a cloned
bovine animal; and (2) is equal in age or older than an animal
selected from the group consisting of pre-and post-pubertal
animals.
[0156] In yet another aspect the invention relates to a cloned
mammalian animal, where the animal is one member of a plurality of
animals, and where the plurality of animals have a substantially
similar nuclear DNA sequence. The term "substantially similar" in
relation to nuclear DNA sequences is defined previously herein.
[0157] In preferred embodiments, (1) the plurality consists of five
or more animals, ten or more animals, one-hundred or more animals,
and one-thousand or more animals; and (2) the plurality of animals
can have an identical nuclear DNA sequence. The term "identical" in
reference to nuclear DNA sequences is described previously
herein.
[0158] In another aspect, the invention relates to a cloned
mammalian animal having one or more cells that comprise modified
nuclear DNA. All of the preferred embodiments relating to modified
nuclear DNA described previously apply to cloned bovine animals of
the invention.
[0159] In yet another aspect, the invention features a method of
using a cloned mammalian animal, comprising the step of isolating
at least one component from the mammalian animal.
[0160] The term "component" as used herein refers to any portion of
an animal. A component can be selected from the group consisting of
fluid, biological fluid, cell, tissue, organ, gamete, embryo, and
fetus. Precursor cells may arise from fluids, biological fluids,
cells, tissues, organs, gametes, embryos, and fetuses isolated from
cloned organisms of the invention.
[0161] The term "gamete" as used herein refers to any cell
participating, directly or indirectly, to the reproductive system
of an animal. Examples of gametes are spermatocytes, spermatogonia,
oocytes, and oogonia. Gametes can be present in fluids, tissues,
and organs collected from animals (e.g., sperm is present in
semen). For example, methods of collecting semen for the purposes
of artificial insemination are well known to a person of ordinary
skill in the art. See, e.g., Physiology of Reproduction and
Artificial Insemination of Cattle (2nd edition), Salisbury et al.,
copyright 1961, 1978, W H Freeman & Co., San Francisco.
However, the invention relates to the collection of any type of
gamete from an animal.
[0162] The term "tissue" is defined previously. The term "organ"
refers to any organ isolated from an animal or any portion of an
organ. Examples of organs and tissues are neuronal tissue, brain
tissue, spleen, heart, lung, gallbladder, pancreas, testis, ovary
and kidney. These examples are not limiting and the invention
relates to any organ and any tissue isolated from a cloned animal
of the invention.
[0163] In a preferred embodiments, the invention relates to (1)
fluids, biological fluids, cells, tissues, organs, gametes,
embryos, and fetuses can be subject to manipulation; (2) the
manipulation can comprise the step of cryopreserving the gametes,
embryos, and/or fetal tissues; (3) the manipulation can comprise
the step of thawing the cryopreserved items; (4) the manipulation
can comprise the step of separating the semen into X-chromosome
bearing semen and Y-chromosome bearing semen; (5) the manipulation
comprises methods of preparing the semen for artificial
insemination; (6) the manipulation comprises the step of
purification of a desired polypeptide(s) from the biological fluid
or tissue; (7) the manipulation comprises concentration of the
biological fluids or tissues; and (8) the manipulation can comprise
the step of transferring one or more cloned cells, cloned tissues,
cloned organs, and/or portions of cloned organs to a recipient
organism (e.g., the recipient organism may be of a different specie
than the donor source).
[0164] The term "separating" as used herein in reference to
separating semen refers to methods well known to a person skilled
in the art for fractionating a semen sample into sex-specific
fractions. This type of separation can be accomplished by using
flow cytometers that are commercially available. Methods of
utilizing flow cytometers from separating sperm by genetic content
are well known in the art. In addition, semen can be separated by
its sex-associated characteristics by other methods well known to a
person of ordinary skill in the art. See, U.S. Pat. Nos. 5,439,362,
5,346,990, and 5,021,244, entitled "Sex-Associated Membrane
Proteins and Methods for Increasing the Probability that Offspring
Will Be of a Desired Sex," Spaulding, issued on Aug. 8, 1995, Sept.
13, 1994, and Jun. 4, 1991 respectively, all of which are
incorporated herein by reference in their entireties including all
figures, tables, and drawings.
[0165] Semen preparation methods are well known to someone of
ordinary skill in the art. Examples of these preparative steps are
described in Physiology of Reproduction and Artificial Insemination
of Cattle (2nd. edition), Salisbury et al., copyright 1961, 1978,
W. H. Freeman & Co., San Francisco.
[0166] The term "purification" as used herein refers to increasing
the specific activity of a particular polypeptide or polypeptides
in a sample. In preferred embodiments, specific activity is
expressed as the ratio between the activity of the target
polypeptide and the concentration of total polypeptide in the
sample. Activity can be catalytic activity and/or binding activity,
for example. In other preferred embodiments, specific activity is
expressed as the ratio between the concentration of the target
polypeptide and the concentration of total polypeptide.
Purification methods include dialysis, centrifugation, and column
chromatography techniques, which are well-known procedures to a
person of ordinary skill in the art. See, e.g., Young et al., 1997,
"Production of biopharmaceutical proteins in the milk of transgenic
dairy animals," BioPharm 10(6): 34-38.
[0167] The term "transferring" as used herein refers to shifting a
group of cells, tissues, organs, and/or portions of organs to an
animal. The cells, tissues, organs, and/or portions of organs can
be, for example, (a) developed in vitro and then transferred to an
animal, (b) removed from an animal and transferred to another
animal of a different specie, (c) removed from an animal and
transferred to another animal of the same specie, (d) removed from
one portion of an animal (e.g., the leg of an animal) and then
transferred to another portion of the same animal (e.g., the brain
of the animal), and/or (e) any combination of the foregoing. The
term "transferring" refers to adding cells, tissues, and/or organs
to an animal and can also relate to removing cells, tissues, and/or
organs from an animal and replacing them with cells, tissues,
and/or organs from another source.
[0168] The term "transferring" as used herein also refers to
implanting one or more cells, tissues, organs, and/or portions of
organs from the cloned mammalian animal into another organism. For
example, neuronal tissue from a cloned mammalian organism can be
grafted into an appropriate area in the human nervous system to
treat neurological diseases such as Alzheimer's disease.
Alternatively, cloned cells, tissues, and/or organs originating
from a porcine organism may be transferred to a human recipient.
Surgical methods for accomplishing this preferred aspect of the
invention are well known to a person of ordinary skill in the art.
Transferring procedures may include the step of removing cells,
tissues, or organs from a recipient organism before a transfer
step.
[0169] In other aspects the invention features (1) a cloned
mammalian animal prepared by a process comprising the steps of: (a)
preparation of a cloned mammalian embryo by any one of the methods
described herein for producing such a cloned mammalian embryo, and
(b) manipulation of the cloned mammalian embryo such that it
develops into a live born animal; (2) a process comprising the
steps of: (a) preparation of a cloned mammalian embryo by any one
of the methods described herein for preparing such a cloned
mammalian embryo, and (b) manipulation of the cloned mammalian
embryo such that it develops into a live born animal; and (3) a
cloned mammalian animal, comprising the steps of: (a) preparation
of a cloned mammalian embryo by any one of the methods for
producing such an embryo described herein, and (b) manipulation of
the cloned mammalian embryo such that it develops into a live born
animal.
[0170] In preferred embodiments, (1) the live born animal is
preferably an ungulate animal and more preferably a bovine animal;
(2) the manipulation can comprise the step of implanting the embryo
into a uterus of an animal; (3) the estrus cycle of the animal can
be synchronized to the developmental stage of the embryo; and (4)
the manipulation can comprise the step of implanting the embryo
into an artificial environment.
[0171] The summary of the invention described above is not limiting
and other features and advantages of the invention will be apparent
from the following detailed description of the preferred
embodiments, as well as from the claims.
BRIEF DESCRIPTION OF THE FIGURES
[0172] FIG. 1 illustrates multiple embodiments of the invention
relating to the generation of immortalized, totipotent cells from
precursor cells. The figure indicates that immortalized, totipotent
cells can arise from embryonic stem cells, primordial germ cells,
and cells isolated from an animal. The precursor cell sources
illustrated by FIG. 1 are not limiting and other precursor cell
sources are described herein.
[0173] FIG. 2 illustrates an embodiment of the invention related to
cloning. The figure illustrates a cloning procedure in which (a) a
precursor cell is reprogrammed into an immortalized, totipotent
cell; (b) the immortalized, totipotent cell is utilized as a donor
for a first nuclear transfer, which utilizes a young oocyte; (c)
the embryo arising from the first nuclear transfer is cultured; (d)
a cell isolated from the embryo arising from the first nuclear
transfer is utilized as a nuclear donor for a second nuclear
transfer, which utilizes an aged oocyte; and (e) the embryo
resulting from the second nuclear transfer may be cultured and then
allowed to develop into a live born animal. The embryos resulting
from the nuclear transfers may be cultured and/or cryopreserved and
thawed.
[0174] FIG. 3 illustrates multiple embodiments of the invention
related to pathways for establishing totipotent cell lines and
cloned animals. Fibroblast cells can be isolated from any source
described herein. This figure is described in further detail in the
Examples section.
[0175] FIG. 4 illustrates multiple embodiments of the invention for
creating cloned transgenic cell lines and cloned transgenic
animals. This figure is described in further detail in the Examples
section.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0176] The present invention relates to cloning technologies. The
present invention provides multiple advantages over the tools and
methods currently utilized in the field of cloning technology. For
example, the invention relates in part to immortalized, totipotent
cells useful for cloning animals. These immortalized, totipotent
cells can give rise to methods of producing cloned animals by
utilizing virtually any type of cell. For example, cells isolated
from a live born animal can be reprogrammed into immortalized,
totipotent cells. This feature of the invention provides the
ability to assess the phenotype of an existing animal and then
readily establish a permanent cell line for cloning that animal. As
described previously herein, no methods in the art have allowed for
such advantages.
[0177] In addition, the immortalized, totipotent cells of the
invention allow for creating permanent cell lines from virtually
any type of cell. This reprogramming method is described previously
herein. These permanent cell lines offer a nearly unlimited source
of donor cells for nuclear transfer cloning techniques. Moreover,
this feature provides the advantage of enhancing cloning efficiency
due to the tower differentiation rates of these cell lines than
existing cell lines used for cloning. For example, embryonic stem
cell lines can harbor multiple colonies of cells that are not
totipotent. The totipotent, immortalized cells of the invention
harbor a higher percentage of totipotent cells than cell lines
previously reported.
[0178] Moreover, the methods and processes for creating the
immortalized, totipotent cells, totipotent cloned embryos, and
cloned animals of the invention demonstrate the enhanced cloning
efficiency over cloning tools and techniques previously reported.
In particular, the totipotent, immortalized cell lines and the
refined nuclear transfer techniques of the invention provide for
this enhanced cloning efficiency. This enhanced efficiency
satisfies a long felt need in the art.
[0179] I. Immortalized and Totipotent Cells
[0180] A. Generation of Immortalized and Totipotent Cells
[0181] Immortalized, totipotent cells of the invention can be
produced from virtually any type of precursor cell. Preferred
embodiments of the invention relate to the following types of
precursor cells: (1) embryos arising from the union of two gametes
in vitro or in vivo; (2) embryonic stem cells (ESC's) arising from
embryos (e.g., pre-blastocyst cells and inner cell mass cells); (3)
cultured and non-cultured cells derived from the inner cell mass of
embryos; (4) cultured and non-cultured cells arising from a fetus;
(5) primordial germ cells arising from a developing cell mass
(e.g., genital ridge cells); (6) immortalized cultured cells
arising from primordial germ cells, where the immortalized cells
are referred to as embryonic germ cells (EGCs) in the art; (7)
cultured and non-cultured cells obtained from amniotic fluid; (8)
cultured and non-cultured cells arising from an ovarian follicle
(e.g., cumulus cells); (9) cultured and non-cultured cells arising
from a liver (e.g., hepatocytes); and (10) cultured and
non-cultured cells isolated from an animal.
[0182] ESCs and EGCs can be readily generated from methods known in
the art. See, e.g., Stice et al., 1996, Biology of Reproduction 54:
100-110, hereby incorporated by reference herein in its entirety
including all figures, tables, and drawings. See also, Strelchenko,
1996, Theriogenology 45: 130-141. ESCs have been demonstrated to
give rise to fetuses, from which primordial germ cells and EGCs can
be derived. Therefore, ESCs are a nearly unlimited source for
primordial germ cells and EGCs.
[0183] Cells derived from an animal can be isolated from nearly any
type of tissue. For example, an ear-punch can be taken from an
animal, the cells from the sample can be separated, and the
separated cells can be subsequently cultured in vitro by using cell
culture techniques well known to a person of ordinary skill in the
art. Preferably, cells of the invention are extracted from bovine
animals. Examples of materials and methods for reprogramming
primary culture cells into immortalized, totipotent cells are
described in exemplary embodiments hereafter.
[0184] Although exemplary embodiments of the invention are directed
to bovine animals, materials and methods of the invention can be
applied to the generation of immortalized, totipotent cells using
precursor cells isolated from any mammal. Preferably immortalized,
totipotent cells are extracted from ungulates. Examples of
preferred ungulates envisioned for the invention are described
previously.
[0185] Immortalized, totipotent cells of the invention are
preferably generated from the examples of cells indicated in the
preceding paragraph after treatment with a receptor ligand
cocktail. Examples of receptor ligands are well known to a person
of ordinary skill in the art. Cytokines and/or growth factors are
preferred receptor ligands of the invention. See, e.g., R&D
Systems Catalog, 614 McKinley Place N.E., Minneapolis, Minn. 55413.
In exemplary embodiments, varying amounts of human recombinant
leukemia inhibitory factor (hrLIF) and basic bovine fibroblast
growth factor (bFGF) can be added to the culture medium to
reprogram the precursor cells into immortalized, totipotent cells.
Varying concentrations of these two cytokines can be added to the
culture medium, preferably in concentrations of 1 -1000 ng/mL, more
preferably in concentrations between 10-500 ng/mL, and most
preferably about 100 ng/mL. Exogenous soluble and
membrane-associated forms of steel factor are not required for
converting precursor cells into totipotent, immortalized cells.
[0186] These examples are not meant to be limiting and any cytokine
or combination of cytokines can be added or deleted from those
described in exemplary embodiments described hereafter. Preferred
cytokines for generating immortalized, totipotent cells can be
selected from the group consisting of fibroblast growth factor
(FGF), leukemia inhibitor factor (LIF), cardiotrophin 1 (CT-1),
ciliary neurotrophic factor (CNTF), stem cell factor (SCF),
oncostatin M (OSM), and any member of the interleukin (IL) family,
including IL-6, IL-11, and IL-12.
[0187] Other cytokines and other molecules besides cytokines can be
added or deleted from the receptor ligand cocktail described in the
exemplary embodiments described hereafter to create immortalized,
totipotent cells from any of the cells described in the previous
paragraph. Any of the conditions for generating immortalized,
totipotent cells can be modified from those described herein. The
ability of these modified conditions to generate immortalized,
totipotent cells can be monitored by methods defined in the section
"Identification of Immortalized and Totipotent Cells" described
hereafter.
[0188] B. Culturing Immortalized and Totipotent Cells
[0189] A variety of methods for culturing cells exist in the art.
See, e.g. Culture of animal cells: a manual of basic technique
(2nd. edition), Freshney, copyright 1987, Alan R. Liss, Inc., New
York. Particularly the cells that are precursor cells for
immortalized, totipotent cells, as well as the immortalized,
totipotent cells themselves, can be grown on feeder layers.
Examples of feeder layers are well known to a person of ordinary
skill in the art, and can arise from a number of different cells
that are cultured in vitro. See, e.g., Strelchenko, 1996,
Theriogenology 45: 130-141, as well as exemplary embodiments
described hereafter. However, precursor cells for immortalized,
totipotent cells as well as the immortalized, totipotent cells
themselves need not be grown on feeder layers.
[0190] C. Identification of Immortalized and Totipotent Cells
[0191] Identification of Immortalized Cells
[0192] Immortalized cells can be identified as those that are not
confined to the Hayflick limit. The Hayflick limit is defined by
cells that divide for more than 60 cell divisions. Hence, cells
that have divided for more than 60 cell divisions are immortalized
cells. In addition, immortalized cells typically can be passaged at
lower cell densities than non-immortalized cells.
[0193] The materials and methods described above (e.g., culturing
the cells with cytokines) can convert non-immortalized cells into
immortalized cells. Other methods exist in the art for generating
immortalized cell lines from primary cells. For example,
manipulating the activity of telomerase within the cells can
immortalize cells. See, e.g., U.S. Pat. No. 5,645,986, entitled
"Therapy and Diagnosis of Conditions Related to Telomere Length
and/or Telomerase Activity," West et al., issued Jul. 8, 1997, and
hereby incorporated by reference herein in its entirety including
all figures, drawings, and tables.
[0194] Moreover, cellular immortality can be determined by
identifying both low molecular weight and macromolecular markers
that are specific for immortalized cells. The existence or lack of
existence of a marker can be a determination of cell
immortalization. In addition, a phenomenon associated with a marker
can be an indication of immortality. For example, if the marker is
an enzyme, an indication of the presence of the enzyme and/or a
certain level of catalytic activity of that enzyme may be a
suitable indication that a certain cell type is immortalized.
[0195] Low molecular weight markers include specific nucleosides,
lipid associated sialic acids, polyamines, and pseudouridine. These
examples are not limiting and the invention relates to any other
low molecular weight markers known in the art.
[0196] Macromolecular markers can be separated into several classes
including nucleic acid polymers, peptides, polypeptides, proteins,
enzymes, growth factors, growth factor receptors, hormones, hormone
receptors, oncogenes, oncogene products, and specific
glycoproteins. Macromolecular markers can be selected from the
group consisting of extracellular proteins, membrane associated
proteins, and/or intracellular proteins, which may be membrane
associated or soluble. One such marker for immortalized cells is
telomerase or its associated activity, for example. See, U.S. Pat.
No. 5,645,986, supra. Other examples of markers specific for
immortalized cells can be selected from the following list:
[0197] 1) Epidermal growth factor (EGF) and its receptor
(EGF-R)
[0198] 2) Transforming growth factor-alpha (TGF-alpha) and its
receptor
[0199] 3) c-erbB2 receptor tyrosine kinase (HER2 product)
[0200] 4) Hyaluronan receptor (probably CD44, an integral membrane
glycoprotein)
[0201] 5) Carcinoembryonic antigen (CEA) family of tumor markers
(for example T1, a glycosylated protein)
[0202] 6) Telomerase, a ribonucleoprotein which maintains telomere
length in immortalized cells
[0203] 7) Phosphatases: placental alkaline phosphatase (PLAP), germ
cell alkaline phosphatase, prostate acid phosphatase (PAS)
[0204] 8) Cathepsin D (catalyzes degradation of laminin).
[0205] 9) Omithine decarboxylase (ODC) (catalyzes the rate-limiting
step in polyamine synthesis)
[0206] 10) Beta-glucuronidase
[0207] 11) Alpha-6 integrin
[0208] 12) Keratin K8
[0209] 13) Oncogene products: ras oncogenes (k-ras, Ha-ras, p21),
v-src, c-myc
[0210] 14) Cyclin D1, cyclin A, and Retinoblastoma Gene Protein
(Rb)
[0211] 15) Changes in p53 expression or p53 mutations
[0212] 16) Heterogeneous ribonucleoprotein-A2 (hnRNP-A2)
overexpression
[0213] 17) L-plastin
[0214] 18) Ganglioside fucosyl-GM1
[0215] 19) Mob-1 expression (mob-1) (homology to proinflammatory
cytokines)
[0216] These examples are not limiting and the invention relates to
any markers specific for immortalized cells that are known in the
art.
[0217] In addition to markers for immortalization known in the art,
markers for immortalization can be identified using methods well
known in the art. For example, immortalization markers can be
identified by analyzing particular molecules (e.g., nucleic acid
molecules and polypeptide molecules) that are unique to specific
cell types.
[0218] In examples pertaining to nucleic acid immortalization
markers, immortalized and non-immortalized cells may be subjected
to analysis for nucleic acid sequence content (e.g., hybridization
techniques with nucleic acid probes). Nucleic acid samples from
particular immortalized cells and nucleic acid samples from
particular non-immortalized cells can be screened for particular
nucleic acid sequences. If samples from non-immortalized cells lack
a nucleic acid sequence present in immortalized cells, then this
nucleic acid sequence could be a marker for distinguishing
immortalized cells from non-immortalized cells. Similarly, if
samples from non-immortalized cells harbor a nucleic acid sequence
that immortalized cells lack, this nucleic acid sequence could be a
marker for distinguishing immortalized cells from non-immortalized
cells. Similar methods can elucidate polypeptide markers by
utilizing polypeptide analytical techniques (e.g., PAGE, SDS-PAGE,
procedures comprising antibodies, and HPLC techniques known in the
art).
[0219] Identification of Totipotent Cells
[0220] Totipotent cells can be identified by a number of tests.
Examples of these tests include:
[0221] (1) identifying a marker specific for totipotent cells;
[0222] (2) performing one or more nuclear transfer cycles with a
cell (as described hereafter) and developing the resulting embryo
into an animal.
[0223] Markers can be utilized to distinguish totipotent cells from
non-totipotent cells. Markers can be selected from the group of low
molecular weight markers, macromolecular markers, cell surface
markers, and intracellular markers. Examples of markers that may be
suitable for identifying totipotent cells can be selected from the
group consisting of alkaline phosphatase, cytokeratin, vimentin,
laminin, and c-kit. These markers are well known to a person of
ordinary skill in the art and these examples are not meant to be
limiting.
[0224] Some of these markers have been tested for cultured bovine
cells being identified for totipotency. As noted previously,
totipotent, immortalized bovine cells of the invention generally do
not appreciably stain for alkaline phosphatase. Therefore the cells
of the invention are to be contrasted with pluripotent cells
discussed in previously referenced publications. It should be noted
that some of the exemplary markers listed previously may not be
specific for totipotent cells as some of these markers may exist in
pluripotent cells as well as in totipotent cells. For example,
although immortalized, totipotent bovine cells do not appreciably
stain for alkaline phosphatase, immortalized, totipotent porcine
cells may appreciably stain for alkaline phosphatase. The invention
relates to any markers specific for totipotent cells that are known
to a person of ordinary skill in the art.
[0225] Markers for totipotency that are not clearly defined in the
art can be elucidated by processes defined in the previous section,
which illustrates methods for elucidating immortalization cell
markers.
[0226] A preferred test for determining totipotency of cells is
determining whether cells give rise to totipotent embryos and
eventually cloned animals. This test represents a definitive test
for cellular totipotency. An example of such a test includes the
following steps: (1) utilizing a potentially totipotent cell for
nuclear transfer with an enucleated oocyte; (2) allowing the
resulting cybrid to develop; (3) separating an embryo that
developed from the cybrid into individual cells and subjecting one
or more of the individual cells to a second round of nuclear
transfer; (4) allowing a resulting cybrid from step (3) to develop
into an embryo; (5) implanting the embryo from step (2) or (4) into
a uterine environment; and (6) allowing the embryo to develop. If
the ensuing fetus develops past the first trimester of pregnancy
then the cells initially used for nuclear transfer are most likely
totipotent cells. If the cells utilized for nuclear transfer
develop into a live born cloned animal then the cells are
definitively totipotent. Examples of the techniques utilized for
this exemplary test (e.g., enucleation of oocytes and nuclear
transfer) are described completely in the art and in exemplary
embodiments defined hereafter.
[0227] Hence, the materials and methods provided herein are the
first to feature immortalized, totipotent cells for cloning a
bovine animal. As described above these materials and methods can
be applied to other ungulates due to the high degree of nuclear DNA
sequence homology among ungulates. Using the tests for identifying
immortalized, totipotent cells, the methods and materials described
herein can be modified by a person of ordinary skill in the art to
produce immortalized, totipotent cells from any type of precursor
cell. Hence, the invention covers any of the materials and methods
described herein as well as modifications to these methods for
generating immortalized, totipotent cells, since a person of
ordinary skill in the art can readily produce immortalized,
totipotent cells by utilizing the materials and methods described
herein in conjunction with methods for identifying immortalized,
totipotent cells.
[0228] II. Transgenic Immortalized and Totipotent Cells
[0229] Materials and methods readily available to a person of
ordinary skill in the art can be utilized to convert immortalized,
totipotent cells of the invention into transgenic immortalized,
totipotent cells. Once the nuclear DNA is modified in the
immortalized, totipotent cells of the invention, embryos and
animals arising from these cells can also comprise the modified
nuclear DNA. Hence, materials and methods readily available to a
person of ordinary skill in the art can be applied to the
immortalized, totipotent cells of the invention to produce
transgenic animals and chimeric animals. See, e.g., EPO 264 166,
entitled "Transgenic Animals Secreting Desired Proteins Into Milk";
WO 94/19935, entitled "Isolation of Components of Interest From
Milk"; WO 93/22432, entitled "Method for Identifying Transgenic
Pre-implantation Embryos"; and WO 95/17085, entitled "Transgenic
Production of Antibodies in Milk," all of which are incorporated by
reference herein in their entirety including all figures, drawings
and tables.
[0230] Methods for generating transgenic cells typically include
the steps of (1) assembling a suitable DNA construct useful for
inserting a specific DNA sequence into the nuclear genome of a
cell; (2) transfecting the DNA construct into the cells; (3)
allowing random insertion and/or homologous recombination to occur.
The modification resulting from this process may be the insertion
of a suitable DNA construct(s) into the target genome; deletion of
DNA from the target genome; and/or mutation of the target
genome.
[0231] DNA constructs can comprise a gene of interest as well as a
variety of elements including regulatory promoters, insulators,
enhancers, and repressors as well as elements for ribosomal binding
to the RNA transcribed from the DNA construct. DNA constructs can
also encode ribozymes and anti-sense DNA and/or RNA, identified
previously herein. These examples are well known to a person of
ordinary skill in the art and are not meant to be limiting.
[0232] Due to the effective recombinant DNA techniques available in
conjunction with DNA sequences for regulatory elements and genes
readily available in data bases and the commercial sector, a person
of ordinary skill in the art can readily generate a DNA construct
appropriate for establishing transgenic cells using the materials
and methods described herein.
[0233] Transfection techniques are well known to a person of
ordinary skill in the art and materials and methods for carrying
out transfection of DNA constructs into cells are commercially
available. Materials typically used to transfect cells with DNA
constructs are lipophilic compounds, such as Lipofectin.TM. for
example. Particular lipophilic compounds can be induced to form
liposomes for mediating transfection of the DNA construct into the
cells.
[0234] Target sequences from the DNA construct can be inserted into
specific regions of the nuclear genome by rational design of the
DNA construct. These design techniques and methods are well known
to a person of ordinary skill in the art. See, U.S. Pat. No.
5,633,067, "Method of Producing a Transgenic Bovine or Transgenic
Bovine Embryo," DeBoer et al., issued May 27, 1997; U.S. Pat. No.
5,612,205, "Homologous Recombination in Mammalian Cells," Kay et
al., issued Mar. 18, 1997; and PCT publication WO 93/22432, "Method
for Identifying Transgenic Pre-Implantation Embryos," both of which
are incorporated by reference herein in their entirety, including
all figures, drawings, and tables. Once the desired DNA sequence is
inserted into the nuclear genome, the location of the insertion
region as well as the frequency with which the desired DNA sequence
has inserted into the nuclear genome can be identified by methods
well known to those skilled in the art.
[0235] Once the transgene is inserted into the nuclear genome of
the immortalized, totipotent cell, that cell can be used as a
nuclear donor for cloning a transgenic animal. A description of the
embodiments related to transgenic animals are described in more
detail hereafter.
[0236] A. Diseases and Parasites
[0237] Desired DNA sequences can be inserted into the (nuclear
cellular) genome to enhance the resistance of a cloned transgenic
animal to particular parasites and diseases. Examples of parasites
include worms, flies, ticks, and fleas. Examples of infectious
agents include bacteria, fungi, and viruses. Examples of diseases
include Johne's, BVD, tuberculosis, foot and mouth, BLV, BSE and
brucellosis. These examples are not limiting and the invention
relates to any disease or parasite or infectious agent known in the
art. See, e.g., Hagan & Bruners Infectious Diesases of Domestic
Animals (7th edition), Gillespie & Timoney, copyright 1981,
Cornell University Press, Ithaca N.Y.
[0238] A transgene can confer resistance to a particular parasite
or disease by completely abrogating or partially alleviating the
symptoms of the disease or parasitic condition, or by producing a
protein which controls the parasite or disease.
[0239] B. Elements of DNA Constructs and Production of DNA
Constructs
[0240] A wide variety of transcriptional and translational
regulatory sequences may be employed, depending upon the nature of
the host. The transcriptional and translational regulatory signals
may be derived from viral sources, such as adenovirus, bovine
papilloma virus, cytomegalovirus, simian virus, or the like,
whereas the regulatory signals are associated with a particular
gene sequence possessing potential for high levels of expression.
Alternatively, promoters from mammalian expression products, such
as actin, casein, alpha-lactalbumin, uroplakin, collagen, myosin,
and the like, may be employed. Transcriptional regulatory signals
may be selected which allow for repression or activation, so that
expression of the gene product can be modulated. Of interest are
regulatory signals which can be repressed or initiated by external
factors such as chemicals or drugs. Other examples of regulatory
elements are described herein.
[0241] C. Examples of Preferred Recombinant Products
[0242] A variety of proteins and polypeptides can be encoded by a
gene harbored within a DNA construct suitable for creating
transgenic cells. Those proteins or polypeptides include hormones,
growth factors, enzymes, clotting factors, apolipoproteins,
receptors, drugs, pharmaceuticals, bioceuticals, nutraceuticals,
oncogenes, tumor antigens, tumor suppressors, cytokines, viral
antigens, parasitic antigens, bacterial antigens and chemically
synthesized polymers and polymers biosynthesized and/or modified by
chemical, cellular and/or enzymatic processes. Specific examples of
these compounds include proinsulin, insulin, growth hormone,
androgen receptors, insulin-like growth factor I, insulin-like
growth factor II, insulin growth factor binding proteins, epidermal
growth factor, TGF-.alpha., TGF-.beta., dermal growth factor
(PDGF), angiogenesis factors (acidic fibroblast growth factor,
basic fibroblast growth factor and angiogenin), matrix proteins
(Type IV collagen, Type VII collagen, laminin), oncogenes (ras,
fos, myc, erb, src, sis, jun), E6 or E7 transforming sequence, p53
protein, cytokine receptor, IL-1, IL-6, IL-8, IL-2, .alpha.,
.beta., or .gamma.IFN, GMCSF, GCSF, viral capsid protein, and
proteins from viral, bacterial and parasitic organisms. Other
specific proteins or polypeptides which can be expressed include:
phenylalanine hydroxylase, .alpha.-1-antitrypsin,
cholesterol-7.alpha.-hy- droxylase, truncated apolipoprotein B,
lipoprotein lipase, apolipoprotein E, apolipoprotein A1, LDL
receptor, scavenger receptor for oxidized lipoproteins, molecular
variants of each, VEGF, and combinations thereof. Other examples
are clotting factors, apolipoproteins, drugs, tumor antigens, viral
antigens, parasitic antigens, monoclonal antibodies, and bacterial
antigens. One skilled in the art readily appreciates that these
proteins belong to a wide variety of classes of proteins, and that
other proteins within these classes can also be used. These are
only examples and are not meant to be limiting in any way.
[0243] It should also be noted that the genetic material which is
incorporated into the cells from DNA constructs includes (1)
nucleic acid sequences not normally found in the cells; (2) nucleic
acid molecules which are normally found in the cells but not
expressed at physiological significant levels; (3) nucleic acid
sequences normally found in the cells and normally expressed at
physiological desired levels; (4) other nucleic acid sequences
which can be modified for expression in cells; and (5) any
combination of the above.
[0244] In addition, DNA constructs may become incorporated into the
nuclear DNA of cells, where the incorporated DNA can be transcribed
into ribonucleic acid molecules that can cleave other RNA molecules
at specific regions. Ribonucleic acid molecules which can cleave
RNA molecules are referred to in the art as ribozymes, which are
RNA molecules themselves. Ribozymes can bind to discrete regions on
a RNA molecule, and then specifically cleave a region within that
binding region or adjacent to the binding region. Ribozyme
techniques can thereby decrease the amount of polypeptide
translated from formerly intact message RNA molecules.
[0245] Furthermore, DNA constructs can be incorporated into the
nuclear complement of cells and when transcribed produce RNA that
can bind to both specific RNA or DNA sequences. The nucleic acid
sequences are utilized in anti-sense techniques, which bind to the
message (MRNA) and block the translation of these messages.
Anti-sense techniques can thereby block or partially block the
synthesis of particular polypeptides in cells.
[0246] III. Nuclear Transfer
[0247] Nuclear transfer (NT) techniques using non-immortalized and
non-totipotent cells are well known to a person of ordinary skill
in the art. See, U.S. Pat. No. 4,994,384 (Prather et al.) and
5,057,420 (Massey et al.). All of the advantages inherent to using
the immortalized, totipotent cells as described above are also
advantages for NT techniques, specifically the fact that the
immortalized, totipotent cells are a nearly unlimited source of
nuclear donors and that these cells increase the efficiency of NT.
Exemplary embodiments define a two-cycle NT technique that provides
for efficient production of totipotent bovine embryos. This
technique can be applied to any mammal, preferably ungulates.
[0248] A. Nuclear Donors
[0249] Immortalized, totipotent cells of the invention can be used
as nuclear donors in a NT process for generating a cloned embryo.
As described above, the immortalized, totipotent cells can be
generated from nearly any type of cell. For NT techniques, a donor
cell may be separated from a growing cell mass or isolated from a
cell line. The entire cell may be placed in the perivitelline space
of a recipient oocyte or may be directly injected into the
recipient oocyte by aspirating the nuclear donor into a needle,
placing the needle into the recipient oocyte, releasing the nuclear
donor and removing the needle without significantly disrupting the
plasma membrane of the oocyte. Alternatively, a nucleus
(karyoplast) may be isolated from a nuclear donor and placed into
the perivitelline space of or injected directly into the recipient
oocyte, for example.
[0250] B. Recipient Oocytes
[0251] A recipient oocyte is typically an oocyte with a portion of
its ooplasm removed, where the removed ooplasm comprises the oocyte
nucleus. Enucleation techniques are well known to a person of
ordinary skill in the art. See e.g., U.S. Pat. Nos. 4,994,384 and
5,057,420.
[0252] Oocytes can be isolated from either oviducts and/or ovaries
of live animals by oviductal recovery procedures or transvaginal
oocyte recovery procedures well known in the art and described
herein. Furthermore, oocytes can be isolated from deceased animals.
For example, ovaries can be obtained from abattoirs and the oocytes
aspirated from these ovaries. The oocytes can also be isolated from
the ovaries of a recently sacrificed animal or when the ovary has
been frozen and/or thawed.
[0253] Oocytes can be matured in a variety of media well known to a
person of ordinary skill in the art. One example of such a medium
suitable for maturing oocytes is depicted in an exemplary
embodiment described hereafter. Oocytes can be successfully matured
in this type of medium within an environment comprising 5% CO.sub.2
at 39.degree. C. Oocytes may be cryopreserved and then thawed
before placing the oocytes in maturation medium. Cryopreservation
procedures for cells and embryos are well known in the art as
discussed herein.
[0254] The nuclear donor may be incorporated into either a young or
an aged oocyte. The age of the oocyte can be determined by the time
that has elapsed since the oocyte was placed in maturation medium
and the time it was activated. A young oocyte can be defined as an
oocyte that is cultured in vitro less than 28 hours before
activation. An aged oocyte is defined as an oocyte that is cultured
in vitro for more than 28 hours before activation.
[0255] The age of the oocytes can be functionally identified by the
appearance of their ooplasm. For example, because certain cellular
materials have not yet dispersed within the ooplasm of a young
oocyte, young oocytes have a pycnotic appearance. Aged oocytes, in
comparison, are characterized by a more homogeneous cytoplasm. A
publication discussing the use of aged oocytes for NT is WO
97/07662, entitled "Inactivated Oocytes as Cytoplast Recipients for
Nuclear Transfer."
[0256] The nuclear donor cell and the recipient oocyte can arise
from the same specie or different species. For example, a bovine
immortalized, totipotent cell can be inserted into a bovine
enucleated oocyte. Alternatively, an immortalized, totipotent cell
derived from a bison can be inserted into a bovine enucleated
oocyte. Any nuclear donor/recipient oocyte combinations are
envisioned by the invention. Preferably the nuclear donor and
recipient oocyte arise from one specie or different species of
ungulates. Cross-species NT techniques can be utilized to produce
cloned animals that are endangered.
[0257] The oocytes can be activated by electrical and/or
non-electrical means before, during, and/or after fusion of the
nuclear donor and recipient oocyte. For example, the oocyte can be
placed in a medium containing one or more components suitable for
non-electrical activation prior to fusion. Alternatively, a fused
cybrid can be placed in a medium containing one or more components
suitable for non-electrical activation. The activation process is
discussed in greater detail hereafter.
[0258] C. Injection/Fusion
[0259] A nuclear donor can be translocated into an oocyte using a
variety of materials and methods that are well known to a person of
ordinary skill in the art. In one example, a nuclear donor may be
directly injected into a recipient oocyte. This direct injection
can be accomplished by gently pulling a nuclear donor into a
needle, piercing a recipient oocyte with that needle, releasing the
nuclear donor into the oocyte, and removing the needle from the
oocyte without significantly disrupting its membrane. Appropriate
needles can be fashioned from glass capillary tubes, as defined in
the art and specifically by publications incorporated herein by
reference.
[0260] In another example, at least a portion of plasma membrane
from a nuclear donor and recipient oocyte can be fused together
using techniques well known to a person of ordinary skill in the
art. See, Willadsen, 1986, Nature 320:63-65, hereby incorporated
herein by reference in its entirety including all figures, tables,
and drawings. Typically, lipid membranes can be fused together by
electrical and chemical means, as defined previously and in other
references incorporated by reference herein.
[0261] Other examples of non-electrical means of cell fusion
involve incubating cybrids in solutions comprising polyethylene
glycol (PEG), and/or Sendai virus. Various molecular weights of PEG
can be utilized for cell fusion.
[0262] Although the efficiency of NT as a process is sensitive to
minor modifications, other variables for fusion can be determined
without undue experimentation. For example, modifications to cell
fusion techniques can be monitored for their efficiency by viewing
the degree of cell fusion under a microscope. The resulting embryo
can then be cloned and identified as a totipotent embryo by the
same methods as those previously described herein for identifying
immortalized, totipotent cells, which can include tests for
selectable markers and/or tests for developing an animal.
[0263] D. Activation
[0264] Methods of activating oocytes and cybrids are known to those
of ordinary skill in the art. See, U.S. Pat. No. 5,496,720,
"Parthenogenic Oocyte Activation," Susko-Parrish et al., issued on
Mar. 5, 1996, hereby incorporated by reference herein in its
entirety including all figures, tables, and drawings.
[0265] Both electrical and non-electrical means can be used for
activating the cybrids. Although use of a non-electrical means for
activation is not always necessary, non-electrical activation can
enhance the developmental potential of cybrids, particularly when
young oocytes are utilized as recipients.
[0266] Examples of electrical techniques for activating cells are
well known in the art. See, U.S. Pat. Nos. 4,994,384 and 5,057,420.
Non-electrical means for activating cells can include any method
known in the art that increases the probability of cell division.
Examples of non-electrical means for activating a nuclear donor
and/or recipient can be accomplished by introducing cells to
ethanol; inositol trisphosphate (IP.sub.3); Ca.sup.++ionophore and
protein kinase inhibitors such as 6-dimethylaminopurine;
temperature change; protein synthesis inhibitors (e.g.,
cyclohexamide); phorbol esters such as phorbol 12-myristate
13-acetate (PMA); mechanical techniques, thapsigargin, and sperm
factors. Sperm factors can include any component of a sperm. Other
non-electrical methods for activation include subjecting the cell
or cells to cold shock and/or mechanical stress.
[0267] Examples of preferred protein kinase inhibitors are protein
kinase A, G, and C inhibitors such as 6-dimethylaminopurine (DMAP),
staurosporin, 2-aminopurine, sphingosine. Potentially, tyrosine
kinase inhibitors may also be utilized to activate cells.
[0268] Although the NT process is sensitive to minor modifications,
other variables for activation can be determined without undue
experimentation. Other activation materials and methods can be
identified by modifying the specified conditions defined in the
exemplary protocols described hereafter and in U.S. Pat. No.
5,496,720.
[0269] The result of any modifications upon efficiency and
totipotency of the activated embryo can be identified by the
methods described previously in the section entitled
"Identification of Immortalized and Totipotent Cells." Methods for
identifying totipotent embryos can include one or more tests, such
as (a) identifying specific markers for totipotent cells in
embryos, and (b) by determining whether the embryos are totipotent
by allowing them to develop into an animal. Therefore, the
invention relates to any modifications to the activation procedures
described herein even though these modifications may not be
explicitly stated herein.
[0270] F. Manipulation of Embryos Resulting from Nuclear
Transfer
[0271] An embryo resulting from a NT can be manipulated in a
variety of manners. The invention relates to cloned embryos that
arise from at least one NT.
[0272] Exemplary embodiments of the invention demonstrate that two
or more NT procedures may enhance the efficiency for the production
of totipotent embryos. The exemplary embodiments indicate that
incorporating two or more NT procedures into methods for producing
cloned totipotent embryos may enhance placental development. In
addition, increasing the number of NT cycles involved in a process
for producing totipotent embryos may represent a necessary factor
for converting non-totipotent cells into totipotent cells. The
effect of incorporating two or more NT cycles on the totipotency of
resulting embryos is a surprising result, which was not previously
identified or explored in the art.
[0273] Incorporating two or more NT cycles into methods for cloned
totipotent embryos can provide another advantage. Incorporation of
multiple NT procedures into methods for creating cloned totipotent
embryos provides a method for multiplying the number of cloned
totipotent embryos.
[0274] When multiple NT procedures are utilized for the formation
of a cloned totipotent embryo, young or aged oocytes can be
utilized as recipients in the first, second or subsequent NT
procedures. For example, if a first NT and then a second NT are
performed, the first NT can utilize a young enucleated oocyte as a
recipient and the second NT may utilize an aged enucleated oocyte
as a recipient. Alternatively, the first NT may utilize an aged
enucleated oocyte as a recipient and the second NT may utilize a
young enucleated oocyte as a recipient for the same two-cycle model
for NT. In addition, both NT cycles may utilize young enucleated
oocytes as recipients or both NT cycles may utilize aged enucleated
oocytes as recipients in the two-cycle NT example.
[0275] For NT techniques that incorporate two or more NT cycles,
one or more of the NT cycles may be preceded, followed, and/or
carried out simultaneously with an activation step. As defined
previously herein, an activation step may be accomplished by
electrical and/or non-electrical means as defined herein.
Exemplified embodiments described hereafter describe NT techniques
that incorporate an activation step after one of the NT cycles.
However, activation steps may also be carried out in conjunction
with NT cycles (e.g., simultaneously with the NT cycle) and/or
activation steps may be carried out prior to a NT cycle.
[0276] A preferred embodiment of the invention, for example,
relates to a first NT utilizing a young enucleated oocyte as a
recipient followed by activation. This, in turn, is followed by a
second NT utilizing an aged enucleated oocyte as a recipient. This
second NT procedure is not followed by activation. This example is
not meant to be limiting and the invention relates to any number of
NT cycles that are optionally preceded by, followed by,
simultaneously carried out with an activation procedure.
[0277] NT techniques may utilize virtually any cell as a nuclear
donor. For example, in a preferred embodiment, a first NT may
utilize an immortalized, totipotent cell of the invention as a
nuclear donor and a second NT may utilize an embryonic cell as a
nuclear donor. The second NT cycle in this example may utilize a
blastomere (a cell isolated from an embryo), a cell isolated from a
fetus (e.g., a primordial germ cell) as a nuclear donor, a cell
isolated from a cell line, or a synchronized cell (described
herein). The invention pertains in part to utilizing nearly any
type of cell as a nuclear donor in any NT. The effect of using
different nuclear donors on the overall efficiency for producing
cloned totipotent embryos can be tested by practicing the tests for
totipotency described in the preceding section entitled
"Identification of Immortalized and Totipotent Cells."
[0278] The cloned totipotent embryos resulting from NTs can be (1)
disaggregated or (2) allowed to develop further.
[0279] If the embryos are disaggregated, these embryonic derived
cells can be utilized to establish cultured cells. Any type of
embryonic cell can be utilized to produce cultured cells. These
cultured cells are sometimes referred to as embryonic stem cells or
embryonic stem-like cells in the scientific literature. The
embryonic stem cells can be derived from early embryos, morulae,
and blastocyst stage embryos. Multiple methods are known to a
person of ordinary skill in the art for producing cultured
embryonic cells. These methods are enumerated in specific
references previously incorporated by reference herein.
[0280] If the embryos are allowed to develop in utero, cells
isolated from the developing fetus can be utilized to produce
cultured cells. In preferred embodiments, primordial germ cells are
isolated from the genital ridge of 28 to 75 day old developing cell
masses for the establishment of cell lines. These cultured cells
are sometimes referred to as embryonic germ cells (EG). These
cultured cells can be generated using methods well known to a
person of ordinary skill in the art. The methods are enumerated in
references previously incorporated by reference herein.
[0281] The cloned totipotent embryos resulting from NT can also be
manipulated by cryopreserving and/or thawing the embryos. See, U.S.
Pat. No. 5,160,312, entitled "Cryopreservation Process for Direct
Transfer of Embryos," Voelkel, and issued on Nov. 3, 1992; and U.S.
Pat. No. 4,227,381, entitled "Wind Tunnel Freezer," Sullivan et
al., issued on Oct. 14, 1980, all of which are hereby incorporated
by reference herein in their entireties including all tables,
figures, and drawings. Other embryo manipulation methods include
culturing, performing embryo transfer, dissociating for NT,
dissociating for establishing cell lines for use in NT, splitting,
aggregating, sexing, and biopsying the embryos resulting from NT,
which are described hereafter. The exemplary manipulation
procedures are not meant to be limiting and the invention relates
to any embryo manipulation procedure known to a person of ordinary
skill in the art.
[0282] IV. Development of Cloned Embryos
[0283] A. Totipotency
[0284] Totipotent embryos can be identified by the methods
described in the section "Identification of Immortalized and
Totipotent Cells." Individual cells can be isolated and subjected
to these similar tests. The tests relate to similar markers for
identifying totipotent cells, as well as a test for determining
totipotency by allowing an embryo to develop until it passes the
second trimester of gestation, or preferably, gives rise to a live
born animal. Methods for identifying other markers for totipotency
are also described in that section.
[0285] B. Culture of Embryos in vitro
[0286] Methods for culturing embryos in vitro are well known to
those skilled in the art. See, U.S. Pat. No. 5,213,979, entitled
"In vitro Culture of Bovine Embryos," First et al., issued on May
25, 1993, and U.S. Pat. No. 5,096,822, entitled "Bovine Embryo
Medium," Rosenkrans, Jr. et al., issued on Mar. 17, 1992, both of
which are incorporated by reference herein in its entirety,
including all figures, tables, and drawings. In addition, exemplary
embodiments for media suitable for culturing cloned embryos in
vitro are described hereafter. Feeder cell layers may or may not be
utilized for culturing cloned embryos in vitro. Feeder cells are
described previously and in exemplary embodiments hereafter.
[0287] The present invention is superior to existing materials and
methods for cloning organisms, because embodiments of the invention
allow for culturing all cells and embryos in vitro prior to
implantation. For example, cloning methods described for cloning
ovine organisms require an in vivo development step in the oviducts
of an ovine host animal before the embryos are implanted in a
suitable host. Because embodiments of the present invention do not
require in vivo development steps prior to implantation into the
uterus, the materials and methods of the present invention
represent an inventive step over cloning methods previously
described by others.
[0288] C. Development of Embryos in utero
[0289] Cloned embryos can be cultured in an artificial or natural
uterine environment after NT procedures. Examples of artificial
development environments are being developed and some are known to
those skilled in the art. Components of the artificial environment
can be modified with little experimentation, for example, by
modifying one component and monitoring the growth rate of the
embryo.
[0290] Methods for implanting embryos into the uterus of an animal
are also well known in the art. Preferably, the developmental stage
of the embryo(s) is correlated with the estrus cycle of the
animal.
[0291] Embryos from one specie can be placed into the uterine
environment of an animal from another specie. For example it has
been shown in the art that bovine embryos can develop in the
oviducts of sheep. Stice & Keefer, 1993, "Multiple generational
bovine embryo cloning," Biology of Reproduction 48: 715-719. The
invention relates to any combination of ungulate embryo in any
other ungulate uterine environment. The cross-species relationship
between embryo and uterus can allow for efficient production of
cloned animals of an endangered species. For example, a bison
embryo can develop in the uterus of a domestic bovine. In another
example, a big-horn sheep embryo can develop in the uterus of a
large domesticated sheep.
[0292] Once the embryo is placed in the uterus of an animal, the
embryo can develop to term. Alternatively, the embryo can be
allowed to develop in the uterus and then can be removed at a
chosen time. Surgical methods are well known in the art for
removing fetuses from uteri before they are born.
[0293] V. Cloned Animals
[0294] A. Bovine Cloned Animals
[0295] As described previously herein, the invention provides the
advantages of being able to assess the phenotype of an animal
before cloning. This is an advantage of the invention since
previous reports have only allowed the cloning of bovine animals
from blastomeres, a method that does not allow for phenotype
assessment.
[0296] Multiple products can be isolated from a cloned animal. For
example, semen can be collected from an animal, such as a bovine
bull. Semen can be cryopreserved as well as separated sperm into
sex-specific fractions. See, U.S. Pat. Nos. 5,439,362, 5,346,990,
and 5,021,244, entitled "Sex-associated Membrane Proteins and
Methods for Increasing the Probability that Offspring Will be of a
Desired Sex," Spaulding, and issued on Aug. 8, 1995, Sept. 13,
1994, and Jun. 4, 1991, respectively, all of which are hereby
incorporated by reference herein in their entireties including all
figures, drawings, and tables. Methods of collecting semen are well
known to a person of ordinary skill in the art. Physiology of
Reproduction and Artificial Insemination of Cattle (2nd. edition),
Salisbury et al., copyright 1961, 1978, W. H. Freeman & Co.,
San Francisco.
[0297] The invention relates in part to any products collected from
a cloned animal, preferably a cloned bovine animal. The products
can be any body fluids or organs isolated from the animal, or any
products isolated from the fluids or organs. In preferred
embodiments, products such as milk and meat may be collected from
cloned animals, preferably cloned bovine animals. In another
embodiment, the invention relates to determining the phenotype of a
bovine steer, which is a neutered animal, and then cloning this
animal such that the cloned animals are reproductively functional
and can be used to produce semen. Other preferred embodiments of
the invention relate to such products as xenograft materials,
sperm, embryos, oocytes, any type of cells, and offspring harvested
from cloned animals of the invention, preferably cloned bovine
animals.
[0298] Xenograft materials, which are described previously herein,
can relate to any cellular material extracted from one organism and
placed into another organism. Medical procedures for extracting the
cellular material from one organism and grafting it into another
organism are well known to a person of ordinary skill in the art.
Examples of preferable xenograft cellular materials can be selected
from the group consisting of liver, lung, heart, nerve,
gallbladder, and pancreas cellular material.
[0299] B. Non-Bovine Cloned Animals
[0300] Due to the high DNA sequence homology between bovine animals
and other ungulates, the materials and methods of the invention can
be utilized to clone other ungulates. The materials and methods of
the invention are the most efficient means for cloning a mammal as
known in the state of the art.
[0301] In preferred embodiments the materials and methods of the
invention can be utilized to clone endangered species, such as
bison. In addition, the materials and methods of the invention can
be utilized to clone commercially relevant ungulates, such as pigs.
Due to the methods for reprogramming primary cells isolated from an
animal into immortalized, totipotent cells, the more closely
related the animal species is to cattle, the higher probability
that the cloning methods of the invention will have greater
success. Exemplary embodiments are described hereafter for cloning
non-bovine animals.
[0302] C. Cloned Animals with Modified Nuclear DNA
[0303] As discussed in a previous section, transgenic animals can
be generated from the methods of the invention by using transgenic
techniques well known to those of ordinary skill in the art.
Preferably, cloned transgenic bovine animals are produced from
these methods. These cloned transgenic animals can be engineered
such that they are resistant or partially resistant to diseases and
parasites endemic to such animals. Examples of these diseases and
parasites are outlined in a preceding section.
[0304] Moreover, the cloned transgenic animals can be engineered
such that they produce a recombinant product. Examples of
recombinant products are outlined in a preceding section. The
expression of these products can be directed to particular cells or
regions within the cloned transgenic animals by selectively
engineering a suitable promoter element and other regulatory
elements to achieve this end.
[0305] For example, human recombinant products can be expressed in
the urine of cattle by operably linking a uroplakin promoter to the
DNA sequence encoding a recombinant product. Alternatively,
examples are well known to a person of ordinary skill in the art
for selectively expressing human recombinant products in the milk
of a bovine animal.
[0306] Once the recombinant product or products have been expressed
in a particular tissue or fluid of the cloned transgenic animal,
the suitable tissue or fluid can be collected using methods well
known in the art. Recombinant products can be purified from that
fluid or tissue by using standard purification techniques well
known to a person of ordinary skill in the art.
EXAMPLES
[0307] The examples below are non-limiting and are merely
representative of various aspects and features of the present
invention.
Example 1: Feeder Layer Preparation
[0308] A feeder cell layer was prepared from mouse fetuses that
were from 10 to 20 days gestation. The head, liver, heart and
alimentary tract were removed and the remaining tissue washed and
incubated at 37.degree. C. in 0.025% trypsin-0.02%, EDTA (Difco,
Cat # 0153-61-1). Loose cells were cultured in tissue culture
dishes containing MEM-alpha supplemented with penicillin,
streptomycin, 10% fetal calf serum and 0.1 mM 2-mercaptoethanol.
The feeder cell cultures were established over a two to three week
period at 37.4.degree. C., 3.5% CO.sub.2 and humidified air. Before
being used as feeder cells, mouse fibroblasts were pre-treated with
mitomycin C (Calbiochem, Cat # 47589) at a final concentration of
10 .mu.g/ml for 3 hours and washed 5 times with PBS before
pre-equilibrated growth media was added.
[0309] Feeder cells can be established from bovine fetuses from 30
to 70 days using the same procedure. Bovine fetal cells may be
optionally treated with mitomycin C.
Example 2: Establishing Cultured Cells From Non-Embryonic
Tissue
[0310] One advantage provided by the materials and methods defined
herein is the ability to create an immortalized and totipotent cell
from virtually any type of precursor cell. These precursor cells
can be embryonic cells, cultured embryonic cells, primordial germ
cells, fetal cells, and cells isolated from the tissues of adult
animals, for example. Cells isolated from the kidney and ear of an
adult grown bovine have been utilized as precursor cells for the
generation of imortalized, totipotent cells.
[0311] After cells are isolated from their respective tissues, the
cells can be subjected to the materials and methods defined in
Example 3.
[0312] A first step towards generating immortalized, totipotent
cells from tissues of grown animals includes a primary culture of
isolated cells. A protocol for culturing cells isolated from the
tissues of grown animals is provided hereafter. Although the
illustrative protocol relates to ear punch samples, this protocol
can apply to cells isolated from any type of tissue.
[0313] The following steps are preferably performed utilizing
sterile procedures:
[0314] 1) Wash each ear sample twice with 2 mL of trypsin/EDTA
solution in two separate 35 mm Petri dishes. Process each ear
sample separately. Mince the ear sample with sterile scissors and
scalpel in a 35 mm Petri dish containing 2 mL of trypsin/EDTA
solution. The minced pieces are preferably less than 1 mm in
diameter.
[0315] 2) Incubate minced ear pieces in the trypsin/EDTA solution
for 40-50 min. in a 37.degree. C. incubator with occasional
swirling. The trypsin/EDTA solution is described in more detail
hereafter. The dish may be wrapped with a stretchable material,
such as Parafilm.RTM., to reduce CO.sub.2 accumulation.
[0316] 3) Transfer digested ear pieces to a 15 mL sterile tube.
Wash the dish from which the digested ear pieces were recovered
with 2 mL of the trypsin/EDTA solution and transfer this wash
solution to the sterile tube.
[0317] 4) Vortex the tube at high speed for 2 min.
[0318] 5) Add 5 mL of media (defined below) to inactivate the
trypsin.
[0319] 6) Centrifuge the 15 mL tube at 280 xg for 10 minutes.
[0320] 7) Decant the supernatant and re-suspend the cell pellet in
residual solution by gently taping the side of the tube.
[0321] 8) Add 2 mL of media to the tube and then centrifuge as
described in step (6).
[0322] 9) Decant the supernatant, re-suspend the pellet as
described in step (7), then add 2 mL of media.
[0323] 10) Keep 2-3 pieces of the ear for DNA analysis and store at
-20.degree. C. in a 15 mL tube.
[0324] 11) Transfer resuspended cells into a 35 mm Nunc culture
dish and incubate at 37.degree. C. in a humidified 5% CO.sub.2/95%
air atmosphere.
[0325] 12) Change media every 2 days.
[0326] Trypsin/EDTA solution:
[0327] 0.025% trypsin (w/v) (Bacto trypsin, Difco # cat
0153-61-1)
[0328] 0.02% EDTA (Sigma) (w/v)
[0329] Add the trypsin and EDTA to Ca.sup.2+-free and
Mg.sup.2+-free Dulbecco's phosphate-buffered saline (PBS) (Gibco
cat# 450-1600EA) and sterilize by filtration through a 0.2 .mu.m
filter.
[0330] Media:
[0331] Combine Alpha minimum essential medium (MEM) (Biowhittaker)
with 10% fetal bovine serum (Hyclone), 4 mM L-glutamine, 100 U/mL
penicillin, 100 .mu.g/mL streptomycin, 0.25 .mu.g/mL amphotercin B
(Fungizone).
[0332] This protocol has been also successfully utilized to
establish cultures of kidney and liver cells isolated from grown
bovine animals. As discussed above, the protocol can be utilized to
create cell cultures from any type of cell isolated from a grown
animal, for any species or family of animals.
EXAMPLE 3: Reprogramming and Establishment of Immortalized and
Totipotent Cells from Precursor Cells
[0333] The reprogramming procedures described hereafter can utilize
any cell type of cells as precursor cells for the generation of
immortalized, totipotent cells. As an example, the cell cultures
described previously can be utilized as precursor cells for the
reprogramming procedures described below. As another example, the
following procedure describes one embodiment of the invention,
where primordial germ cells were utilized as precursor cells for
the generation of immortalized, totipotent cells. An embodiment of
the reprogramming process is illustrated in FIG. 2.
[0334] A bovine fetus approximately 40 days old was obtained from a
pregnant animal. The genital ridges were located at the
caudo-ventral part of the abdominal cavity. Genital ridges were
removed aseptically and washed in phosphate buffered saline (PBS)
(Gibco, Cat # 14287-015) with 500 U/mL penicillin/500 .mu.g/ml
streptomycin. The tissue was sliced into 1-1.5 mm pieces and placed
into a solution containing pronase E (3 mg/ml; Sigma Cat # P6911)
in Tyrodes Lactate (TL) HEPES (Biowhittaker, Cat # 04-616F) for
30-45 minutes at 35-37.degree. C. The proteolytic action of pronase
E disaggregated the slices of genital ridges to a cell suspension.
Pronase E was removed by dilution and centrifugation in TL HEPES
solution. After this step, the cell suspension was frozen and
stored at -196.degree. C.
[0335] A thawed cell suspension (final concentration 100,000
cells/ml) was placed into a 35 mm Petri dish containing a murine
primary embryonic fibroblast feeder layer. The culture media used
was MEM alpha (Biowhittaker, Cat # 12-169F) supplemented with 0.1
mM 2-mercaptoethanol (Gibco, Cat # 21985-023), 4 mM glutarnine, 100
ng/ml human recombinant leukemia inhibitory factor (hrLIF; R&D
System, Cat # 250-L), 100 ng/ml bovine basic fibroblast growth
factor (bFGF; R&D System, Cat # 133-FB) and 10% fetal calf
serum (FCS, HyClone, Cat # A1111 D) at 37.5.degree. C. and 3.5%
C.sub.2. Exogenous steel factor (e.g., membrane associated steel
factor and soluble steel factor) was not added to the culture
media. After 24 hours, and again at 48 hour intervals, supplemented
culture media was replaced. After an initial culture of 6 days,
concentrations of hrLIF and bFGF were lowered to 40 ng/ml,
respectively. After nine days in culture, hrLIF and bFGF were
removed from the medium entirely.
[0336] At the beginning of in vitro culture of genital ridge cells,
simple embryonic bodies were occasionally observed. These bodies
eventually disappeared with subsequent passages. The rate of
establishing immortalized, totipotent cell lines from genital ridge
cells was 100% and did not appear to be sex dependent. Table 1
contains data from establishment of seven immortalized, totipotent
cell lines. Established immortalized, totipotent cell lines were
maintained in MEM-alpha supplemented with 10% FCS which was
replaced every second or third day. High density population cells
were passaged every week at a dilution ratio of 1:4 to 1:8. Cells
were passaged by incubating with 0.025% trypsin-0.02% EDTA mixture
and preparing new cultures in fresh growth medium. The growth
promoting capacity of MEM-alpha media for immortalized, totipotent
cells was enhanced by adding insulin-transferrin, sodium selenite
supplement, diluted to 1:100 (Sigma Cat # 11884). As a preventive
measure against mycoplasma contamination, short term cultivation
with tylosine tartrate (Sigma, Cat T3151) was carried out. Before
NT, cell lines were tested for presence of mycoplasma by PCR
performed with DNA primers specific for mycoplasma sequences
(Stratagene, Cat 302007).
1TABLE 1 Characterization of Established Bovine Immortalized and
Totipotent Cell Lines Cell line Weight of fetus (gm) Days in
culture Sex of Cell line EG 14.2 >400 male EG-1 20.2 >300
male EG-2 3.9 >30 female EG-3 4.8 >30 male EG-4 39.6 >100
female EG-5 3.9 >250 male EG-6 8.6 >30 male
EXAMPLE 4: Embryo Construction
[0337] The following embodiment of the invention describes
materials and methods utilized to produce totipotent embryos of the
invention. Immortalized embryos of the invention can be produced by
utilizing immortalized and totipotent cells of the invention as
nuclear donors in NT procedures. As described previously, multiple
NT procedures can be utilized to create a totipotent embryo. The
following two examples describe a multiple NT procedure, which
describes the use of two NTs.
[0338] Mycoplasma free immortalized, totipotent cells used in the
NT procedure, were prepared by cutting out a group of immortalized,
totipotent cells from the feeder layer using a glass needle. The
isolated immortalized, totipotent cells were then incubated in a TL
HEPES solution containing from 1 to 3 mg/ml pronase Eat
approximately 32.degree. C. for 1 to 4 hours, the amount of time
which was needed in this example to disaggregate the cells. Once
the cells were in a single cell suspension they were used for NT
within a 2-3 hour period.
[0339] Oocytes aspirated from ovaries were matured overnight (16
hours) in maturation medium. Medium 199 (Biowhittaker, Cat #
12-119F) supplemented with luteinizing hormone 101U/ml (LH; Sigma,
Cat # L9773), 1 mg/ml estradiol (Sigma, Cat # E8875) and 10% FCS or
estrus cow serum, was used. Within 16 hours of maturation, the
cumulus layer expanded and the first polar bodies were
extruded.
[0340] In the first NT procedure, young oocytes were stripped of
their cumulus cell layers and nuclear material stained with Hoechst
33342 5 mg/ml (Sigma, Cat # 2261) in TL HEPES solution supplemented
with cytochalasin B (7.mu.g/ml, Sigma, Cat # C6762) for 15 min.
Oocytes were then enucleated in TL HEPES solution under mineral
oil. A single immortalized, totipotent cell of optimal size (12 to
15 .mu.m) was then inserted from a cell suspension and injected
into the perivitelline space of the enucleated oocyte. The
immortalized, totipotent cell and oocyte membranes were then
induced to fuse by electrofusion in a 500 .mu.m chamber by
application of an electrical pulse of 90V for 15 .mu.s.
[0341] Cybrid activation was induced by a 4 min exposure to 5 .mu.M
calcium ionophore A23187 (Sigma Cat. # C-7522) or ionomycin Ca-salt
in HECM (hamster embryo culture medium) containing 1 mg/ml BSA
followed by a 1:1000 dilution in HECM containing 30 mg/ml BSA for 5
min. For HECM medium, see, e.g., Seshagiri & Barister, 1989,
"Phosphate is required for inhibition of glucose of development of
hamster eight-cell embryos in vitro," Biol. Reprod. 40: 599-606.
This step is followed by incubation in CR2 medium containing 1.9 mM
6-dimethylaminopurine (DMAP; Sigma product, Cat # D2629) for 4 hrs
followed by a wash in HECM and then cultured in CR2 media with BSA
(3 mg/ml) under humidified air with 5% CO.sub.2 at 39.degree. C.
For CR2 medium, see, e.g., Rosenkrans & First, 1994, "Effect of
free amino acids and vitamins on cleavage and developmental rate of
bovine zygotes in vitro," J. Anim. Sci. 72: 434-437. Mitotic
divisions of the cybrid formed an embryo. Three days later the
embryos were transferred to CR2 media containing 10% FCS for the
remainder of their in vitro culture.
[0342] Table 2 shows the effect of oocyte age on blastocyst
development. The data was obtained utilizing blastomeres from in
vitro produced embryos or immortalized, totipotent cells as donor
nuclei in the NT procedure. Developmental potential was measured in
young versus aged oocytes.
2TABLE 2 Effect of Oocyte Timing for Different Cell Sources
Immortalized and occyte age (hours) Totipotent Cells (n = 174)
Blastomeres (n = 192) 16-28 (n = 175) 17.9% blastocyst (n = 140) no
development (n = 35) 28-48 (n = 191) no development (n = 34) 17.3%
blastocyst (n = 157)
[0343] The data presented in Table 2 shows that oocytes maintained
in culture for 16-28 h were more suitable recipients for
immortalized, totipotent cells, while aged oocytes maintained in
culture for 28-48 h were a more suitable recipient for blastomeres
derived from embryos. In addition, activation procedures differed
between young and aged oocytes. Young oocytes, when used in the NT
procedure, appear to require chemical activation with ionomycin and
DMAP from these studies. Aged oocytes, on the other hand, appear to
be easily activated by electrofusion according to these
studies.
EXAMPLE 5: Second Nuclear Transfer (Recloning)
[0344] Cells obtained from fetuses and embryos produced by the NT
procedures described herein can be used in a second NT, or
recloning, procedure. For example, a fetus can be harvested from a
maternal host, the head, vicera, and genital ridge removed, and the
remaining fetal cells used to establish a cell line to provide
nuclear donor material for a subsequent NT procedure. The following
example describes obtaining and using a blastomere from an NT
embryo as a nuclear donor in a recloning procedure.
[0345] Embryos from the first generation NT at the morula stage
were disaggregated either by pronase E (1-3 mg/ml in TL HEPES) or
mechanically after treatment with cytochalasin B. Single
blastomeres were placed into the perivitelline space of enucleated
aged oocytes (28-48 hours of incubation). Aged oocytes were
produced by incubating matured "young" oocytes for an additional
time in CR2 media with 3 mg/ml BSA in humidified air with 5%
CO.sub.2 at 39.degree. C.
[0346] A blastomere from an embryo produced from an immortalized,
totipotent cell was fused into the enucleated oocyte via
electrofusion in a 500 .mu.m chamber with an electrical pulse of
105V for 15 .mu.s in an isotonic sorbitol solution (0.25M) at
30.degree. C. Aged oocytes were simultaneously activated with a
fusion pulse, not by chemical activation as with young oocytes.
[0347] After blastomere-oocyte fusion, the cybrids from second
generation NT were cultured in CR2 media supplemented with BSA (3
mg/ml) under humidified air with 5% CO.sub.2 at 39.degree. C. On
the third day of culture, developing embryos were evaluated and
cultured further until day seven in CR2 media containing 10% FCS.
Morphologically good to fair quality embryos were non-surgically
transferred into recipient females. Table 3 shows the increased
gestation length achieved by use of recloned (double NT)
immortalized, totipotent cells.
3TABLE 3 Development of Immortalized and Totipotent Cells Derived
Fetuses after Double NT No. of No. of pregnant embryos No. of
recipients recipients after No. of transferred transferred into 140
days calves Exper #1 15 5 1 1 Exper #2 18 6 1 (two fetuses) 2
EXAMPLE 6: Cloning Non-Bovine Ungulates
[0348] The specification provides for methods of cloning non-bovine
ungulates. Examples of such ungulates can be selected from the
group consisting of bovids, ovids, cervids, suids, equids and
camelids, such as bison, sheep, big-horn sheep, caribou, antelope,
deer, goat, water buffalo, camel, and pig.
[0349] Immortalized, totipotent cell lines can be prepared from
multiple types of cells isolated from the non-bovine ungulate by
using the methods described in previous examples relating to bovine
animals, or by using the screening procedures for these methods as
described in the specification. Virtually any type of cell isolated
from the non-bovine ungulate can be utilized to establish an
immortalized, totipotent cell line. For example, an ear-punch
sample taken from a bison can be cultured in vitro using a variety
of cell culture media such as MEM-alpha medium.
[0350] Bison-derived primary cells can then be converted or
reprogrammed into immortalized, totipotent bison cells by
supplementing the cell culture medium with hrLIF and bFGF as
described in previous examples and in the specification.
Alternatively, the bison-derived primary cells can be converted
into immortalized, totipotent cells by supplementing the growth
medium with other types of molecules identified by methods for
identifying such reprogramming molecules as described in the
specification. The reprogrammed bison-derived cells can then be
tested for totipotency by analyzing selected markers, such as
alkaline phosphatase, laminin, and c-kit. In addition, the
bison-derived cells can be considered permanent if the number of
cell divisions exceeds the Hayflick limit and/or if the cells can
grow to confluency after being replated under conditions where the
cells are not in physical contact with one another, for
example.
[0351] Once totipotent, immortalized cells have been established as
nuclear donors, proper enucleated oocytes can be prepared for NT.
Oocytes from the same or different specie as the nuclear donor can
be used for NT. For example, a bison-derived nuclear donor cell can
be fused or directly injected into a bison-derived enucleated
oocyte or an enucleated oocyte from another specie, such as a
bovine.
[0352] As described in the specification, the oocytes can be
derived from any ungulate in a variety of ways, such as sacrificing
an animal and retrieving oocytes from its oviducts, or spaying the
animals by ovarian hysterectomy and isolating the oocytes from the
oviducts or ovaries. Oocytes can also be obtained from live animals
by utilizing such methods as transvaginal oocyte recovery. The
oocytes can then be enucleated by using methods described herein as
applied to sheep or cattle. These methods can be easily applied to
oocytes derived from other ungulates.
[0353] Nuclear transfer techniques can be performed after
enucleated oocytes and nuclear donor cells are prepared. Young or
aged oocytes can be utilized for the NT procedure, and the number
of NTs can vary as described in the specification. In addition the
parameters that define the fusion step for a NT may be varied as
described herein. An activation step can be applied to one or more
of the NT cycles. For example, the NT cycles defined in a previous
exemplary embodiment can be applied to the generation of cloned
bison. The embryo resulting from the NT can be tested for
totipotency by utilizing tests for one or more markers, such as
alkaline phosphatase, cytokeratin, vimentin, laminin, and c-kit. In
addition, the embryo can be tested for totipotency by implanting it
into the uterus of an animal and allowing development to term.
[0354] Once a cloned totipotent embryo is produced from the methods
described above for a non-bovine ungulate, the embryo can be
further manipulated. Such manipulations include cryopreserving,
thawing, culturing, disaggregating the embryo into single cells,
and implanting the embryo. The embryo may be cultured in an
artificial development environment (as described previously) or may
be placed in utero of a properly synchronized female animal. An
embryo derived from one specie may be placed in a uterus of the
same or different specie. For example, a bison-derived embryo can
be placed in the uterus of a bovine. The embryo can be allowed to
develop until term, or may be retrieved from the uterine
environment before birth.
EXAMPLE 7: Multiple Pathways for Cloning Animals
[0355] FIG. 3 illustrates multiple embodiments of the invention.
Animals can be cloned from cells that are reprogrammed into
totipotent and immortalized cells.
[0356] Fibroblast cell cultures were prepared as defined above from
ear punches extracted from an adult bovine animal. However, the
cell cultures could be established from any type of differentiated
cell. Individual cells isolated from these cultures were utilized
as nuclear donors in a nuclear transfer process, labeled as step 2
in FIG. 3. Although one nuclear transfer cycle was utilized to
obtain embryos (labeled as step 3 in FIG. 3), multiple nuclear
transfer cycles could be applied to obtain these embryos. Also
optional is (1) the addition of a stimulus before or after nuclear
transfer, and (2) an activation step before or after nuclear
transfer.
[0357] The embryo of step 3 in FIG. 3 was implanted into a
recipient bovine female as described herein and a fetus (step 7)
was isolated from that female. Cells isolated from embryos of step
3 may be utilized to establish embryonic stem cell cultures (step
4). In addition, the embryos of step 3 may be implanted into a
female host and allowed to develop into a cloned animal (step
5).
[0358] The steps labelled 8, 9, 10, 11, and 12 in FIG. 3 were
performed to establish totipotent and immortalized cells. The fetus
of step 7 was manipulated in three manners. The manipulation in
step 8 involved the isolation of genital ridge cells, specifically
primordial germ cells, from the fetus of step 7. In step 9, the
primordial germ cells were placed in co-culture with feeder cells.
The feeder cells were either established from mouse fibroblast
cells or from the rest of the fetus from which the primordial germ
cells were extracted. Example 1 defines a method for establishing
feeder cells. The head region and body cavity contents were removed
from the fetus before the fetus was digested into a consistency
suitable for establishing feeder cells. However, the fetus may be
digested before the head region and contents of the body cavity are
removed. In addition, feeder cells may be established from a fetus
other than the fetus from which the primordial germ cells are
isolated.
[0359] In step 10, a cell culture was established with a digested
fetus from which the primordial germ cells, head region, and body
cavity contents were removed. Step 11 illustrates that cell
cultures may be established utilizing fetuses from which no cell
types have been removed.
[0360] In step 12, cell cultures were either (1) subjected to a
mechanical stimulus, or (2) not subjected to a mechanical stimulus.
When applied, the mechanical stimulus was effected by supplementing
the culture medium with a receptor ligand cocktail comprising 100
ng/ml human recombinant leukemia inhibitory factor (hrLIF; R&D
System, Cat # 250-L) and 100 ng/ml bovine basic fibroblast growth
factor (bFGF; R&D System, Cat # 133-FB). After step 12, cells
were isolated from the cell cultures and utilized as nuclear donors
in nuclear transfer processes, which are defined previously.
Although one nuclear transfer cycle was utilized for step 13, more
than one nuclear transfer cycle could be utilized.
[0361] Embryos developed after the nuclear transfer process of step
13. The embryos of step 14 may be implanted into a bovine recipient
female and develop into a cloned bovine animal.
[0362] Cells isolated from any of the developing cell masses of
steps 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, and 16 in FIG. 3 may be
transfected with a DNA construct to form transgenic cells suitable
for cloning transgenic animals. One embodiment for cloning
transgenic animals is defined in the next example.
EXAMPLE 8: Cloning Transgenic Animals
[0363] Transgenic cells suitable for creating a cloned transgenic
animal can be prepared from cells isolated from an adult animal.
FIG. 4 illustrates processes that can be utilized to create such
transgenic cells. Although transgenic cells can be created from
nearly any cell type by using the teachings of the invention, FIG.
4 illustrates procedures for establishing transgenic embryonic stem
cells and transgenic immortalized and totipotent cells.
[0364] Fibroblast cell cultures can be established from ear punches
extracted from a bovine animal as defined previously. Individual
cells can be isolated from this cell culture and utilized as
nuclear donors in a nuclear transfer process. A single nuclear
transfer cycle or multiple nuclear transfer cycles can be applied.
Other optional steps are defined in the previous example.
[0365] Pre-blastocyst stage embryos and/or blastocyst stage embryos
developed from the nuclear transfer process can be utilized to
establish embryonic stem cells. Materials and methods for preparing
embryonic stem cells are described by Stice et al., 1996, Biology
of Reproduction 54: 100-110, hereby incorporated by reference
herein in its entirety, including all figures, tables, and
drawings. Immortalized and totipotent cells can be established
according to the procedures defined in previous examples.
[0366] Cells can then be transfected with a DNA construct. Cells
can be transfected at multiple steps, as indicated in FIG. 4.
Materials and methods for preparing transgenic cells are defined in
publications referenced previously. Immortalized and totipotent
cells of the invention were successfully transfected with a DNA
construct comprising (a) a neomycin gene, which encodes a product
that renders cells resistant to a compound designated G418; (b) a
gene encoding the enzyme .alpha.-glucosidase; and (c) a casein
promoter element. The transfected cells were selected for
transgenic modification by selecting for transgenic cells in cell
culture conditions harboring G418. The transgenic cells are then
screened for transgenic modification by utilizing one or more
screening techniques. Examples of these techniques are: (1)
polymerase chain reaction, (2) Southern blotting, and (3)
FISH-filter procedures. These techniques are well known to a person
of ordinary skill in the art. The latter two techniques are
utilized to determine the number of copies of an inserted gene
sequence in embryonic stem cell nuclear DNA.
[0367] These screening procedures can be applied to transfected
cells at any of the steps indicated in FIG. 4. Cloned transgenic
animals may be created from transgenic fetuses. Table 4 shows the
cloned bovine animals produced by the methods described in Examples
1-8.
4TABLE 4 Development of Immortalized and Totipotent Cells Bovine
Animals Produced Age of Type of Type of Fetus Cell Cell Harvested
Used Used Date of Birth Gender Breed (days) (1.degree. NT)
(2.degree. NT) Stimulus 2/6/97 M H 60 (est) EG Blastomere LIF, FGF
7/7/97 (*) M H 60 (est) EG Blastomere LIF, FGF 7/7/97 (*) M H 60
(est) EG Blastomere LIF, FGF 2/7/98 F H 55 (est) EG Blastomere LIF,
FGF, FSK 2/26/98 F H 55 (est) EG Blastomere LIF, FGF, FSK 10/26/98
F H 55 (est) EG Blastomere LIF, FGF, FSK 10/28/98 F H 58 EG
Blastomere LIF, FGF, FSK 12/2/98 F BS 58 EG n/a LIF, FGF, FSK
12/6/98 F H 58 EG n/a Culture 12/22/98 (*) F H 58 EG n/a Culture
12/22/98 F H 58 EG n/a Culture 12/22/98 (*) F H 58 EG (t) n/a LIF,
FGF 12/30/98 F H 58 EG n/a Culture 12/30/98 F H 58 EG n/a Culture
12/31/98 F H 58 EG n/a Culture 1/6/99 F H 58 EG (t) n/a LIF, FGF
1/7/99 F H 58 EG n/a Culture 1/7/99 F H 58 EG n/a Culture 1/15/99 F
H 58 EG n/a Culture 1/15/99 F H 58 EG (t) n/a LIF, FGF 1/19/99 F H
58 EG (t) n/a LIF, FGF (*) - Stillborn H - Holstein BS - Brown
Swiss Age of fetus harvested - Age (in days) of fetus used as a
source of precursor cells in 1.degree. NT (t) - transgenic nuclear
donor: a nuclear donor cell transfected with a DNA construct having
a human .alpha. glucosidase gene EGF - Epidermal growth factor LIF
- Leukemia inhibitor factor FSK - Forskolin
[0368] While the invention has been described and exemplified in
sufficient detail for those skilled in this art to make and use it,
various alternatives, modifications, and improvements should be
apparent without departing from the spirit and scope of the
invention.
[0369] One skilled in the art readily appreciates that the present
invention is well adapted to carry out the objects and obtain the
ends and advantages mentioned, as well as those inherent therein.
The cell lines, embryos, animals, and processes and methods for
producing them are representative of preferred embodiments, are
exemplary, and are not intended as limitations on the scope of the
invention. Modifications therein and other uses will occur to those
skilled in the art. These modifications are encompassed within the
spirit of the invention and are defined by the scope of the
claims.
[0370] It will be readily apparent to a person skilled in the art
that varying substitutions and modifications may be made to the
invention disclosed herein without departing from the scope and
spirit of the invention.
[0371] All patents and publications mentioned in the specification
are indicative of the levels of those of ordinary skill in the art
to which the invention pertains. All patents and publications are
herein incorporated by reference to the same extent as if each
individual publication was specifically and individually indicated
to be incorporated by reference.
[0372] The invention illustratively described herein suitably may
be practiced in the absence of any element or elements, limitation
or limitations which is not specifically disclosed herein. Thus,
for example, in each instance herein any of the terms "comprising",
"consisting essentially of" and "consisting of" may be replaced
with either of the other two terms. The terms and expressions which
have been employed are used as terms of description and not of
limitation, and there is no intention that in the use of such terms
and expressions of excluding any equivalents of the features shown
and described or portions thereof, but it is recognized that
various modifications are possible within the scope of the
invention claimed. Thus, it should be understood that although the
present invention has been specifically disclosed by preferred
embodiments and optional features, modification and variation of
the concepts herein disclosed may be resorted to by those skilled
in the art, and that such modifications and variations are
considered to be within the scope of this invention as defined by
the appended claims.
[0373] In addition, where features or aspects of the invention are
described in terms of Markush groups, those skilled in the art will
recognize that the invention is also thereby described in terms of
any individual member or subgroup of members of the Markush group.
For example, if X is described as selected from the group
consisting of bromine, chlorine, and iodine, claims for X being
bromine and claims for X being bromine and chlorine are fully
described.
[0374] Other embodiments are set forth within the following
claims.
* * * * *