Cloning using donor nuclei from a non-quiesecent somatic cells

Methods and cell lines for cloning bovine embryos and offspring are provided. The resultant embryos or offspring are especially useful for the expression of desired heterologous DNAs.

FIELD OF THE INVENTION
 The present invention relates to cloning procedures in which cell nuclei
 derived from differentiated fetal or adult bovine cells, which include
 non-serum starved differentiated fetal or adult bovine cells, are
 transplanted into enucleated oocytes of the same species as the donor
 nuclei. The nuclei are reprogrammed to direct the development of cloned
 embryos, which can then be transferred to recipient females to produce
 fetuses and offspring, or used to produce cultured inner cell mass cells
 (CICM). The cloned embryos can also be combined with fertilized embryos to
 produce chimeric embryos, fetuses and/or offspring.
 REFERENCES
 The following publications, patent applications and patents are cited in
 this application as superscript numbers:
 1 Bain, et al., Dev. Biol. 168:342-357 (1995)
 2 Bradley, et al., Nature 309:255-256 (1984)
 3 Campbell, et al., Theriogenology 43:181 (1995)
 4 Campbell, et al., Nature 380:64-68 (1996)
 5 Cherny, et al., Theriogenology 41:175 (1994)
 6 Cheong, et al., Biol. Reprod. 48:958 (1993)
 7 Collas and Barnes, Mol. Reprod. Dev. 38:264-267 (1994)
 8 Cundiff, L. V., Bishop M. D. and Johnson, R. K. Challenges and
 opportunities for integrating genetically modified animals into
 traditional animal breeding plans. Journal of Animal Science 71(Suppl.3)
 20-25 (1993).
 9 Doetschman, T., Gene transfer in embryonic stem cells. In Pinkert. C.
 (ed.) Transgenic Animal Technology: A Laboratory Handbook. Academic Press,
 Inc., New York, pp. 115-146 (1994).
 10 Evans, et al., Nature 29:154-156 (1981)
 11 Fissore, et al., Mol. Reprod. Dev. 46:176-189 (1997)
 12 Friedrich, G. and Soriano, P., Promoter traps in embyronic stem cells: A
 genetic screen to identify and mutate developmental genes in mice. Genes
 and Development 5:1513-1523 (1991).
 13 Gerfen, et al., Anim. Biotech. 6(1):1-14 (1995)
 14 Graham, Wister Inot. Symp. Monogr. 9:19 (1969)
 15 Handyside, et al., Roux's Arch. Dev. Biol. 196:185-190 (1987)
 16 Keefer, et al., Biol. Reprod. 50:935-939 (1994)
 17 MacQuitty, Nature Biotech. 15:294 (1987)
 18 Martin, Proc. Natl. Acad. Sci., USA 78:7634-7638 (1981)
 19 Notarianni, et al., J. Reprod. Fert. Suppl. 41:51-56 (1990)
 20 Notarianni, et al., J. Reprod. Fert. Suppl. 43:255-260 (1991)
 21 Palacios, et al., Proc. Natl. Acad. Sci., USA 92:7530-7537 (1995)
 22 Pedersen, J. Reprod. Fertil. Dev. 6:543-552 (1994)
 23 Prather, et al., Differentiation 48:1-8 (1991)
 24 Purcel, V. G. and Rexroad, Jr., C. E., Status of research with
 transgenic farm animals, Journal of Animal Science 71(Suppl.3). 10-19
 (1993).
 25 Saito, et al., Roux's Arch. Dev. Biol. 201:134-141 (1992)
 26 Seidel, G. E., Jr., Resource requirements for transgenic livestock
 research. Journal of Animal Science
 71(Suppl. 3). 26-33 (1993).
 27 Sims, et al., Proc. Natl. Acad. Sci., USA 90:6143-6147 (1993)
 28 Smith, et al., Dev. Biol. 121:1-9 (1987)
 29 Smith, et al., Biol. Reprod. 40:1027-1035 (1989)
 30 Stice and Robl, Mol. Reprod. Dev. 25:272-280 (1990)
 31 Stice, et al., Biol. Reprod. 54:100-110 (1996)
 32 Van Stekelenburg-Hamers, et al., Mol. Reprod. Dev. 40:444-454 (1995)
 33 Wall, et al., Development of porcine ova that were centrifuged to permit
 visualization of pronuclei and nuclei, Biol. Reprod. 32:645-651 (1985)
 34 Wilmut, I., Schnieke, A. E., McWhir, J., Kind, A. J., Campbell, K. H.
 S., Viable offspring derived from fetal and adult mammalian cells, Nature
 385:810-813 (1997).
 35 Evans, et al., WO 90/03432, published Apr. 5, 1990.
 36 Smith, et al., WO 94/24274, published Oct. 27, 1994.
 37 Wheeler, et al., WO 94/26884, published Nov. 24, 1994.
 38 Prather, et al., U.S. Pat. No. 4,994,384, issued Feb. 19, 1991.
 39 Wheeler, U.S. Pat. No. 5,057,420, issued Oct. 15, 1991.
 40 Rosenkrans, Jr., et al., U.S. Pat. No. 5,096,822, issued Mar. 17, 1992.
 All of the above publications, patent applications and patents are herein
 incorporated by reference in their entirety to the same extent as if each
 individual publication, patent application or patent was specifically and
 individually indicated to be incorporated by reference in its entirety.
 BACKGROUND OF THE INVENTION
 Genetic modification of cattle could be useful in increasing the efficiency
 of meat and milk production. An ideal system for producing transgenic
 animals for agricultural applications would be highly efficient and use
 small numbers of recipient animals to produce transgenics. It would allow
 the insertion of a transgene into a specific genotype. The insertion would
 preferably be into a predetermined site which would confer high expression
 and not affect general viability and productivity of the animal.
 Furthermore, the identification of a locus for insertion would allow
 multiple lines to be produced and crossed to produce homozygotes and new
 genetic background could easily be added to the transgenic line by the
 production of new transgenics at any time. Therefore, the ideal system
 would likely require the transfection and selection of cells that could be
 easily grown in culture yet retain the potency to form germ cells and pass
 the gene to subsequent generations.
 Various methods have been utilized in an attempt to genetically modify
 cattle so as to introduce superior agricultural qualities including
 pronuclear microinjection. One of the limitations of pronuclear
 microinjection is that the gene insertion site is random. This typically
 results in variations in expression levels and several transgeniclines
 must be produced to obtain one line with appropriate levels of expression
 to be useful. Because integration is random, it is advantageous that a
 line of transgenic animals be started from one founder animal, to avoid
 difficulties in monitoring zygosity and potential difficulties that might
 occur with interactions among multiple insertion sites..sup.8 Furthermore,
 starting a transgenic line from one hemizygous animal with a random insert
 would require breeding several generations and significant time for
 introgression of the transgene into the population before breeding and
 testing homozygotes if inbreeding is to be avoided..sup.8 Even without
 concern for inbreeding, it would take 6.5 years before reproduction could
 be tested in homozygous animals..sup.26 Finally, the quality of the
 genetics of a monozygous transgenic line would lag behind that of the
 general population because of the reduced population within which to
 select future generations of transgenic animals and the difficulty of
 bringing new genetics into a population in which the transgene is fixed.
 A second limitation of the pronuclear microinjection procedure is its
 efficiency; which ranges from 0.34 to 2.63% of the gene-injected embryos
 developing into transgenic animals and a fraction of these appropriately
 expressing the gene..sup.24 This inefficiency results in a high cost of
 producing transgenic cattle because of the large number of recipients
 needed and, more importantly, unpredictability in the genetic background
 into which the gene is inserted because of the large number of embryos
 needed for microinjection. For agricultural purposes, a high quality
 genetic background is essential, therefore, long-term backcrossing
 strategies must be used with pronuclear microinjection. Thus, the ability
 to clone, or to make numerous identical genetic copies, of an animal
 comprising a desired genetic modification would be advantageous.
 Another such system for producing transgenic animals has been developed and
 widely used in the mouse and involves the use of embryonic stem (ES)
 cells.
 Embryonic stem cells in mice have enabled researchers to select for
 transgenic cells and perform gene targeting. This allows more genetic
 engineering than is possible with other transgenic techniques. Mouse ES
 cells are relatively easy to grow as colonies in vitro. The cells can be
 transfected by standard procedures and transgenic cells clonally selected
 by antibiotic resistance..sup.9 Furthermore, the efficiency of this
 process is such that sufficient transgenic colonies (hundreds to
 thousands) can be produced to allow a second selection for homologous
 recombinants..sup.9 Mouse ES cells can then be combined with a normal host
 embryo and, because they retain their potency, can develop into all the
 tissues in the resulting chimeric animal, including the germ cells. The
 transgenic modification can then be transmitted to subsequent generations.
 Methods for deriving embryonic stem (ES) cell lines in vitro from early
 preimplantation mouse embryos are well known..sup.10, 18 ES cells can be
 passaged in an undifferentiated state, provided that a feeder layer of
 fibroblast cells.sup.10 or a differentiation inhibiting source.sup.28 is
 present.
 ES cells have been previously reported to possess numerous applications.
 For example, it has been reported that ES cells can be used as an in vitro
 model for differentiation, especially for the study of genes which are
 involved in the regulation of early development. Mouse ES cells can give
 rise to germline chimeras when introduced into preimplantation mouse
 embryos, thus demonstrating their pluripotency..sup.2
 In view of their ability to transfer their genome to the next generation,
 ES cells have potential utility for germline manipulation of livestock
 animals by using ES cells with or without a desired genetic modification.
 Some research groups have reported the isolation of purportedly
 pluripotent embryonic cell lines. For example, Notarianni, et al..sup.20
 reports the establishment of purportedly stable, pluripotent cell lines
 from pig and sheep blastocysts which exhibit some morphological and growth
 characteristics similar to that of cells in primary cultures of inner cell
 masses isolated immunosurgically from sheep blastocysts. Also, Notarianni,
 et al..sup.19 discloses maintenance and differentiation in culture of
 putative pluripotential embryonic cell lines from pig blastocysts. Gerfen,
 et al..sup.13 discloses the isolation of embryonic cell lines from porcine
 blastocysts. These cells are stably maintained without mouse embryonic
 fibroblast feeder layers and reportedly differentiate into several
 different cell types during culture.
 Further, Saito, et al..sup.25 reports cultured, bovine embryonic stem
 cell-like cell lines which survived three passages, but were lost after
 the fourth passage. Handyside, et al..sup.15 discloses culturing of
 immunosurgically isolated inner cell masses of sheep embryos under
 conditions which allow for the isolation of mouse ES cell lines derived
 from mouse ICMs. Handyside, et al. also reports that under such
 conditions, the sheep ICMs attach, spread, and develop areas of both ES
 cell-like and endoderm-like cells, but that after prolonged culture only
 endoderm-like cells are evident.
 Recently, Cherny, et al..sup.5 reported purportedly pluripotent bovine
 primordial germ cell-derived cell lines maintained in long-term culture.
 These cells, after approximately seven days in culture, produced ES-like
 colonies which stained positive for alkaline phosphatase (AP), exhibited
 the ability to form embryoid bodies, and spontaneously differentiated into
 at least two different cell types. These cells also reportedly expressed
 mRNA for the transcription factors OCT4, OCT6 and HES1, a pattern of
 homeobox genes which is believed to be expressed by ES cells exclusively.
 Also recently, Campbell, et al..sup.4 reported the production of live lambs
 following nuclear transfer of cultured embryonic disc (ED) cells from day
 nine ovine embryos cultured under conditions which promote the isolation
 of ES cell lines in the mouse. The authors concluded that ED cells from
 day nine ovine embryos are totipotent by nuclear transfer and that
 totipotency is maintained in culture.
 Van Stekelenburg-Hamers, et al..sup.32 reported the isolation and
 characterization of purportedly permanent cell lines from inner cell mass
 cells of bovine blastocysts. The authors isolated and cultured ICMs from 8
 or 9 day bovine blastocysts under different conditions to determine which
 feeder cells and culture media are most efficient in supporting the
 attachment and outgrowth of bovine ICM cells. They concluded that the
 attachment and outgrowth of cultured ICM cells is enhanced by the use of
 STO (mouse fibroblast) feeder cells (instead of bovine uterus epithelial
 cells) and by the use of charcoal-stripped serum (rather than normal
 serum) to supplement the culture medium. Van Stekelenburg, et al.
 reported, however, that their cell lines resembled epithelial cells more
 than pluripotent ICM cells.
 Smith, et al..sup.36, Evans, et al..sup.35, and Wheeler, et al..sup.37
 report the isolation, selection and propagation of animal stem cells which
 purportedly may be used to obtain transgenic animals. Evans, et al. also
 reported the derivation of purportedly pluripotent embryonic stem cells
 from porcine and bovine species which assertedly are useful for the
 production of transgenic animals. Further, Wheeler, et al. disclosed
 embryonic stem cells which are assertedly useful for the manufacture of
 chimeric and transgenic ungulates.
 Alternatively, ES cells from a transgenic embryo could be used in nuclear
 transplantation. The use of ungulate inner cell mass (ICM) cells for
 nuclear transplantation has also been reported. In the case of livestock
 animals, e.g., ungulates, nuclei from like preimplantation livestock
 embryos support the development of enucleated oocytes to term..sup.16,29
 This is in contrast to nuclei from mouse embryos which beyond the
 eight-cell stage after transfer reportedly do not support the development
 of enucleated oocytes..sup.6 Therefore, ES cells from livestock animals
 are highly desirable because they may provide a potential source of
 totipotent donor nuclei, genetically manipulated or otherwise, for nuclear
 transfer procedures. Collas, et al..sup.7 discloses nuclear
 transplantation of bovine ICMs by microinjection of the lysed donor cells
 into enucleated mature oocytes. Collas, et al. disclosed culturing of
 embryos in vitro for seven days to produce fifteen blastocysts which, upon
 transferral into bovine recipients, resulted in four pregnancies and two
 births. Also, Keefer, et al..sup.27 disclosed the use of bovine ICM cells
 as donor nuclei in nuclear transfer procedures, to produce blastocysts
 which, upon transplantation into bovine recipients, resulted in several
 live offspring. Further, Sims, et al..sup.27 disclosed the production of
 calves by transfer of nuclei from short-term in vitro cultured bovine ICM
 cells into enucleated mature oocytes.
 Thus, based on the foregoing, it is evident that many groups have attempted
 to produce ES cell lines, e.g., because of their potential application in
 the production of cloned or transgenic embryos and in nuclear
 transplantation.
 However, embryonic stem cell lines and other embryonic cell lines must be
 maintained in an undifferentiated state that requires feeder layers and/or
 the addition of cytokines to media. Even if these precautions are
 followed, these cells often undergo spontaneous differentiation and cannot
 be used to produce transgenic offspring by currently available methods.
 Also, some embryonic cell lines have to be propagated in a way that is not
 conducive to gene targeting procedures. Thus, genetic modification using
 differentiated cells would be advantageous.
 The production of live lambs following nuclear transfer of cultured
 embryonic disc cells has also been reported..sup.4 Still further, the use
 of bovine pluripotent embryonic cells in nuclear transfer and the
 production of chimeric fetuses has been reported.sup.7,31 Collas, et
 al..sup.7 demonstrated that granulosa cells (adult cells) could be used in
 a bovine cloning procedure to produce embryos. However, there was no
 demonstration of development past early embryonic stages (blastocyst
 stage). Also, granulosa cells are not easily cultured and are only
 obtainable from females. Collas, et al..sup.7 did not attempt to propagate
 the granulosa cells in culture or try to genetically modify those cells.
 Wilmut, et al..sup.34 produced nuclear transfer sheep offspring derived
 from fetal fibroblast cells, and one offspring from a cell derived from an
 adult sheep.
 Cloning sheep cells is easier in comparison with cells of other species.
 This phenomenon is illustrated by the following table:

SPECIES (from hardest to CELL TYPE OFFSPRING
 easiest to clone) CLONED PRODUCED
 Pig (Prather, Biol. Report, 2 and 4 cell yes
 41:414-418, 1989) stage embryo
 Pig (Prather, Id., 1989; greater than 4 no
 cell stage
 Mouse (Cheong, et al., 2, 4 and 8 cell yes
 Biol. Reprod., 48:958-963, stage embryo
 1993)
 Mouse (Tsunoda, et al., J. greater than 8 no
 Reprod. Fertil., 98:537- cell stage
 540, 1993)
 Cattle (Keefer, et al., 64 to 128 cell yes
 Biol. Reprod., 50:935-939, stage (ICM)
 1994)
 Cattle (Stice, et al., embryonic cell no
 Biol. Repro., 54:100-110, line from ICM
 1996)
 Sheep (Campbell, et al., embryonic cell yes
 Nature, 380:64-66, 1996) line from ICM
 Sheep (Wilmut, et al., BARC fetal and yes
 Symposia, 20:145-150, 1997) adult cells
 However, there exist problems in the area of producing transgenic cows. By
 current methods, heterologous DNA is introduced into either early embryos
 or embryonic cell lines that differentiate into various cell types in the
 fetus and eventually develop into a transgenic animal. One limitation is
 that many early embryos are required to produce one transgenic animal and,
 thus, this procedure is very inefficient. Also, there is no simple and
 efficient method of selecting for a transgenic embryo before going through
 the time and expense of putting the embryos into surrogate females. In
 addition, gene targeting techniques cannot be easily accomplished with
 early embryo transgenic procedures.
 Therefore, notwithstanding what has previously been reported in the
 literature, there exists a need for improved methods of cloning cows using
 cultured differentiated cells as donor nuclei.
 OBJECTS AND SUMMARY OF THE INVENTION
 It is an object of the invention to provide novel and improved methods for
 producing cloned cows using cultured differentiated bovine cells, in
 particular non-serum starved differentiated bovine cells as donor nuclei.
 It is a more specific object of the invention to provide a novel method for
 cloning cows which involves transplantation of the nucleus of a
 differentiated cow cell, in particular a non-serum starved differentiated
 bovine cell, into an enucleated cow oocyte.
 It is another object of the invention to provide a method for multiplying
 adult cows having proven genetic superiority or other desirable traits.
 It is another object of the invention to provide an improved method for
 producing genetically engineered or transgenic cows (i.e., NT units,
 fetuses, offspring). The invention also provides genetically engineered or
 transgenic cows, including those made by such a method.
 It is a more specific object of the invention to provide a method for
 producing genetically engineered or transgenic cows by which a desired DNA
 sequence is inserted, removed or modified in a differentiated cow cell or
 cell nucleus, which may be non-serum starved, prior to use of that
 differentiated cell or cell nucleus for formation of a NT unit. The
 invention also provides genetically engineered or transgenic cows made by
 such a method.
 It is another object of the invention to provide a novel method for
 producing cow CICM cells which involves transplantation of a nucleus of a
 serum or non-serum starved differentiated cow cell into an enucleated cow
 oocyte, and then using the resulting NT unit to produce CICM cells. The
 invention also provides cow CICM cells produced by such a method.
 It is another object of the invention to use such cow CICM cells for
 therapy or diagnosis.
 It is a specific object of the invention to use such cow CICM cells for
 treatment or diagnosis of any disease wherein cell, tissue or organ
 transplantation is therapeutically or diagnostically beneficial. The CICM
 cells may be used within the same species or across species.
 It is another object of the invention to use cells or tissues derived from
 cow NT units, fetuses or offspring for treatment or diagnosis of any
 disease wherein cell, tissue or organ transplantation is therapeutically
 or diagnostically beneficial. Such diseases and injuries include
 Parkinson's, Huntington's, Alzheimer's, ALS, spinal cord injuries,
 multiple sclerosis, muscular dystrophy, diabetes, liver diseases, heart
 disease, cartilage replacement, burns, vascular diseases, urinary tract
 diseases, as well as for the treatment of immune defects, bone marrow
 transplantation, cancer, among other diseases. The tissues may be used
 within the same species or across species.
 It is another specific object of the invention to use cells or tissues
 derived from cow NT units, fetuses or offspring, or cow CICM cells
 produced according to the invention for the production of differentiated
 cells, tissues or organs.
 It is another specific object of the invention to use cells or tissues
 derived from cow NT units, fetuses or offspring, or cow CICM cells
 produced according to the invention in vitro, e.g. for study of cell
 differentiation and for assay purposes, e.g. for drug studies.
 It is another object of the invention to use cells, tissues or organs
 produced from such tissues derived from cow NT units, fetuses or
 offspring, or cow CICM cells to provide improved methods of
 transplantation therapy. Such therapies include by way of example
 treatment of diseases and injuries including Parkinson's, Huntington's,
 Alzheimer's, ALS, spinal cord injuries, multiple sclerosis, muscular
 dystrophy, diabetes, liver diseases, heart disease, cartilage replacement,
 burns, vascular diseases, urinary tract diseases, as well as for the
 treatment of immune defects, bone marrow transplantation, cancer, among
 other diseases.
 It is another object of the invention to provide genetically engineered or
 transgenic tissues derived from cow NT units, fetuses or offspring, or cow
 CICM cells produced by inserting, removing or modifying a desired DNA
 sequence in a differentiated cow cell or cell nucleus prior to use of that
 differentiated cell or cell nucleus for formation of a NT unit.
 It is another object of the invention to use the transgenic or genetically
 engineered tissues derived from cow NT units, fetuses or offspring, or cow
 CICM cells produced according to the invention for gene therapy, in
 particular for the treatment and/or prevention of the diseases and
 injuries identified, supra.
 It is another object of the invention to use the tissues derived from cow
 NT units, fetuses or offspring, or cow CICM cells produced according to
 the invention, or transgenic or genetically engineered tissues derived
 from cow NT units, fetuses or offspring, or cow CICM cells produced
 according to the invention as nuclear donors for nuclear transplantation.
 It is another object of the invention to use transgenic or genetically
 engineered cow offspring produced according to the invention in order to
 produce pharmacologically important proteins.
 Thus, in one aspect, the present invention provides a method for cloning a
 cow (e.g., embryos, fetuses, offspring). The method comprises:
 (i) inserting a desired serum or non-serum starved differentiated cow cell
 or cell nucleus into an enucleated cow oocyte, under conditions suitable
 for the formation of a nuclear transfer (NT) unit to yield a fused NT
 unit;
 (ii) activating the fused NT unit to yield an activated NT unit; and
 (iii) transferring said activated NT unit to a host cow such that the NT
 unit develops into a fetus.
 Optionally, the activated nuclear transfer unit is cultured until greater
 than the 2-cell developmental stage.
 The cells, tissues and/or organs of the fetus are advantageously used in
 the area of cell, tissue and/or organ transplantation, or production of
 desirable genotypes.
 The present invention also includes a method of cloning a genetically
 engineered or transgenic cow, by which a desired DNA sequence is inserted,
 removed or modified in the differentiated cow cell or cell nucleus prior
 to insertion of the differentiated cow cell or cell nucleus into the
 enucleated oocyte. Genetically engineered or transgenic cows produced by
 such a method are advantageously used in the area of cell, tissue and/or
 organ transplantation, production of desirable genotypes, and production
 of pharmaceutical proteins.
 Also provided by the present invention are cows obtained according to the
 above method, and offspring of those cows.
 In another aspect, the present invention provides a method for producing
 cow CICM cells. The method comprises:
 (i) inserting a desired serum or non-serum starved differentiated cow cell
 or cell nucleus into an enucleated cow oocyte, under conditions suitable
 for the formation of a nuclear transfer (NT) unit to yield a fused NT
 unit;
 (ii) activating the fused NT unit to yield an activated NT unit; and
 (iii) culturing cells obtained from said activated NT unit to obtain cow
 CICM cells.
 Optionally, the activated nuclear transfer unit is cultured until greater
 than the 2-cell developmental stage.
 The cow CICM cells are advantageously used in the area of cell, tissue and
 organ transplantation.
 With the foregoing and other objects, advantages and features of the
 invention that will become hereinafter apparent, the nature of the
 invention may be more clearly understood by reference to the following
 detailed description of the preferred embodiments of the invention and to
 the appended claims.
 DETAILED DESCRIPTION OF THE INVENTION
 This invention provides improved to cloning procedures in which cell nuclei
 derived from differentiated fetal or adult bovine cells which may be serum
 or non-serum starved are transplanted into enucleated oocytes of the same
 species as the donor nuclei. However, prior to discussing this invention
 in further detail, the following terms will first be defined.
 Definitions
 As used herein, the following terms have the following meanings:
 The term "differentiated" refers to cells having a different character or
 function from the surrounding structures or from the cell of origin.
 Differentiated cow cells are those cells which are past the early
 embryonic stage. More particularly, the differentiated cells are those
 from at least past the embryonic disc stage (day 10 of bovine
 embryogenesis). The differentiated cells may be derived from ectoderm,
 mesoderm or endoderm.
 The term "nuclear transfer" or "nuclear transplantation" refers to a method
 of cloning wherein the nucleus from a donor cell is transplanted into
 enucleated oocytes. Nuclear transfer techniques or nuclear transplantation
 techniques are known in the literature..sup.3,7,16,27,35-37 Also, U.S.
 Pat. Nos. 4,994,384 and 5,057,420 describe procedures for bovine nuclear
 transplantation. In the subject application, nuclear transfer or nuclear
 transplantation or NT are used interchangeably.
 The term "nuclear transfer unit" or "NT unit" refers to the product of
 fusion between a differentiated cow cell or cell nucleus and an enucleated
 cow oocyte, and is sometimes referred to herein as a fused NT unit.
 The term "non-serum starved bovine differentiated cells" refers to cells
 cultured in the presence of serum greater than about 1%.
 The term "fetus" refers to the unborn young of a viviparous animal after it
 has taken form in the uterus. In cows, the fetal stage occurs from 35 days
 after conception until birth.
 The term "adult" refers to a mammal from birth until death.
 According to the invention, cell nuclei derived from differentiated cow
 cells are transplanted into enucleated cow oocytes. The nuclei are
 reprogrammed to direct the development of cloned embryos, which can then
 be transferred into recipient females to produce fetuses and offspring, or
 used to produce CICM cells. The cloned embryos can also be combined with
 fertilized embryos to produce chimeric embryos, fetuses and/or offspring.
 Prior art methods have used embryonic cell types in cloning procedures.
 This includes work by Campbell, et al..sup.4 and Stice, et al..sup.31 In
 both of those studies, embryonic cell lines were derived from embryos of
 less than 10 days of gestation. In both studies, the cells were maintained
 on a feeder layer to prevent overt differentiation of the donor cell to be
 used in the cloning procedure. The present invention uses differentiated
 cells.
 Adult cells and fetal fibroblast cells from a sheep have purportedly been
 used to produce sheep offspring..sup.34 However, of the mammalian species
 studied, cloning of sheep appears to be the easiest, and pig cloning
 appears to be the most difficult. The successful cloning of cows using
 differentiated cell types according to the present invention was quite
 unexpected.
 Thus, according to the present invention, multiplication of superior
 genotypes of cows is possible. This will allow the multiplication of adult
 cows with proven genetic superiority or other desirable traits. Genetic
 progress will be accelerated in the cow. By the present invention, there
 are potentially billions of fetal or adult cow cells that can be harvested
 and used in the cloning procedure. This will potentially result in many
 identical offspring in a short period.
 It was unexpected that cloned embryos with fetal or adult donor nuclei
 could develop to advanced embryonic and fetal stages. The scientific dogma
 has been that only early embryonic cell types could direct this type of
 development. It was unexpected that a large number of cloned embryos could
 be produced from fetal or adult cells. Also, the fact that new transgenic
 embryonic cell lines could be readily derived from transgenic cloned
 embryos was unexpected.
 Adult cells and fetal fibroblast cells from a sheep have purportedly been
 used to produce a sheep offspring (Wilmut et al, 1997). In that study,
 however, it was emphasized that the use of a serum starved, nucleus donor
 cell in the quiescent state was important for success of the Wilmut
 cloning method. No such requirement for serum starvation or quiescence
 exists for the present invention. On the contrary, cloning is achieved
 using non-serum starved, differentiated mammalian cells. Moreover, cloning
 efficiency according to the present invention can be the same regardless
 of whether fetal or adult donor cells are used, whereas Wilmut et al
 (1997) reported that lower cloning efficiency was achieved with adult
 donor cells.
 There has also been speculation that the Wilmut, et al. method will lead to
 the generation of transgenic animals..sup.17 However, there is no reason
 to assume, for example, that nuclei from adult cells that have been
 transfected with exogenous DNA will be able to survive the process of
 nuclear transfer. In this regard, it is known that the properties of mouse
 embryonic stem (ES) cells are altered by in vitro manipulation such that
 their ability to form viable chimeric embryos is effected. Therefore,
 prior to the present invention, the cloning of transgenic animals could
 not have been predicted.
 The present invention also allows simplification of transgenic procedures
 by working with a cell source that can be clonally propagated. This
 eliminates the need to maintain the cells in an undifferentiated state,
 thus, genetic modifications, both random integration and gene targeting,
 are more easily accomplished. Also by combining nuclear transfer with the
 ability to modify and select for these cells in vitro, this procedure is
 more efficient than previous transgenic embryo techniques. According to
 the present invention, these cells can be clonally propagated without
 cytokines, conditioned media and/or feeder layers, further simplifying and
 facilitating the transgenic procedure. When transfected cells are used in
 cloning procedures according to the invention, transgenic cow embryos are
 produced which can develop into fetuses and offspring. Also, these
 transgenic cloned embryos can be used to produce CICM cell lines or other
 embryonic cell lines. Therefore, the present invention eliminates the need
 to derive and maintain in vitro an undifferentiated cell line that is
 conducive to genetic engineering techniques.
 The present invention can also be used to produce cloned cow fetuses,
 offspring or CICM cells which can be used, for example, in cell, tissue
 and organ transplantation. By taking a fetal or adult cell from a cow and
 using it in the cloning procedure a variety of cells, tissues and possibly
 organs can be obtained from cloned fetuses as they develop through
 organogenesis. Cells, tissues, and organs can be isolated from cloned
 offspring as well. This process can provide a source of "materials" for
 many medical and veterinary therapies including cell and gene therapy.
 If the cells are transferred back into the animal in which the cells were
 derived, then immunological rejection is averted. Also, because many cell
 types can be isolated from these clones, other methodologies such as
 hematopoietic chimerism can be used to avoid immunological rejection among
 animals of the same species as well as between species.
 Thus, in one aspect, the present invention provides a method for cloning a
 cow. In general, the cow will be produced by a nuclear transfer process
 comprising the following steps:
 (i) obtaining desired differentiated cow cells, which may be serum or
 non-serum starved, to be used as a source of donor nuclei;
 (ii) obtaining oocytes from a cow;
 (iii) enucleating said oocytes;
 (iv) transferring the desired differentiated cell or cell nucleus into the
 enucleated oocyte, e.g., by fusion or injection, to form an NT unit;
 (v) activating the NT unit to yield an activated NT unit; and
 (vii) transferring said activated NT unit to a host cow such that the NT
 unit develops into a fetus.
 Optionally, the activated nuclear transfer unit is cultured until greater
 than the 2-cell developmental stage prior to transfer to the host cow.
 The present invention also includes a method of cloning a genetically
 engineered or transgenic cow, by which a desired DNA sequence is inserted,
 removed or modified in the serum or non-serum starved differentiated cow
 cell or cell nucleus prior to insertion of the differentiated cow cell or
 cell nucleus into the enucleated oocyte.
 Also provided by the present invention are cows obtained according to the
 above method, and offspring of those cows.
 In addition to the uses described above, the genetically engineered or
 transgenic cows according to the invention can be used to produced a
 desired protein, such as a pharmacologically important protein, e.g.,
 human serum albumin. That desired protein can then be isolated from the
 milk or other fluids or tissues of the transgenic cow. Alternatively, the
 exogenous DNA sequence may confer an agriculturally useful trait to the
 transgenic cow, such as disease resistance, decreased body fat, increased
 lean meat product, improved feed conversion, or altered sex ratios in
 progeny.
 The present invention further provides for the use of NT fetuses and NT and
 chimeric offspring in the area of cell, tissue and organ transplantation.
 In another aspect, the present invention provides a method for producing
 cow CICM cells. The method comprises:
 (i) inserting a desired serum or non-serum starved differentiated cow cell
 or cell nucleus into an enucleated cow oocyte, under conditions suitable
 for the formation of a nuclear transfer (NT) unit;
 (ii) activating the resultant nuclear transfer unit to yield an activated
 nuclear transfer unit; and
 (iii) culturing cells obtained from said activated NT unit to obtain cow
 CICM cells.
 Optionally, the activated nuclear transfer unit is cultured until greater
 than the 2-cell developmental stage.
 The cow CICM cells are advantageously used in the area of cell, tissue and
 organ transplantation, or in the production of fetuses or offspring,
 including transgenic fetuses or offspring.
 Preferably, the NT units will be cultured to a size of at least 2 to 400
 cells, preferably 4 to 128 cells, and most preferably to a size of at
 least about 50 cells.
 Cow cells may be obtained by well known methods. Cow cells useful in the
 present invention include, by way of example, epithelial cells, neural
 cells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes,
 chondrocytes, lymphocytes (B and T lymphocytes), erythrocytes,
 macrophages, monocytes, mononuclear cells, fibroblasts, cardiac muscle
 cells, and other muscle cells, etc. Moreover, the cow cells used for
 nuclear transfer may be obtained from different organs, e.g., skin, lung,
 pancreas, liver, stomach, intestine, heart, reproductive organs, bladder,
 kidney, urethra and other urinary organs, etc. These are just examples of
 suitable donor cells. Suitable donor cells, i.e., cells useful in the
 subject invention, may be obtained from any cell or organ of the body.
 This includes all somatic or germ cells.
 Fibroblast cells are an ideal cell type because they can be obtained from
 developing fetuses and adult cows in large quantities. Fibroblast cells
 are differentiated somewhat and, thus, were previously considered a poor
 cell type to use in cloning procedures. Importantly, these cells can be
 easily propagated in vitro with a rapid doubling time and can be clonally
 propagated for use in gene targeting procedures. Again the present
 invention is novel because differentiated cell types are used. The present
 invention is advantageous because the cells can be easily propagated,
 genetically modified and selected in vitro.
 Other reported cloning methods (e.g., Wilmut et al, 1997) have relied on
 the use of serum starved cells. The present invention, however, includes
 the use of donor cells which are not in a state of serum starvation.
 According to Wilmut et al (1997), serum starved cells are quiescent, i.e.,
 exiting the growth phase. Other methods (chemical, temperature, etc.) are
 also capable of producing quiescent cells. By contrast, in the present
 invention the donor cells used may or may not be quiescent.
 The stage of maturation of the oocyte at enucleation and nuclear transfer
 has been reported to be significant to the success of NT methods. In
 general, successful mammalian embryo cloning practices use the metaphase
 II stage oocyte as the recipient oocyte because at this stage it is
 believed that the oocyte can be or is sufficiently "activated" to treat
 the introduced nucleus as it does a fertilizing sperm. In domestic
 animals, the oocyte activation period generally ranges from about 16-52
 hours, preferably about 20-45 hours post-aspiration.
 Methods for isolation of oocytes are well known in the art. Essentially,
 this will comprise isolating oocytes from the ovaries or reproductive
 tract of a bovine mammal, e.g., a bovine. A readily available source of
 bovine oocytes is slaughterhouse materials.
 For the successful use of techniques such as genetic engineering, nuclear
 transfer and cloning, oocytes must generally be matured in vitro before
 these cells may be used as recipient cells for nuclear transfer, and
 before they can be fertilized by the sperm cell to develop into an embryo.
 This process generally requires collecting immature (prophase I) oocytes
 from mammalian ovaries, e.g., bovine ovaries obtained at a slaughterhouse,
 and maturing the oocytes in a maturation medium prior to fertilization or
 enucleation until the oocyte attains the metaphase II stage, which in the
 case of bovine oocytes generally occurs about 18-24 hours post-aspiration.
 For purposes of the present invention, this period of time is known as the
 "maturation period." As used herein for calculation of time periods,
 "aspiration" refers to aspiration of the immature oocyte from ovarian
 follicles.
 Additionally, metaphase II stage oocytes, which have been matured in vivo
 have been successfully used in nuclear transfer techniques. Essentially,
 mature metaphase II oocytes are collected surgically from either
 non-superovulated or superovulated cows or heifers 35 to 48 hours past the
 onset of estrus or past the injection of human chorionic gonadotropin
 (hCG) or similar hormone.
 The stage of maturation of the oocyte at enucleation and nuclear transfer
 has been reported to be significant to the success of NT methods. (See
 e.g., Prather et al., Differentiation, 48, 1-8, 1991). In general,
 successful mammalian embryo cloning practices use the metaphase II stage
 oocyte as the recipient oocyte because at this stage it is believed that
 the oocyte can be or is sufficiently "activated" to treat the introduced
 nucleus as it does a fertilizing sperm. In domestic animals, and
 especially cattle, the oocyte activation period generally ranges from
 about 16-52 hours, preferably about 28-42 hours post-aspiration.
 For example, immature oocytes may be washed in HEPES buffered hamster
 embryo culture medium (HECM) as described in Seshagine et al., Biol.
 Reprod., 40, 544-606, 1989, and then placed into drops of maturation
 medium consisting of 50 microliters of tissue culture medium (TCM) 199
 containing 10% fetal calf serum which contains appropriate gonadotropins
 such as luteinizing hormone (LH) and follicle stimulating hormone (FSH),
 and estradiol under a layer of lightweight paraffin or silicon at
 39.degree. C.
 After a fixed time maturation period, which ranges from about 10 to 40
 hours, and preferably about 16-18 hours, the oocytes will be enucleated.
 Prior to enucleation the oocytes will preferably be removed and placed in
 HECM containing 1 milligram per milliliter of hyaluronidase prior to
 removal of cumulus cells. This may be effected by repeated pipetting
 through very fine bore pipettes or by vortexing briefly. The stripped
 oocytes are then screened for polar bodies, and the selected metaphase II
 oocytes, as determined by the presence of polar bodies, are then used for
 nuclear transfer. Enucleation follows.
 Enucleation may be effected by known methods, such as described in U.S.
 Pat. No. 4,994,384 which is incorporated by reference herein. For example,
 metaphase II oocytes are either placed in HECM, optionally containing 7.5
 micrograms per milliliter cytochalasin B, for immediate enucleation, or
 may be placed in a suitable medium, for example an embryo culture medium
 such as CR1aa, plus 10% estrus cow serum, and then enucleated later,
 preferably not more than 24 hours later, and more preferably 16-18 hours
 later.
 Enucleation may be accomplished microsurgically using a micropipette to
 remove the polar body and the adjacent cytoplasm. The oocytes may then be
 screened to identify those of which have been successfully enucleated.
 This screening may be effected by staining the oocytes with 1 microgram
 per milliliter 33342 Hoechst dye in HECM, and then viewing the oocytes
 under ultraviolet irradiation for less than 10 seconds. The oocytes that
 have been successfully enucleated can then be placed in a suitable culture
 medium, e.g., CR1aa plus 10% serum.
 In the present invention, the recipient oocytes will preferably be
 enucleated at a time ranging from about 10 hours to about 40 hours after
 the initiation of in vitro maturation, more preferably from about 16 hours
 to about 24 hours after initiation of in vitro maturation, and most
 preferably about 16-18 hours after initiation of in vitro maturation.
 A single mammalian cell of the same species as the enucleated oocyte will
 then be transferred into the perivitelline space of the enucleated oocyte
 used to produce the NT unit. The mammalian cell and the enucleated oocyte
 will be used to produce NT units according to methods known in the art.
 For example, the cells may be fused by electrofusion. Electrofusion is
 accomplished by providing a pulse of electricity that is sufficient to
 cause a transient breakdown of the plasma membrane. This breakdown of the
 plasma membrane is very short because the membrane reforms rapidly. Thus,
 if two adjacent membranes are induced to breakdown and upon reformation
 the lipid bilayers intermingle, small channels will open between the two
 cells. Due to the thermodynamic instability of such a small opening, it
 enlarges until the two cells become one. Reference is made to U.S. Pat.
 No. 4,997,384 by Prather et al., (incorporated by reference in its
 entirety herein) for a further discussion of this process. A variety of
 electrofusion media can be used including e.g., sucrose, mannitol,
 sorbitol and phosphate buffered solution. Fusion can also be accomplished
 using Sendai virus as a fusogenic agent (Graham, Wister Inot. Symp.
 Monogr., 9, 19, 1969).
 Also, in some cases (e.g. with small donor nuclei) it may be preferable to
 inject the nucleus directly into the oocyte rather than using
 electroporation fusion. Such techniques are disclosed in Collas and
 Barnes, Mol. Reprod. Dev., 38:264-267 (1994), incorporated by reference in
 its entirety herein.
 Preferably, the bovine cell and oocyte are electrofused in a 500 .mu.m
 chamber by application of an electrical pulse of 90-120V for about 15
 .mu.sec, about 24 hours after initiation of oocyte maturation. After
 fusion, the resultant fused NT units are then placed in a suitable medium
 until activation, e.g., CR1aa medium. Typically activation will be
 effected shortly thereafter, typically less than 24 hours later, and
 preferably about 4-9 hours later.
 The NT unit may be activated by known methods. Such methods include, e.g.,
 culturing the NT unit at sub-physiological temperature, in essence by
 applying a cold, or actually cool temperature shock to the NT unit. This
 may be most conveniently done by culturing the NT unit at room
 temperature, which is cold relative to the physiological temperature
 conditions to which embryos are normally exposed.
 Alternatively, activation may be achieved by application of known
 activation agents. For example, penetration of oocytes by sperm during
 fertilization has been shown to activate prefusion oocytes to yield
 greater numbers of viable pregnancies and multiple genetically identical
 calves after nuclear transfer. Also, treatments such as electrical and
 chemical shock may be used to activate NT embryos after fusion. Suitable
 oocyte activation methods are the subject of U.S. Pat. No. 5,496,720, to
 Susko-Parrish et al., herein incorporated by reference in its entirety.
 Additionally, activation may be effected by simultaneously or sequentially:
 (i) increasing levels of divalent cations in the oocyte, and
 (ii) reducing phosphorylation of cellular proteins in the oocyte.
 This will generally be effected by introducing divalent cations into the
 oocyte cytoplasm, e.g., magnesium, strontium, barium or calcium, e.g., in
 the form of an ionophore. Other methods of increasing divalent cation
 levels include the use of electric shock, treatment with ethanol and
 treatment with caged chelators.
 Phosphorylation may be reduced by known methods, e.g., by the addition of
 kinase inhibitors, e.g., serine-threonin kinase inhibitors, such as
 6-dimethylaminopurine, staurosporine, 2-aminopurine, and sphingosine.
 Alternatively, phosphorylation of cellular proteins may be inhibited by
 introduction of a phosphatase into the oocyte, e.g., phosphatase 2A and
 phosphatase 2B.
 In one embodiment, NT activation is effected by briefly exposing the fused
 NT unit to a TL-HEPES medium containing 5 .mu.M ionomycin and 1 mg/ml BSA,
 followed by washing in TL-HEPES containing 30 mg/ml BSA within about 24
 hours after fusion, and preferably about 4 to 9 hours after fusion.
 The activated NT units may then be cultured in a suitable in vitro culture
 medium until the generation of CICM cells and cell colonies. Culture media
 suitable for culturing and maturation of embryos are well known in the
 art. Examples of known media, which may be used for bovine embryo culture
 and maintenance, include Ham's F-10+10% fetal calf serum (FCS), Tissue
 Culture Medium-199 (TCM-199)+10% fetal calf serum,
 Tyrodes-Albumin-Lactate-Pyruvate (TALP), Dulbecco's Phosphate Buffered
 Saline (PBS), Eagle's and Whitten's media. One of the most common media
 used for the collection and maturation of oocytes is TCM-199, and 1 to 20%
 serum supplement including fetal calf serum, newborn serum, estrual cow
 serum, lamb serum or steer serum. A preferred maintenance medium includes
 TCM-199 with Earl salts, 10% fetal calf serum, 0.2 mM Na pyruvate and 50
 pg/ml gentamicin sulphate. Any of the above may also involve co-culture
 with a variety of cell types such as granulosa cells, oviduct cells, BRL
 cells and uterine cells and STO cells.
 Another maintenance medium is described in U.S. Pat. No. 5,096,822 to
 Rosenkrans, Jr. et al., which is incorporated herein by reference. This
 embryo medium, named CR1, contains the nutritional substances necessary to
 support an embryo.
 CR1 contains hemicalcium L-lactate in amounts ranging from 1.0 mM to 10 mM,
 preferably 1.0 mM to 5.0 mM. Hemicalcium L-lactate is L-lactate with a
 hemicalcium salt incorporated thereon. Hemicalcium L-lactate is
 significant in that a single component satisfies two major requirements in
 the culture medium: (i) the calcium requirement necessary for compaction
 and cytoskeleton arrangement; and (ii) the lactate requirement necessary
 for metabolism and electron transport. Hemicalcium L-lactate also serves
 as valuable mineral and energy source for the medium necessary for
 viability of the embryos.
 Advantageously, CR1 medium does not contain serum, such as fetal calf
 serum, and does not require the use of a co-culture of animal cells or
 other biological media, i.e., media comprising animal cells such as
 oviductal cells. Biological media can sometimes be disadvantageous in that
 they may contain microorganisms or trace factors which may be harmful to
 the embryos and which are difficult to detect, characterize and eliminate.
 Examples of the main components in CR1 medium include hemicalcium
 L-lactate, sodium chloride, potassium chloride, sodium bicarbonate and a
 minor amount of fatty-acid free bovine serum albumin (Sigma A-6003).
 Additionally, a defined quantity of essential and non-essential amino
 acids may be added to the medium. CR1 with amino acids is known by the
 abbreviation "CR1aa."
 CR1 medium preferably contains the following components in the following
 quantities:
 sodium chloride--114.7 mM
 potassium chloride--3.1 mM
 sodium bicarbonate--26.2 mM
 hemicalcium L-lactate--5 mM
 fatty-acid free BSA--3 mg/ml
 In one embodiment, the activated NT embryos unit are placed in CR1aa medium
 containing 1.9 mM DMAP for about 4 hours followed by a wash in HECM and
 then cultured in CR1aa containing BSA.
 For example, the activated NT units may be transferred to CR1aa culture
 medium containing 2.0 mM DMAP (Sigma) and cultured under ambient
 conditions, e.g., about 38.5.degree. C., 5% CO.sub.2 for a suitable time,
 e.g., about 4 to 5 hours.
 Afterward, the cultured NT unit or units are preferably washed and then
 placed in a suitable media, e.g., CR1aa medium containing 10% FCS and 6
 mg/ml contained in well plates which preferably contain a suitable
 confluent feeder layer. Suitable feeder layers include, by way of example,
 fibroblasts and epithelial cells, e.g., fibroblasts and uterine epithelial
 cells derived from ungulates, chicken fibroblasts, murine (e.g., mouse or
 rat) fibroblasts, STO and SI-m220 feeder cell lines, and BRL cells.
 In one embodiment, the feeder cells comprise mouse embryonic fibroblasts.
 Preparation of a suitable fibroblast feeder layer is described in the
 example which follows and is well within the skill of the ordinary
 artisan.
 The methods for embryo transfer and recipient animal management in the
 present invention are standard procedures used in the embryo transfer
 industry. Synchronous transfers are important for success of the present
 invention, i.e., the stage of the NT embryo is in synchrony with the
 estrus cycle of the recipient female. This advantage and how to maintain
 recipients are reviewed in Siedel, G. E., Jr. ("Critical review of embryo
 transfer procedures with cattle" in Fertilization and Embryonic
 Development in Vitro (1981) L. Mastroianni, Jr. and J. D. Biggers, ed.,
 Plenum Press, New York, N.Y., page 323), the contents of which are hereby
 incorporated by reference.
 The present invention can also be used to clone genetically engineered or
 transgenic cows. As explained above, the present invention is advantageous
 in that transgenic procedures can be simplified by working with a
 differentiated cell source that can be clonally propagated. In particular,
 the differentiated cells used for donor nuclei, which may or may not be
 serum-starved, have a desired DNA sequence inserted, removed or modified.
 Those genetically altered, differentiated cells are then used for nuclear
 transplantation with enucleated oocytes.
 Any known method for inserting, deleting or modifying a desired DNA
 sequence from a mammalian cell may be used for altering the differentiated
 cell to be used as the nuclear donor. These procedures may remove all or
 part of a DNA sequence, and the DNA sequence may be heterologous. Included
 is the technique of homologous recombination, which allows the insertion,
 deletion or modification of a DNA sequence or sequences at a specific site
 or sites in the cell genome.
 The present invention can thus be used to provide adult cows with desired
 genotypes. Multiplication of adult cows with proven genetic superiority or
 other desirable traits is particularly useful, including transgenic or
 genetically engineered animals, and chimeric animals. Thus, the present
 invention will allow production of single sex offspring, and production of
 cows having improved meat production, reproductive traits and disease
 resistance. Furthermore, cell and tissues from the NT fetus, including
 transgenic and/or chimeric fetuses, can be used in cell, tissue and organ
 transplantation for the treatment of numerous diseases as described below
 in connection with the use of CICM cells. Hence, transgenic cows have uses
 including models for diseases, xenotransplantation of cells and organs,
 and production of pharmaceutical proteins.
 For production of CICM cells and cell lines, the activated NT units are
 cultured under conditions which promote cell division without
 differentiation to provide for cultured NT units. After cultured NT units
 of the desired size are obtained, the cells are mechanically removed from
 the zone and are then used. This is preferably effected by taking the
 clump of cells which comprise the cultured NT unit, which typically will
 contain at least about 50 cells, washing such cells, and plating the cells
 onto a feeder layer, e.g., irradiated fibroblast cells. Typically, the
 cells used to obtain the stem cells or cell colonies will be obtained from
 the inner most portion of the cultured NT unit which is preferably at
 least 50 cells in size. However, cultured NT units of smaller or greater
 cell numbers as well as cells from other portions of the cultured NT unit
 may also be used to obtain ES cells and cell colonies. The cells are
 maintained on the feeder layer in a suitable growth medium, e.g., alpha
 MEM supplemented with 10% FCS and 0.1 mM .beta.-mercaptoethanol (Sigma)
 and L-glutamine. The growth medium is changed as often as necessary to
 optimize growth, e.g., about every 2-3 days.
 This culturing process results in the formation of CICM cells or cell
 lines. One skilled in the art can vary the culturing conditions as desired
 to optimize growth of the particular CICM cells. Also, genetically
 engineered or transgenic cow CICM cells may be produced according to the
 present invention. That is, the methods described above can be used to
 produce NT units in which a desired DNA sequence or sequences have been
 introduced, or from which all or part of an endogenous DNA sequence or
 sequences have been removed or modified. Those genetically engineered or
 transgenic NT units can then be used to produce genetically engineered or
 transgenic CICM cells.
 The resultant CICM cells and cell lines have numerous therapeutic and
 diagnostic applications. Most especially, such CICM cells may be used for
 cell transplantation therapies.
 In this regard, it is known that mouse embryonic stem (ES) cells are
 capable of differentiating into almost any cell type, e.g., hematopoietic
 stem cells. Therefore, cow CICM cells produced according to the invention
 should possess similar differentiation capacity. The CICM cells according
 to the invention will be induced to differentiate to obtain the desired
 cell types according to known methods. For example, the subject cow CICM
 cells may be induced to differentiate into hematopoietic stem cells,
 neural cells, muscle cells, cardiac muscle cells, liver cells, cartilage
 cells, epithelial cells, urinary tract cells, neural cells, etc., by
 culturing such cells in differentiation medium and under conditions which
 provide for cell differentiation. Medium and methods which result in the
 differentiation of CICM cells are known in the art as are suitable
 culturing conditions.
 For example, Palacios, et al..sup.21 teaches the production of
 hematopoietic stem cells from an embryonic cell line by subjecting stem
 cells to an induction procedure comprising initially culturing aggregates
 of such cells in a suspension culture medium lacking retinoic acid
 followed by culturing in the same medium containing retinoic acid,
 followed by transferral of cell aggregates to a substrate which provides
 for cell attachment.
 Moreover, Pedersen.sup.22 is a review article which references numerous
 articles disclosing methods for in vitro differentiation of embryonic stem
 cells to produce various differentiated cell types including hematopoietic
 cells, muscle, cardiac muscle, nerve cells, among others.
 Further, Bain, et al..sup.1 teaches in vitro differentiation of embryonic
 stem cells to produce neural cells which possess neuronal properties.
 These references are exemplary of reported methods for obtaining
 differentiated cells from embryonic or stem cells. These references and in
 particular the disclosures therein relating to methods for differentiating
 embryonic stem cells are incorporated by reference in their entirety
 herein.
 Thus, using known methods and culture mediums, one skilled in the art may
 culture the subject CICM cells, including genetically engineered or
 transgenic CICM cells, to obtain desired differentiated cell types, e.g.,
 neural cells, muscle cells, hematopoietic cells, etc.
 The subject CICM cells may be used to obtain any desired differentiated
 cell type. Therapeutic usages of such differentiated cells are
 unparalleled. For example, hematopoietic stem cells may be used in medical
 treatments requiring bone marrow transplantation. Such procedures are used
 to treat many diseases, e.g., late stage cancers such as ovarian cancer
 and leukemia, as well as diseases that compromise the immune system, such
 as AIDS. Hematopoietic stem cells can be obtained, e.g., by fusing adult
 somatic cells of a cancer or AIDS patient, e.g., epithelial cells or
 lymphocytes with an enucleated oocyte, obtaining CICM cells as described
 above, and culturing such cells under conditions which favor
 differentiation, until hematopoietic stem cells are obtained. Such
 hematopoietic cells may be used in the treatment of diseases including
 cancer and AIDS.
 The present invention can be used to replace defective genes, e.g.,
 defective immune system genes, or to introduce genes which result in the
 expression of therapeutically beneficial proteins such as growth factors,
 lymphokines, cytokines, enzymes, etc.
 DNA sequences which may be introduced into the subject CICM cells include,
 by way of example, those which encode epidermal growth factor, basic
 fibroblast growth factor, glial derived neurotrophic growth factor,
 insulin-like growth factor (I and II), neurotrophin-3, neurotrophin-4/5,
 ciliary neurotrophic factor, AFT-1, cytokines (interleukins, interferons,
 colony stimulating factors, tumor necrosis factors (alpha and beta),
 etc.), therapeutic enzymes, etc.
 The present invention includes the use of cow cells in the treatment of
 human diseases. Thus, cow CICM cells, NT fetuses and NT and chimeric
 offspring (transgenic or nontransgenic) may be used in the treatment of
 human disease conditions where cell, tissue or organ transplantation is
 warranted. In general, CICM cell, fetuses and offspring according to the
 present invention can be used within the same species (autologous,
 syngenic or allografts) or across species (xenografts). For example, brain
 cells from cow NT fetuses may be used to treat Parkinson's disease.
 Also, the subject CICM cells, may be used as an in vitro model of
 differentiation, in particular for the study of genes which are involved
 in the regulation of early development. Also, differentiated cell tissues
 and organs using the subject CICM cells may be used in drug studies.
 Further, the subject CICM cells may be used as nuclear donors for the
 production of other CICM cells and cell colonies.
 In order to more clearly describe the subject invention, the following
 examples are provided.

EXAMPLES
 Materials and Methods for Cow Cloning
 Modified TL-Hepes-PVA Medium (Hepes-PVA)