MXPA00008602A - Embryonic or stem-like cell lines produced by cross-species nuclear transplantation - Google Patents

Abstract

An improved method of nuclear transfer involving the transplantation of differentiated donor cell nuclei into enucleated oocytes of a species different from the donor cell is provided. The resultant nuclear transfer units are useful for the production of isogenic embryonic stem cells, in particular human isogenic embryonic or stem cells. These embryonic or stem-like cells are useful for producing desired differentiated cells and for introduction, removal or modification, of desired genes, e.g., at specific sites of the genome of such cells by homologous recombination. These cells, which may contain a heterologous gene, are especially useful in cell transplantation therapies and for in vitro study of cell differentiation. Also, methods for improving nuclear transfer efficiency by genetically altering donor cells to inhibit apoptosis, select for a specific cell cycle and/or enhance embryonic growth and development are provided.

Description

LINES OF SIMILAR CELLS BASED ON OR EMBR1ON1CAS PRODUCED BY NUCLEAR TRANSPLANT OF CROSS SPECIES FIELD OF THE INVENTION The present invention relates generally to the production of similar cells based or embryonic by transplantation of nuclei of cells derived from animal or human cells in enucleated animal oocytes from a different species of the donor nuclei. The present invention relates more specifically to the production of primate or human embryonic or base similar cells by transplantation of the nucleus of a human primate cell into an enucleated animal oocyte, poj; example, a primate or ungulate oocyte, and in a preferred embodiment an enucleated bovine oocyte. The present invention further relates to the use of the resulting base or embryonic-like cells, preferably cells similar to base or embryonic primate or human for therapy, for diagnostic applications, for the production of differentiated cells which may also be used for therapy or diagnosis, and for the production of differentiated transgenic or embryonic transgenic cells, cell lines, tissues and organs. In addition, the base or embryonic-like cells according to the present invention can be used by themselves as nuclear donors in nuclear transplantation or nuclear transfer methods for the production of chimeras or clones, preferably, transgenic chimeric or cloned animals.

BACKGROUND OF THE INVENTION Methods for deriving embryonic (ES) cell-based cell lines in vitro from early preimplantation mouse embryos are well known. (See, for example, Evans et al., Nature, 29: 154-156 (1981); Martin, Proc. Nati, Acad. Sci., USA, 78: 7634-7638 (1981)). ES cells can be passaged in an undifferentiated state, provided that a fibroblast cell feeder layer is present (Evans et al., Id.) Or a source of inhibition of differentiation (Smith et al., Dev. Biol., 1 21: 1-9 (1987)). ES cells have been previously reported to possess numerous aplicacioryes. 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 cause germline chimeras when introduced into preimplantation mouse embryos, thus demonstrating their pluripotency (Bradley et al., Nature, 309: 2155-256 (1984)). In view of their ability to transfer their genome to the next generation, ES cells have potential utility for germline manipulation of cattle animals by using ES cells with or without a desired genetic modification. Moreover, in the case of livestock animals, for example, ungulates, the nuclei of similar preimplantation cattle embryos support the development of enucleated oocytes at term (Smith et al., Biol. Reprod., 40: 1027-1035 ( 1989) and Keefer et al., Biol. Reprod. , 50: 939-939 (1994)). This is in contrast to nuclei of mouse embryos that beyond the eight cell stage after the transfer, reportedly do not support the development of enucleated oocytes (Cheong et al., Biol. Reprod., 48: 958 (1993 )). Therefore, ES cells from livestock animals are highly desirable because they can provide a potential source of totipotent, genetically engineered or different donor nuclei for nuclear transfer procedures. Some research groups have reported the isolation of putatively pluripotent embryonic cell lines. For example, Notarianni et al. , J. Reprod. Fert. Suppl. , 43: 255-260 (1 991), report the establishment of putatively stable pluripotent cell lines of pig and bighorn blastocysts, which exhibit some morphological and growth characteristics similar to those of cells in primary cultures of cell masses isolated immunosurgically from blastocysts of sheep. (Id.) In addition, Notarianni et al. , J. Reprod. Fert. Suppl. , 41: 51-56 (1990) describe the maintenance and differentiation in culture of putative pluripotent embryonic cell lines of pig blastocysts. In addition, Gerfen et al. , Anim. Biotech, 6 (1): 1-14 (1995) describe the isolation of embryonic cell lines from porcine blastocysts. These cells are stably maintained in feeder layers of mouse embryonic fibroblasts without the use of conditioned medium. These cells differ, it is said, in several different cell types during culture (Gerfen et al., Id.).

In addition, Saito et al., Toux's Arch. Dev. Biol., 201: 1-34-141 (1992) report cell lines similar to embryonic base cells cultured coils, which survive three steps, but were lost after the fourth step . Additionally, Handyside et al. , Roux 'Arch. Dev. Biol., 196: 185-190 (1987) describe the culture of internal cell masses immunosurgically isolated from sheep embryos under conditions that allow the isolation of mouse ES cell lines derived from mouse ICMs. Handyside et al. (1987) (Id.), Report that under such conditions, sheep ICMs bind, spread and develop areas of both ES cell-like cells and endoderm-like cells, but that after prolonged culture only the cells ^ imilar to endoderm. (Id.) Recently, Cherny et al., Theriogenelogy, 41: 175 (1994) reported cell lines derived from bovine primordial germ cells, presumably pluripotent, maintained in long-term culture. These cells, after approximately seven days of culture, produced ES-like colonies, which stain 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 HES 1, a homeobox gene pattern, which is thought to be expressed exclusively by ES cells. In addition, recently Campbell et al. , Nature, 380: 64-68 (1996) reported the production of live lambs following the transfer Nuclear embryonic disc (ED) cells cultured from nine-day old sheep embryos cultured under conditions that promote the isolation of ES cell lines in the mouse. The authors concluded based on their results that the ED cells of nine-day sheep embryos are totipotent by nuclear transfer and that the totipotency is maintained in the culture. Van Stekelenburg-Hamers et al. , Mol. Reprod. Dev., 40: 444-454 (195), reported the isolation and characterization of putatively permanent cell lines of cells from internal cell masses of bovine blastocysts. The authors isolated and cultured ICMs from bovine blastocysts of 8 or 9 days under different conditions to determine which feeder cells and culture media are more efficient to support the binding and outgrowth of bovine ICM cells. They concluded based on their results that the binding and outgrowth of cultured ICM cells is intensified by the use of STO (mouse fibroblast) feeder cells (instead of bovine uterine epithelial cells) and by the use of carbon stripped serum (in place of normal serum) to supplement the culture medium. However, Van Stekelenburg et al reported that their cell lines resembled epithelial cells rather than pluripotent ICM cells. (Id.) Further, Smith et al. , WO 94/24274, published October 27, 1994. Evans et al, WO 90/03432, published April 5, 1990 and Wheeler et al. , WO 94/26889, published on November 24, 1994, reported the isolation, selection and propagation of animal base cells, which supposedly can be used to obtain transgenic animals.

In addition, Evans et al. , WO 90/03432, published on April 5, 1990, reported the derivation of putatively pluripotent embryonic stem cells derived from porcine and bovine species, which were affirmatively useful for the production of transgenic animals. In addition, Wheeler et al. , WO 94/26884, published on November 24, 1994, described embryonic base cells, which were affirmatively useful for the manufacture of chimeric and transgenic ungulates. In this way, based on the above, it is evident that many groups have tried to produce ES cell lines, for example, due to its potential application in the production of cloned or transgenic embryos and in nuclear transplantation. The use of ungulate ICM cells for nuclear transplantation has also been reported. For example, Collas et al. , Mol. Reprod. Dev., 38: 264-267 (1994) describe the nuclear transplantation of cattle ICMs by microinjection of donor cells Used in enucleated mature oocytes. The reference described in vitro embryo culture for seven days to produce fifteen blastocysts, which upon transfer to bovine recipients, resulted in four pregnancies and two births. In addition, Keefer et al. , Biol. Reprod. , 50: 935-939 (1994), describe the use of bovine ICM cells as donor nuclei in nuclear transfer procedures, to produce blastocysts which, upon transplantation into bovine receptors, resulted in several live progeny. In addition, Sims et al. , Proc. Nati Acad. Sci., USA, 90: 6143-6147 (1993), described the production of calves by transfer of nuclei of bovine ICM cells cultured in vitro short term in enucleated mature oocytes. In addition, the production of live lambs following the nuclear transfer of cultured embryonic disc cells has been reported (Campbell et al., Nature, 380: 64-68 (1 996)). Additionally, the use of bovine pluripotent embryonic cells in nuclear transfer and the production of chimeric fetuses has also been reported (Stice et al., Biol. Reprod., 54: 100-1 10 (1996)); Collas et al. , Mol. Reprod. Dev., 38: 264-267 (1994). There have also been several attempts to produce cross-species NT units (Wolfe et al., Theriogenology, 33: 350 (1990).) Specifically, bovine embryonic cells were fused with bison oocytes to produce some NT units of Although embryonic cells, not adult cells, were used as donor nuclei in the nuclear transfer procedure, the dogma has been that embryonic cells are more easily reprogrammed than adult cells. It goes back to early NT studies in the frog (reviewed by DiBerardino, Differentiation, 17: 17-30 (1 980).) In addition, this study involved phylogenetically very similar animals (cattle and oocyte nuclei of bison). when more diverse species were fused during NT (beef cores in hamster oocytes), no internal cell mass structures were obtained. s, cell mass cells had never been reported Internal NT units could be used to form a colony similar to ES cells that could be propagated. In addition, Collas et al. (Id.), Showed the use of granulosa cells (adult somatic cells) to produce transfer embryos nuclear bovines. However, unlike the present invention, these experiments did not involve cross-species nuclear transfer. Furthermore, unlike the present invention, colonies of ES-like cells were not obtained. Very recently, the US patent no. 5,843,780, 0 issued to James A. Thomson on December 1, 1998, transferred to the Wisconsin Alumni Research Foundation, which is to describe a purified preparation of primate embryonic base cells that are (i) capable of proliferation in an in vitro culture during about a year; (ii) they maintain a karyotype in which all the chromosomes characteristic of the primate species and notably not alternating through prolonged culture; (iii) maintain the potential to differentiate into derivatives of endoderm, mesoderm and ectoderm tissues throughout the culture; and (iv) they will not differentiate when cultured in a fibroblast feeder layer. These cells were reportedly negative for the SSEA-1 marker, positive for the SEA-3 marker, positive for the SSEA-4 marker, express alkaline phosphatase activity, are pluripotent, and have karyotypes including the presence of all chromosomes characteristic of the primate species, and in which none of the chromosomes is altered.

•) Additionally, these cells are respectfully positive for the markers TRA-1 -60 and TRA-1 -81. The cells supposedly differentiate into endoderm, mesoderm and ectoderm cells when injected into a SCID mouse. In addition, the putative embryonic base cell lines derived from human or primate blasts are discussed in Thomson et al. , Science 282: 1 145-1 147 and Proc. Nati Acad. Sci. USA 92: 7844-7848 (1995). In this way, Thomson describes what supposedly are similar human-based or embryonic or non-human primate cells and methods for their production. However, there is still a significant need for methods to produce similar human-based or embryonic cells that are autologous to a intended transplant recipient given their significant therapeutic and diagostic potential. In this regard, numerous human diseases have been identified, which can be treated by cell transplantation. For example, Parkinson's disease is caused by degeneration of dopaminergic neurons in the substantia nigra. The standard treatment for Parkinson's disease involves the administration of L-DOPA, which temporarily improves the loss of dopamine, but causes severe side effects and ultimately does not reverse the progress of the disease. A different approach to treating Parkinson's disease, which promises to have wide applicability to the treatment of many brain diseases and central nervous system lesions, involves the transplantation of cells or tissues from fetal or neonatal animals into the adult brain. Fetal neurons from a variety of brain regions can be incorporated into the adult brain. Such grafts have been shown to alleviate experimentally induced behavioral deficits, including complex cognitive functions, in laboratory animals. The results of initial tests from human clinical trials have also been promising. However, supplies of human fetal cells or tissue obtained from abortions is very limited. Moreover, obtaining cells or tissues from aborted fetuses is highly controversial. Currently there is no procedure available to produce "fetal-like" cells of the patient. In addition, allograft tissue is not readily available and both allograft and xenograft tissue are subject to graft rejection. Moreover, in some cases, it would be beneficial to make genetic modifications in cells or tissues before transplantation. However, many cells or tissues where such a modification would be desirable do not divide well in culture and many types of genetic translocation require cells to divide rapidly. Therefore, there is a clear need in the art for a supply of similar undifferentiated human-based or embryonic cells for use in transplants and cell and gene therapies.

OBJECTIVES OF THE INVENTION An object of the invention is to provide novel and improved methods for producing similar base or embryonic cells. A more specific objective of the invention is to provide a novel method for producing similar base or embryonic cells, the which involves transplantation of the nucleus of a mammalian or human cell into an enucleated oocyte of a different species. Another specific object of the invention is to provide a novel method for producing human-like or non-human primate or embryonic-like cells, which involves transplantation of the nucleus of a human or non-human primate cell into an enucleated human or animal oocyte, by example, an enucleated oocyte of ungulate, human or primate. Another objective of the invention is to provide a novel method for producing human-like or non-human primate-based or embryonic cells, of defective lineage, which involves transplantation of the nucleus of a human or non-human primate cell, eg, a cell adult human in an enucleated oocyte of human or non-human primate, where such a cell has been genetically designed to be incapable of differentiation in a specific cell lineage or has been modified so that the cells are "deadly1," and therefore they do not cause a viable progeny, for example, when designing the expression of ribozyme or antisense telomerase gene Still another objective of the invention is to enhance the efficiency of nuclear transfer and specifically to enhance the development of preimplantation embryos produced by nuclear transfer by genetically designing donor somatic cells used for nuclear transfer, to provide for the expression of genes that enhance embryonic development, for example, genes of the MHC I family, and in particular Ped genes, such as Q7 and / or Q9.

Yet another object of the invention is to intensify the production of nuclear transfer embryos by IVP and more specifically nuclear transfer embryos by genetically altering the donor cell used for nuclear transfer, so that it is resistant to apoptosis, for example, by introducing a a DNA construct that provides for the expression of genes that inhibit apoptosis, for example, Bcl-2 or members of the Bcl-2 family and / or by the expression of antisense ribozymes specific for genes that induce apoptosis during early embryonic development. Still another object of the invention is to improve the nuclear transfer efficiency by improved selection of donor cells of a specific cell cycle stage, for example, G 1 phase, by genetically designing donor cells, so that they express a DNA construct that encodes a particular cyclin linked to a detectable marker, for example, one that encodes a visualizable marker protein (for example, fluorescent label). It is also an object of the invention to intensify the development of embryos produced in vitro, by culturing such embryos in the presence of one or more protease inhibitors, preferably one or more capsase inhibitors, thereby inhibiting apoptosis. Another objective of the invention is to provide similar base or embryonic cells produced by core transplantation of an animal or human cell in an enucleated oocyte of a different species. A more specific objective of the invention is to provide similar human or primate-based embryonic cells produced by transplantation of the nucleus of a primate or human cell into an animal enucleated oocyte, for example, an enucleated oocyte of human, primate or ungulate. Another object of the invention is to use such base or embryonic-like cells for therapy or diagnosis. A specific object of the invention is to use such primate or human embryonic-based or embryonic-like cells for the treatment or diagnosis of any disease, wherein the transplantation of cells, tissue or organ is therapeutically or diagnostically beneficial. Another specific objective of the invention is to use the base or embryonic similar cells produced according to the invention for the production of differentiated organs, tissues or cells. A more specific objective of the invention is to use the similar primate or human-based embryonic or similar cells produced according to the invention for the production of differentiated human organs, tissues or cells. Another specific object of the invention is to use the base or embryonic-like cells according to the invention for the production of genetically-designed base or embryonic-like cells, said cells can be used to produce human differentiated or genetically engineered cells, tissues or organs, for example, that have use in gene therapies. Another specific objective of the invention is to use the similar base or embryonic cells produced according to the invention in vitro, by example, for study of cell differentiation and for trial purposes, for example, for drug studies. Another object of the invention is to provide improved methods of transplantation therapy, comprising the use of isogenic or sinegenic cells, tissues or organs produced from the base or embryonic similar cells produced according to the invention. 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, diseases of the urinary tract, as well as for the treatment of immune defects, bone marrow transplantation, cancer, among other diseases. Another objective of the invention is to use genetically engineered or genetically engineered or embryonic-based similar cells produced according to the invention for gene therapy, in particular for the treatment and / or prevention of the diseases and lesions identified, supra. Another objective of the invention is to use the similar base or embryonic cells produced according to the invention or similar base or embryonic, transgenic or genetically engineered cells, produced according to the invention, as nuclear donors for nuclear transplantation. Still another object of the invention is to use genetically engineered ES cells produced according to the invention, for the production of transgenic animals, for example, non-human primates, roosters, ungulates, etc. Such transgenic animals can be used to produce, for example, animal models for the study of human diseases, or for the production of desired polypeptides, for example, therapeutics or nutripharmaceuticals. With the foregoing and other objects, advantages and features of the invention which will become apparent hereafter, the nature of the invention can be understood more clearly by reference to the following detailed description of the preferred embodiments of the invention and the appended claims. .

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a photograph of a nuclear transfer (NT) unit produced by transfer of an adult human cell into an enucleated bovine oocyte. Figures 2 to 5 are photographs of embryonic-based similar cells derived from an NT unit, as shown in Figure 1.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a novel method for producing similar base or embryonic cells, and more specifically, to similar base or embryonic cells of human or non-human primate, by nuclear transfer or nuclear transplantation. In the present application, nuclear transfer, nuclear transplantation or NT are used interchangeably. As discussed above, the isolation of true base or embryonic cells by nuclear transfer or nuclear transplantation has never been reported. Instead, the reported previous isolation of ES-like cells has been from fertilized embryos. In addition, successful nuclear transfer involving cells or DNA from genetically different species, or more specifically adult cells or DNA from one species (eg, human) and oocytes from other unrelated species, has not been reported. Instead, although embryos produced by fusion of cells from closely related species, for example, bovine-goat and bovine-bison, have been reported, they produced no ES cells. (Wolfe et al., Theriogenology, 33 (1): 350 (1990)). In addition, a method for producing primate or human ES cells derived from a source of non-fetal tissue has not been reported. Instead, the limited human fetal cells and tissues that are currently available must be obtained or derived from spontaneous abortion tissues or aborted fetuses. Furthermore, prior to the present invention, no one obtained similar base or embryonic cells by nuclear cross-species transplantation. Quite unexpectedly, the present inventors discovered that human-like or embryonic-like cells and cellulose colonies can be obtained by transplantation of the nucleus of a human cell, for example, an adult differentiated human cell, into a Enucleated animal oocyte, which is used to produce nuclear transfer units (NT), the cells that on the culture give origin to similar human-based or embryonic cells and cell colonies. This result is highly surprising because it is the first demonstration of effective cross-species nuclear transplantation involving the introduction of a differentiated donor nucleus or cell into an enucleated oocyte of a genetically different species, for example, the transplantation of cell nuclei from a human or animal cell differentiated, for example, adult cell, in the enucleated egg of a different animal species, to produce nuclear transfer units containing cells, which when cultivated under appropriate conditions, give origin to similar cells based or embryonic and cellular colonics. Preferably, NT units used to produce ES-like cells will be grown 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. In the present invention, base or embryonic similar cells refer to cells produced in accordance with the present invention. The present application refers to such cells as base-like cells instead of base cells, due to the manner in which they are produced normally, i.e., by cross-species nuclear transfer. Although these cells are expected to possess similar differentiation capacity as normal base cells, they may possess some insignificant differences due to the way they are produced. For example, these base-like cells may possess the mitochondria of the oocytes used for nuclear transfer, and thus do not behave identically to conventional embryonic base cells. The present discovery was made based on the observation that the nuclear transplantation of the nucleus of an adult human cell, specifically a human epithelial cell obtained from the oral cavity of a human donor, when transferred into an enucleated bovine oocyte, resulted in the formation of nuclear transfer units, whose cells on the culture caused embryonic or similar human-based cells and human-based embryonic or similar cell colonies. This result has recently been reproduced by keratinocyte transplantation of an adult human in a bovine oocyte elucidated with the successful production of a blastocyst and ES cell line. Based on this, the hypothesis is formulated by the present inventors that bovine oocytes and human oocytes, possibly mammals in general, must undergo maturation processes during embryonic development, which are sufficiently similar or conserved in order to allow that the bovine oocyte function as an effective substitute for a human oocyte. Apparently, oocytes generally comprise factors, possibly of a proteinaceous or nucleic acid nature, that induce embryonic development under appropriate conditions, and these functions that are the same or very similar in different species. These factors may comprise RNAs of material and / or telomerase.

Based on the fact that the nuclei of human cells can be effectively transplanted into bovine oocytes, it is reasonable to expect that human cells can be transplanted into oocytes from other unrelated species, e.g., other ungulates as well as other animals. In particular, other ungulate oocytes should be suitable, for example, pigs, sheep, horses, goats, etc. In addition, oocytes from other sources should be suitable, for example, oocytes derived from other primates, amphibians, rodents, rabbits, guinea pigs, etc. Furthermore, using similar methods, it should be possible to transfer cells or nuclei of human cells into human oocytes and use the resulting blasts to produce human ES cells. Accordingly, in its broadest embodiment, the present invention involves the transplantation of an animal or human cell nucleus, or animal or human cell into the enucleated oocyte of an animal species different from the donor nucleus, by injection or fusion, to produce an NT unit containing cells that can be used to obtain similar cells on a base or embryonic basis and / or cell cultures. For example, the invention may involve the transplantation of an ungulate cell nucleus or ungulate cell into an enucleated oocyte of another species, eg, another ungulate or ungulate, by injection or fusion, said cells and / or nuclei being combined to produce NT units and these are grown under suitable conditions to obtain multicellular NT units, preferably comprising at least about 2 to 400 cells, more preferably 4 to 1 28 cells, and very preferably at least about 50 cells. The cells of such NT units can be used to produce similar cells based or embryonic or cell colonies on culture. However, the preferred embodiment of the invention comprises the production of human-like or non-human primate-based embryonic or similar cells, by transplantation of the nucleus of a human donor cell or a human cell into an enucleated oocyte of human, primate or non-human animal. primate, for example, a ungulate oocyte, and in a preferred embodiment a bovine enucleated oocyte. In general, base or embryonic-like cells will be produced by a nuclear transfer process comprising the following steps: (i) obtaining human cells or desired animals to be used as a source of donor nuclei (which can be genetically altered); (ii) obtaining oocytes from a suitable source, e.g., a mammal and most preferably a primate or a source of ungulate, e.g., bovine, (iii) enucleating said oocytes; (iv) transferring the human or animal cell or nucleus into the enucleated oocyte of a different animal species than the donor cell or nucleus, for example, by fusion or injection; (v) culturing the resulting NT product or NT unit to produce multiple cell structures; Y (vi) culturing cells obtained from said embryos to obtain similar base or embryonic cells and similar cell-based colonies. Nuclear transfer techniques or nuclear transplantation techniques are known in the literature and are described in many of the references cited in the background of the invention. See, in particular, Campbell et al. , Theriogenology, 43: 1 81 (1995); Collas et al. , Mol. Report Dev., 38: 264-267 (1994); Keefer et al, Biol. Reprod. , 50: 935-939 (1994); Sims et al. , Proc. Nati Acad. Sci., USA, 90: 6143-6147 (1 993); WO 94/26884; WO 94/242474 and WO 90/03432, which are incorporated in their entirety by reference herein. In addition, US patents us. 4,994,384 and 5,057,420 describe procedures for bovine nuclear transplantation. See, also Cibelli et al. , Science, Vol. 280: 1 256-1 258 (1998). The 'human or animal cells, preferably mammalian cells, can be obtained and cultivated by well-known methods. Human and animal cells useful in the present invention include, by way of example, epithelial, neural, epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), other immune cells, erythrocytes, macrophages, melanocytes, monocytes, mononuclear cells, fibroblasts, cardiac muscle cells, and other muscle cells, etc. Moreover, human cells used for nuclear transfer can be obtained from different organs, for example, 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, ie, cells useful in the present invention, can be obtained from any cell or organ of the body. This includes all somatic or germ cells. Preferably, the donor cells or nuclei would comprise cells that are actively dividing, that is, non-quiescent, since it has been reported that they are enhancing the cloning efficiency. In addition, preferably, such donor cells will be in the G 1 cell cycle. The resulting blasts can be used to obtain embryonic base cell lines according to the culture methods reported by Thomson et al. , Science 282: 1 145-1 147 (1981) and Thomson et al. , Proc. Nati Acad. Sci., USA 92: 7544-7848 (1995), incorporated in their entirety by reference herein. In the example that follows, the cells used as donors for nuclear transfer were epithelial cells derived from the oral cavity of a human donor and adult human keratinocytes. However, as discussed, the method described is applicable to other cells or human nuclei. Furthermore, cell nuclei can be obtained from both somatic and germinal human cells. It is also possible to stop the donor cells at mitosis before nuclear transfer, using a suitable technique known in the art. Methods for stopping the cell cycle in several stages have been thoroughly reviewed in US Patent 5,262,409, which is incorporated herein by reference. In Particularly, although it has been reported that cycloheximide has an inhibitory effect on mitosis (Bowen and Wilson (1 955) J. Heredity 45: 3-9), it can also be used for enhanced activation of mature bovine follicular oocytes when combined with treatment of electric pulses (Yang et al. (192) Biol. Reprod 42 (Suppl 1): 1 17). The activation of zygote genes is associated with hyperacetylation of Histone H4. It has been shown that Trichostatin-A inhibits histone deacetylase in a reversible manner (Adenot et al., Differential H4 acetylation of paternal and maternal chromatin precedes DNA replication and differential transcriptional activity in pronuclei of 1 -cell mouse embryos (differential acetylation of paternal chromatin H4) and maternal precedes DNA replication and differential transcription activity in pronuclei of 1-cell mouse embryos.) Development (Nov. 1997) 1 24 (22): 461 5-4625; Yoshida et al. Trichostatin A and trapoxin: novel probes for the role of histone acetylation in chromatin structure and function. (Trichostatin A and trapoxin: novel chemical probes for the role of histone acetylation in chromatin structure and function.) Bioessays (May, 1995) 1 7 (5): 423-430), since it has other compounds. For example, it is also believed that butyrate causes hyper-acetylations of histones by inhibiting histone deacetylase. In general, it seems that butyrate modifies gene expression and almost in all cases its addition to cells in culture seems to stop cell growth. The use of butyrate in this regard is described in U.S. Pat. 5,681, 71 8, which is incorporated herein by reference. In this manner, donor cells can be exposed to Trichostatin-A or another appropriate deacetylase inhibitor prior to fusion, or such a compound can be added to the culture medium before genome activation. Additionally, it is thought that DNA demethylation is a requirement for appropriate access of transcription factors to DNA regulatory sequences. The global demethylation of DNA from the eight cell stage to the blastocyst stage in preimplantation embryos has been previously described (Stein et al., Mol. Reprod. &Dev. 47 (4): 421-429). In addition, Jaenishc et al. (1 997) have reported that 5-azacytidine can be used to reduce the level of DNA methylation in cells, potentially leading to increased access of transcription factors to DNA regulatory sequences. Acingly, the donor cells can be exposed to 5-azacytidine (5-Aza) prior to fusion, or 5-Aza can be added to the culture medium from the 8 cell to blastocyst stage. Alternatively, other known methods for effecting DNA demethylation can be used. Oocytes used for nuclear transfer can be obtained from animals including mammals and amphibians. The mammalian sources suitable for oocytes include rams, cattle, sheep, pigs, horses, rabbits, goats, guinea pigs, mice, hamsters, rats, primates, humans, etc. In preferred embodiments, the oocytes will be obtained from primates or ungulates, for example, a bovine. Oocyte isolation methods are well known in the art. Essentially, this will include isolating oocytes from the ovaries orlexion. reproductive tract of a mammal or amphibian, for example, a bovine. An easily available source of bovine oocytes are the materials of the slaughterhouse. For the successful use of techniques, such as genetic engineering, nuclear transfer and cloning, oocytes must generally be matured in vitro before these cells can be used as receptor cells for nuclear transfer, and before they can be fertilized by the cell of sperm to develop an embryo. This process usually requires collecting immature oocytes (prophase I) from ovaries of animals, for example, bovine ovaries obtained in a slaughterhouse and maturing the oocytes in a maturing medium before fertilization or enucleation until the oocyte reaches the metaphase stage. II, which in the case of bovine oocytes usually occurs around 18-24 hours post-aspiration. For purposes of the present invention, this period is known as the "ripening period". As used herein for calculation of periods, "aspiration" refers to the aspiration of the immature oocyte of the ovarian follicles. Additionally, the metaphase stage I oocytes, which have been matured in vivo, have been used successfully in nuclear transfer techniques. Essentially, mature metaphase II oocytes are surgically collected from either non-superovulated or superovulated cows or heifers 35 to 48 hours after the onset of estrus or after injection of human chorionic gonadotropin (hCG) or similar hormone.

The stage of oocyte maturation in nuclear enucleation and transfer has been reported as significant for the success of NT methods. (See, for example, Prather et al., Differentiation, 48, 1-8, 1991). In general, previous successful mammalian embryo cloning practices use the stage and metaphase II oocyte as the receptor oocyte, because it is believed that at this stage the oocyte may be or is sufficiently "activated" to treat the nucleus. introduced as it is a fertilizing sperm. In domestic animals, and especially cattle, the period of oocyte activation generally varies from about 16-52 hours, preferably around 28-42 hours post-aspiration. For example, immature oocytes can be washed in hamster embryo culture medium buffered with HEPES (HECM), as described in Seshagine et al. , Biol. Reprod. , 40, 544-606, 1989, and then placed in drops of maturation medium consisting of 50 microliters of tissue culture medium (TCM) 199 containing 1 0% fetal calf serum, which contains appropriate gonadotropins, such as luteinizing hormone (LH) a follicle stimulating hormone (FSH) and estradiol under a layer of lightweight paraffin or silicon at 39 ° C. After a fixed period of maturation, which will normally vary from about 10 to 40 hours, and preferably about 16-1 8 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 the removal of accumulated cells. This can be effected by repeated pipetting through very fine perforating pipettes or by brief vortexing. The extracted oocytes are then classified by polar bodies, and the selected metaphase I oocytes I, as determined by the presence of polar bodies, are then used for nuclear transfer. Then follow the enucleation. Enucleation can be effected by known methods, such as those described in U.S. Pat. 4,994,384, which is incorporated herein by reference. For example, metaphase oocytes I I are placed either in H ECM, optionally containing 7.5 micrograms per milliliter of cytochalasin B, for immediate enucleation, or they can be placed in a suitable medium, for example, CR1 aa, plus 10% of estrus cow serum and then they are subsequently enucleated, preferably, not later than 24 hours later, and more preferably 16-1 8 hours later . Enucleation can be achieved with microsurgery using a micropipette to remove the polar body and the adjacent cytoplasm. The oocytes can then be classified to identify those that have been successfully enucleated. This classification can be done by spotting the oocytes with 1 microgram per milliliter of 3342 Hoechst dye in HECM, and then see 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.

In the present invention, the receptor oocytes will be enucleated, preferably, in 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 maturation in vitro, and most preferably around 16-18 hours after the initiation of in vitro maturation. A single animal or human cell or nucleus derived therefrom, which is normally heterologous to the enucleated oocyte, will then be transferred into the perivitelline space of the enucleated oocyte used to produce the NT unit. The animal or human cell or nucleus and the enucleated oocyte will be used to produce NT units according to methods known in the art. For example, cells can be fused by electrofusion. Electrofusion is achieved by providing a pulse of electricity that is sufficient to cause a transient failure of the plasma membrane. This failure of the plasma membrane is very short because the membrane is rapidly reformed. Essentially, if two adjacent membranes are induced to fail, upon reformation the lipid bilayers will intermix and 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. Patent 4,997,384 by Prather et al. , (incorporated by reference in its entirety herein) for further discussion of this process. A variety of electrofusion means can be used including, for example, sucrose, mannitol, sorbitol and solution buffered with phosphate. Fusion can also be achieved using Sendai virus as a fusogenic agent (Graham, Wister Inot, Symp, Monogr., 9, 19, 1969). In addition, in some cases (for example, with small donor nuclei) it may be preferable to inject the nucleus directly into the oocyte instead of using fusion by electroporation. Such techniques are described in Collas and Barnes, Mol. Reprod. Dev., 38: 264-267 (1994), and are hereby incorporated by reference in their entirety. Preferably, the human or animal cell and the oocyte are electrofused in a 500 μm chamber by the application of an electric pulse of 90-120V for about 1.5 μs, approximately 24 hours after the initiation of oocyte maturation. After fusion, the resulting fused NT units are then placed in a suitable medium until activation, for example, one identified infra. Normally, activation will be effected shortly thereafter, generally less than 24 hours later, and preferably around 4-9 hours later. The NT unit can be activated by known methods. Such methods include, for example, cultivating the NT unit at sub-physiological temperature, in essence by applying a cold or effectively cold temperature shock to the NT unit. This can be done very conveniently by cultivating the NT unit at room temperature, which is cold in relation to the physiological temperature conditions to which the embryos are normally exposed.

Alternatively, activation can be achieved by application of known activation agents. For example, it has been shown that penetration of oocytes by sperm during fertilization activates pre-oocyte oocytes to produce larger numbers of viable pregnancies and multiple genetically identical calves after nuclear transfer. In addition, treatments, such as electrical or chemical shock, or cycloheximide treatment, may also be used to activate NT embryos after fusion. Suitable oocyte activation methods are the subject of US Patent no. 5,496,720 for Susko-Parrish et al. , which is incorporated herein by reference. For example, the activation of oocytes can be carried out simultaneously or sequentially by: (i) increasing the levels of divalent cations in the oocyte, and (ii) reducing the phosphorylation of cellular proteins in the oocyte. This will generally be effected by introducing divalent cations into the oocyte cytoplasm, for example, magnesium, strontium, barium or calcium, for example, in the form of an ionophore. Other methods to increase levels of divalent cations include the use of electric shock, treatment with ethanol and treatment with caged chelators. Phosphorylation can be reduced by known methods, for example, by the addition of kinase inhibitors, for example, serine-threonine kinase inhibitors, such as, 6-dimethyl-amino-purine, staurosporine, 2-aminopurine and sphingosine.

Alternatively, phosphorylation of cellular proteins can be inhibited by introducing a phosphatase into the oocyte, for example, phosphatase 2A and phosphatase 2B. The activated NT units can be cultured in a suitable in vitro culture medium until the generation of similar base or embryonic cells and cell colonies. Culture media suitable for embryo culture and maturation are well known in the art. Examples of known media, which can be used for bovine embryo culture and maintenance, include Ham's F-1 0 + fetal calf serum 10% (FCS), Tissue Culture Medium-199 (TCM-199) + calf serum fetal 10%, Tyrodes-Albumin-Lactate-Pyruvate (TALP), Dulbecco's buffered saline solution (PBS), Eagle's medium and Whitten's medium. One of the most common means used for the collection and maturation of oocytes is TCM-1 99 and 1 to 20% of serum complement including fetal calf serum, newborn serum, estrual cow serum, lamb serum or serum of beef. A preferred maintenance medium includes TCM-199 with Earl's salts, 10% fetal calf serum, 0.2 MM Ma pyruvate and 50 μg / ml gentamicin sulfate. 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. In particular, human endometrial epithelial cells secrete leukemia inhibitory factor (LI F) during preimplantation and implantation period. Consequently, the addition of LI F to the culture medium could be important to intensify the in vitro development of the reconstructed embryos. The use of LI F for cultures of similar base or embryonic cells has been described in US Patent 5,71,256, which is incorporated herein by reference. Another means of maintenance is described in U.S. Patent 5,096,822 to Rosenkrans, Jr. et al. , which is incorporated herein by reference. This embryo medium, called CR1, contains the nutritional substances necessary to support an embryo. CR1 contains hemicalcium L-lactate in amounts ranging from 1.0mM to 10mM, preferably 1.0mM to 5.0mM. The Hemicalcium L-Iactate is an L-lactate with a hemicalcium salt incorporated therein. In addition, the culture medium suitable for maintaining human embryonic cells in culture is discussed in Thomson et al. , Science 282: 1 145-1 147 (1998) and Proc. Nati Acad. Sci., USA 92: 7844-7848 (1,995).

Subsequently, the cultivated NT unit or units are preferably washed, and then placed in a suitable medium, for example, medium CRIaa, Ham's F-10, Tissue Culture Media-199 (TCM-199), Tyrodes-Albumin-Lactate -Pyrovate (TALP), Dulbecco's phosphate buffered saline (PBS), Eagle's or Whitten's medium, preferably containing about 10% FCS. Preferably, such cultivation will be effected in cavity plates, which 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 fibroblasts (e.g., mouse or rat), SI-m220 and STO feeder cell lines and BRL cells. In the preferred embodiment, the feeder cells will comprise mouse embryonic fibroblasts. Means for the preparation of a suitable fibroblast feeder layer are described in the following example and are within the skill of the ordinary technician. The NT units are grown in the feeder layer until the NT units reach a suitable size to obtain cells that can be used to produce similar embryonic-based cells or cell colonies. Preferably, these NT units will be cultured until they reach a size of at least about 2 to 400 cells, more preferably about 4 to 1 28 cells, and most preferably at least about 50 cells. The cultivation will be carried out under suitable conditions, that is, approximately 38.5 ° C and 5% CO2, changing the culture medium in order to optimize the growth normally close to every 2-5 days, preferably, around every 3 days . In the case of NT units derived from enucleated bovine oocyte / human cell, enough cells to produce a colony of ES cells, normally in the order of approximately 50 cells, will be obtained about 1 2 days after the initiation of the activation of oocytes However, this may vary depending on the particular cell used as the nuclear donor, the particular oocyte species and culture conditions. A person skilled in the art can easily investigate visually when a sufficient number has been obtained of cells based on the morphology of the NT units grown. In the case of human / human nuclear transfer embryos, it may be advantageous to use culture medium known to be useful for maintaining human cells in tissue culture. Examples of a culture medium suitable for human embryo culture include the medium reported in Jones et al. , Human Reprod. , 1 3 (1): 169-177 (1,998), medium P1-catalog # 99242, and medium P-1 catalog # 99292, both available from Irvine Scientific, Santa Ana, California, and those used by Thomson et al. to the. (1,998) and (1,995). (Id.). As discussed above, the cells used in the present invention will preferably comprise mammalian somatic cells, most preferably cells derived from a culture of actively proliferating (non-quiescent) mammalian cells. In an especially preferred embodiment, the donor cell will be genetically modified by the addition, deletion or substitution of a desired DNA sequence. For example, the donor cell, e.g., a keratinocyte or fibroblast, e.g., of human, primate or bovine origin, can be transfected or transformed with a DNA construct that provides expression of a desired gene product, e.g. , therapeutic polypeptide. Examples thereof include limpocins, for example, IGF-I, IGF-II, interferons, colony stimulating factors, connective tissue polypeptides, such as, collagens, genetic factors, coagulation factors, enzymes, enzyme inhibitors, etc.

In addition, as discussed above, donor cells can be modified prior to nuclear transfer to achieve other desired effects, eg, development of impaired cell lineage, enhanced embryonic development and / or inhibition of apoptosis. Examples of desirable modifications are discussed below. One aspect of the invention will involve genetic modification of the donor cell, eg, a human cell, so that it is of deficient lineage, and therefore, when used for nuclear transfer will be unable to give rise to viable progeny. This is especially desirable in the context of human nuclear transfer embryos, where for ethical reasons, the production of a viable embryo can be an undesired effect. This can be done by genetically designing a human cell, so that it is incapable of differentiation in lineages of specific cells when used for nuclear transfer. In particular, the cells can be genetically modified, so that when they are used as nuclear transfer donors, the resulting "embryos" do not contain or substantially lack at least one tissue of mesoderm, endoderm or ectoderm. It is anticipated that this can be achieved by leaving out or impairing the expression of one or more specific genes of mesoderm, endoderm or ectoderm. Examples thereof include: Mesoderm: SRF, MESP-1, HNF-4, beta-I integrin, MSD; Endoderm: GATA-6, GATA-4; Ectoderm: RNA helicase A, H beta 58.

The above list is intended to be exemplary and not exhaustive of known genes that are involved in the development of mesoderm, endoderm and ectoderm. The generation of embryos and deficient mesoderm cells, deficient endoderm and deficient ectoderm has been previously reported in the literature. See, for example, Arsenian et al., EMBO J., Vol. 17 (2): 6289-6299 (1998); Saga Y, Mech. Dev., Vol.75 (1-2): 53-66 (1998); Holdener et al., Development, Voll 120 (5): 1355-1346 (1994); Chen et al., Genes Dev. Vol. 8 (20): 2466-2477 (1994); Rohwedel et al., Dev. Biol., 201 (2): 167-189 (1998) (mesoderm); Morrisey et al., Genes, Dev., Vol. 12 (22): 3579-3590 (1998); Soudais et al., Development, Vol. 121 (11): 3877-3888 (1995) (endoderm); and Lee et al., Proc. Nati Acad. Sci. USA, Vol. 95: (23): 13709-13713 (1998); and Radice et al, Development, Vol. 111 (3): 801-811 (1991) (ectoderm). In general, a desired somatic cell, for example, a human keratinocyte, epithelial cell or fibroblast, will be genetically engineered so that one or more specific genes for particular cell lineages are "flushed" and / or the expression of such genes is significantly impaired. This can be done by known methods, for example, homologous recombination. A preferred genetic system for effecting "emptying" of desired genes is described by Capecchi et al., U.S. Patent Nos. 5,631,153 and 5,464,764, which report positive-negative selection vectors (PNS) that allow focused modification of DNA sequences in a genome of desired mammal. Such genetic modification will result in a cell that is unable to differentiate into a particular cell line when used as a nuclear transfer donor. This genetically modified cell will be used to produce a defective lineage nuclear transfer embryo, that is, not develops at least one of a mesoderm, endoderm or functional ectoderm. Therefore, the resulting embryos, even if implanted, for example, in a human uterus, would not give rise to a viable progeny. However, the ES cells resulting from such nuclear transfer will still be useful as it will produce cells from one or two undamaged lineages remaining. For example, a human nuclear transfer embryo of deficient ectoderm will still give rise to differentiated cells derived from mesoderm and endoderm. A deficient ectoderm cell can be produced by suppression and / or damage of one or both of the RNA genes helicase A or H beta 58. 15 These deficient lineage donor cells can also be genetically modified to express another desired DNA sequence. In this way, the genetically modified donor cell will give rise to a deficient lineage blastocyst which, when plated, will at most differentiate two of the embryonic germ layers. Alternatively, the donor cell can be modified from mantea that is "deadly". This can be achieved by expressing ribozyme or anti-sense telomerase genes. This can be done by known genetic methods that will provide expression of ribozymes or anti-sense DNA, or by gene emptying. These "deadly" cells, when used for nuclear transfer, they will not be able to differentiate into viable progeny. Another preferred embodiment of the present invention is the production of nuclear transfer embryos that grow more efficiently in tissue culture. This is advantageous since it should reduce the time required and mergers necessary to produce ES and / or progeny cells (if the blastocysts are to be implanted in a substitute). This is also desirable because it has been observed that the blastocysts and ES cells resulting from nuclear transfer may have impaired development potential. Although these problems can often be improved by altering tissue culture conditions, an alternative solution is to intensify embryonic development by enhancing the expression of genes involved in embryonic development. For example, it has been reported that gene products of the type Ped, which are members of the MHC I family, are of significant importance for embryonic development. More specifically, it has been reported in the case of mouse preimplantation embryos that the Q7 and Q9 genes are responsible for the "rapid growth" phenotype. Accordingly, it is anticipated that the introduction of DNAs that provide for the expression of these and other related genes, or their human or other mammalian counterparts in the donor cells, will give rise to nuclear transfer embryos that grow more rapidly. This is particularly desirable in the context of cross-species nuclear transfer embryos, which may develop less 3 efficiently in tissue culture than nuclear transfer embryos produced by fusion of cells or nuclei of the same species. ft? In particular, a DNA construct containing the Q7 and / or Q9 gene will be introduced into donor somatic cells prior to transfer nuclear. For example, an expression construct can be constructed containing a strong constitutive mammalian promoter operably linked to the Q7 and / or Q9 genes, an IRES, one or more suitable selectable markers, eg, neomycin, ADA, DHFR, and a sequence poly-A, for example, bGH poly A sequence. In addition, it can be It is advantageous to further intensify the expression of Q7 and Q9 genes by including isolates. It is anticipated that these genes will soon be expressed in a blastocyst development, since these genes are highly conserved in different species, for example, cattle, goats, pigs, dogs, cats and humans. In addition, it is anticipated that donor cells can be designed to affect other genes that enhance embryonic development. Thus, these genetically modified donor cells should produce blastocysts and embryos in the preimplantation stage more efficiently. Still another aspect of the invention involves the reconstruction of donor cells that are resistant to apoptosis, that is, programmed cell death. It has been reported in the literature that genes related to cell death occur in embryos in the preimplantation stage. (Adams et al., Science, 281 (5381): 1 322-1 326 (1998)). Genes reported to induce apoptosis include, for example, Bad, Bok, BH3, Bik, Hrk, BNI P3, BimL, Bad, Bid and EGL-1. In contrast, genes that reportedly protect cells from programmed cell death include, by way of example, BcL-XL, Bcl-w, Mcl-1, A1, Nr-13, BH RF-1, LMW5-HL, ORF16, Ks- Bel-2, E1 B-19K and CED-9. In this way, donor cells can be constructed where the genes that induce apoptosis are "emptied" or where the expression of genes that protect the cells from apoptosis is intensified or ignited during embryonic development. For example, this can be done by introducing a DNA construct that provides regulated expression of such protective genes, for example, Bcl-2 or related genes during embryonic development. Therefore, the gene can be "turned on" when cultivating the embryo under specific growth conditions. Alternatively, it can be linked to a constitutive promoter. More specifically, a DNA construct containing a Bcl-2 gene operably linked to a regulatable or constitutive promoter, eg, PGK, SV40, CMV, ubiquitin, or beta-actin, an IRES, a suitable selectable marker, and a sequence of poly-A, can be constructed and introduced into a desired mammalian donor cell, for example, human keratinocyte or fibroblast. These donor cells, when used to produce nuclear transfer embryos, should be resistant to apoptosis, and therefore differentiate more efficiently in tissue culture. Therefore, the speed and / or number of suitable preimplantation embryos produced by nuclear transfer can be increased.

Another means to achieve the same result is to impair the expression of one or more genes that induce apoptosis. This will be effected by emptying or by using antisense or ribozymes against genes that are expressed in, and which induce early apoptosis in embryonic development. Examples of them are identified before. Still alternatively, donor cells can be constructed containing both modifications, i.e., deterioration of genes that induce apoptosis and enhanced expression of genes that prevent or prevent apoptosis. The construction and selection of genes that affect apoptosis and cell lines expressing such genes is described in U.S. Pat. 5,646,008, Craig B. Thompson et al., And assigned to the University of Michigan. This patent is incorporated herein by reference. One means for enhancing efficiency is to select cells from a particular cell cycle stage, such as the donor cell. It has been reported that this can have significant effects on nuclear transfer efficiency. (Barnes et al, Mol. Reprod. Devel., 36 (1): 33-41 (1993). Different methods have been reported for selecting cells from a particular cell cycle stage and include starvation of serum (Campbell et al., Nature, 380: 64-66 (1996), Wilmut et al, Nature, 385: 810-81 3 (1997), and chemical synchronization (Urbani et al, Exp. Cell Res., 219 (1): 1 59-168 (1995) For example, a particular cyclin DNA can be operably linked to a regulatory sequence, together with a detectable marker, for example, green fluorescent protein (GFP), segregated by the cyclin destruction box, and optionally sequences of isolation to intensify the expression of marker protein and cyclin. Therefore, cells of a desired cell cycle can be detected in visual form easily and selected for use as a nuclear transfer donor. An example of this is the cyclin D 1 gene in order to select cells that are in G 1. However, any cyclin gene should be suitable for use in the claimed invention. (See, for example, King et al., Mol. Biol. Cell, Vol. 7 (9): 1 343-1 357 (1996)). However, less invasive or more efficient methods are needed to produce cells of a desired cell cycle stage. It is anticipated that this can be effected by genetically modifying donor cells, so that they express specific cyclins under detectable conditions. Therefore, the cells of a specific cell cycle can be easily discerned from other cell cycles. 'Cyclins are proteins that are expressed only during specific stages of the cell cycle. They include cyclin D1, D2 and D3 in the G1 phase, cyclin B1 and B2 in the G2 / M phase and cyclin E, A and H in the S phase. These proteins are easily transferred and destroyed in cytolyglolol. This "transient" expression of such proteins is attributable, in part, to the presence of a "destruction box," which is a short amino acid sequence, which is part of the protein that functions as a tag to direct early destruction. of these proteins via the ubiquitin route. (Adams et al., Science, 281 (5321): 1322-1 326 (1998)). In this invention, donor cells will be constructed that express one or more such cyclin genes under conditions easily detectable, preferably viewable, for example, by the use of a fluorescent label. For example, a particular cyclin DNA can be operably linked to a regulatory sequence, together with a detectable marker, for example, green fluorescent protein (GFP), followed by the cyclin destruction box, and optionally isolation sequences to enhance the marker protein and / or cyclin expression. Therefore, the cells of a desired cell cycle can be detected visually easily and selected for use as a nuclear transfer donor. An example thereof is the cyclin D gene that can be used to select cells that are in G 1. However, a cyclin gene should be suitable for use in the claimed invention. (See, for example, King et al., Mol. Biol. Cell, Vol. 7 (9): 1 343-1 357 (1996)). Yet another aspect of the invention is a method for enhancing nuclear transfer efficiency, preferably in a cross-species nuclear transfer process. Although the present inventors have shown that the nuclei or cells of a species, when they are inserted or fused with an enucleated oocyte of a different species, can give rise to nuclear transfer embryos that produce blastocysts, said embryos can give rise to ES cell lines. , the efficiency of such process being quite low. Therefore, many fusions are usually required to produce a blastocyst, whose cells can be grown to produce ES cells and ES cell lines.

Yet another means to intensify the development of in vitro nuclear transfer embryos is by optimizing culture conditions. One way to achieve this result will be to cultivate NT embryos under conditions that prevent apoptosis. With respect to this embodiment of the invention, it has been found that proteases, such as capsases, can cause the death of oocytes by apoptosis similar to other cell types. (See, Jurisicosva et al., Mol. Reprod. Devel., 51 (3): 243-253 (1998).) It is anticipated that the development of blastocyst will be intensified by including in the culture means used for nuclear transfer and to maintain blastocysts or preimplantation culture stage embryos one or more capsase inhibitors. Such inhibitors include, by way of example, capsase-4 inhibitor I, capsase-3 inhibitor I, capsase-6 inhibitor I, capsase-9 inhibitor I and capsase-1 inhibitor I. The amount >of them will be an effective amount to inhibit apoptosis, for example, 0.00001 to 5.0% by weight of medium; more preferably 0.01% to 1.0% by weight of medium. In this way, the above methods can be used to increase the efficiency of nuclear transfer by enhancing the development of subsequent embryos or bastocysts in tissue culture. After NT units of the desired size are obtained, the cells are mechanically removed from the area and then used to produce similar base or embryonic cells and cell lines. This is effected, preferably, by taking the grouping of cells comprising the NT unit, which normally will contain at least about 50 cells, washing such cells and plating the cells on a feeder layer, for example, irradiated fibroblast cells. Normally, the cells used to obtain the base-like cells or cell colonies will be obtained from the innermost portion of the NT unit grown, which is preferably at least 50 cells in size. However, NT units of lower or higher number of cells, as well as cells of other portions of the NT unit can also be used to obtain ES-like cells and cell colonies. It may be that increased exposure of donor cell DNA to the cytosol of the oocyte will facilitate the process of dedifferentiation. In this regard, recloning could be achieved by taking blastomeres from a reconstructed embryo and fusing them with a new enucleated oocyte. Alternatively, the donor cell can be fused with an enucleated oocyte and 4 to 6 hours later, without activation, the chromosomes can be removed and fused with a younger oocyte. The activation would happen later. The cells are maintained in the feeder layer in a suitable growth medium, for example, alpha MEM supplemented with 10% FCS and 0.1 mM beta-mercaptoethanol (Sigma) and L-glutamine. The growth medium is changed as frequently as necessary to optimize growth, for example, approximately every 2-3 days. This culture process results in the formation of similar cells based on embryonic or cell lines. In the case of NT embryos derived from bovine oocyte / human cell, colonies are observed approximately on the second day of culture in the measured alpha MEM. However, this time may vary depending on the particular nuclear donor cell, specific oocyte and culture conditions. One skilled in the art can vary the culture conditions as desired to optimize the growth of the particular base or embryonic-like cells. Embryonic or base-like cells and cell colonies obtained will normally exhibit an appearance similar to base or embryonic similar cells of the species used as the nuclear cell donor in place of the donor oocyte species. For example, in the case of base-like or embryonic cells obtained by the transfer of a human nuclear donor cell in an enucleated bovine oocyte, the cells exhibit a morphology more similar to mouse embryonic base cells than bovine ES-like cells. More specifically, the individual cells of the ES cell line colony are not well defined, and the colony perimeter is refractive and smooth in appearance. In addition, the cell colony has a longer cell folding time, approximately twice that of mouse ES cells. In addition, unlike the ES cells derived from bovine and porcine, the colony does not have an appearance similar to epithelial. As discussed above, it has been reported by Thomson, in U.S. Patent 5,843,780, that the primate base cells are SSEA-1 (-), SSEA-4 (+). TRA-1 -60 (+); TRA-1 -81 (+) and alkaline phosphatase (+). It is anticipated that primate and human ES cells produced in accordance with the present methods they will exhibit identical or similar marker expression. Alternative way, that such cells are effectively embryonic human or primate base cells, will be confirmed based on their capacity to give origin to all mesoderm, ectoderm and endoderm tissues. This will be demonstrated by cultivating ES cells produced according to the invention under appropriate conditions, for example, as described by Thomsen, U.S. Patent 5,843,780, incorporated herein by reference in its entirety herein. Embryonic or base-like cells and resulting cell lines, preferably similar or embryonic-like cells and human cell lines, have numerous therapeutic and diagnostic applications. Very especially, such base or embryonic-like cells can be used for cell transplantation therapies. Human-like or embryonic-like cells have application in the treatment of numerous disease conditions. In this regard, it is known that mouse embryonic (ES) base cells are capable of differentiating into almost any cell type, e.g., hematopoietic base cells. Therefore, human-based or embryonic-like cells produced according to the invention should possess similar differentiation capacity. Embryonic-based or similar cells according to the invention will be induced to differentiate to obtain the desired cell types according to known methods. For example, the present human-like or embryonic-like cells can be induced to differentiate into hematopoietic base cells, muscle cells, cardiac muscle cells, liver cells, cartilage cells, f "epithelial cells, urinary tract cells, etc. , by cultivating such cells in differentiation medium and under conditions that provide differentiation. The means and methods that result in the differentiation of embryonic base cells are known in the art, since they are suitable culture conditions. For example, Palacios et al. , Proc. Nati Acad. Sci., USA, 92: 7530-7537 (1,995) show the production of hematopoietic base cells at Starting from an embryonic cell line by subjecting the base cells to an induction process comprising initially culturing aggregates of such cells in a suspension culture medium lacking retinoic acid, followed by cultivating in the same medium containing retinoic acid, followed by transfer of aggregates cells to a substrate that provides cell binding. Furthermore, Pedersen, J. Reprod. Fertile. Dev., 6: 543-552 (1 994) is a review article that references numerous articles describing methods for in vitro differentiation of embryonic base cells to produce various types of differentiated cells including hematopoietic cells, muscle cells, heart muscle, nerve cells, among others. In addition, Bain et al. , Dev. Biol., 168: 342-357 (1995) shows in vitro differentiation of embryonic base cells to produce neural cells, which possess neuronal properties. These references are examples of reported methods to obtain differentiated cells from cells similar to base or embryonic. These references, and in particular the descriptions therein that relate to methods for differentiating embryonic base cells, are incorporated by reference herein in their entirety. 1 In this way, using known methods and culture media, one skilled in the art can cultivate the present base or embryonic-like cells to obtain desired differentiated cell types, eg, neural cells, muscle cells, hematopoietic cells, etc. In addition, the use of Bcl-2 or inducible Bcl-xl could be useful for Intensify the in vitro development of lineages of specific cells. In vivo, Bcl-2 prevents many, but not all, forms of apoptotic cell death that occur during lymphoid and neural development. A . A thorough discussion of how Bcl-2 expression could be used to inhibit the »apoptosis of lineages of relevant cells following the transfection of donor cells, is described in US Patent no. 5,646,008, which is incorporated herein by reference. The present base-like or embryonic cells can be used to obtain any type of desired differentiated cell. The therapeutic uses of such differentiated human cells are unequaled. For example, human hematopoietic base cells can be used in medical treatments that require bone marrow transplantation. Such procedures are used to treat many diseases, for example, late stage cancer, such as ovarian cancer and leukemia, as well as diseases that compromise the immune system, such as, AI DS. Hematopoietic-based cells can be obtained, for example, by fusing adult somatic cells from a cancer patient or AIDS, for example, epithelial cells or lymphocytes with a gummed oocyte, for example, bovine oocyte, by obtaining similar base or embryonic cells as described above, and culturing such cells under conditions that favor differentiation, until the hematopoietic base cells are obtained. Such hematopoietic cells can be used in the treatment of diseases including cancer and AI DS. Alternatively, adult somatic cells from a patient with a neurological disorder can be fused with an enucleated animal oocyte, for example, a primate or bovine oocyte, human based or human embryonic stem cells obtained therefrom, and such cells cultured under differentiation conditions to produce neural cell lines. Specific diseases treatable by transplantation of such human neural cells include, by way of example, Parkinson's disease, Alzheimer's disease, ALS and cerebral palsy, among others. In the specific case of Parkinson's disease, it has been demonstrated that transplanted fetal brain neural cells make appropriate connections with the surrounding cells and produce dopamine. This can result in a long-term reversal of the symptoms of Parkinson's disease. To allow for the specific selection of differentiated cells, donor cells can be transfected with selectable markers expressed via inducible promoters, thereby allowing the selection or enrichment of lineages of particular cells when differentiation is induced. For example, CD34-neo can be used for the selection of hematopoietic cells, Pwl-neo for muscle cells, Mash-1 -neo for sympathetic neurons, Mal-neo for human CNS neurons of the gray matter of the cerebral cortex, etc. The great advantage of the present invention is that it provides an essentially unlimited supply of isogenic or sinegenic human cells suitable for transplantation. Consequently, the significant problem associated with current transplantation methods, ie, rejection of transplanted tissue, which may occur due to host-versus-graft or graft-vs-host rejection, will be obvious. Conventionally, rejection is prevented or reduced by the administration of anti-rejection drugs, such as cyclosporin, however, such drugs have significant adverse side effects, for example, immunosuppression, carcinogenic properties, as well as being very expensive. The present invention should eliminate, or at least greatly reduce, the need for anti-rejection drugs. Other diseases and conditions treatable by isogenic cell therapy include, by way of example, spinal cord injuries, multiple sclerosis, muscular dystrophy, diabetes, liver diseases, i.e., hypercholesterolemia, heart disease, cartilage replacement, burns, ulcers standing, gastrointestinal diseases, vascular diseases, kidney diseases, diseases of the urinary tract and diseases and conditions related to aging.

In addition, human-based or embryonic-like cells produced in accordance with the invention can be used to produce genetically engineered or genetically engineered human differentiated cells. In essence, this will be effected by introducing a desired gene or genes, which may be heterologous, or removing all or part of an endogenous gene or genes from human-based or embryonic-like cells produced according to the invention, and allowing such cells differ in the type of cell desired. A preferred method to achieve such modification is by homologous recombination because such a technique can be used to insert, delete or modify a gene or genes at a specific site or sites in the genome of similar cells at the base. This methodology can be used to replace defective genes, for example defective immune em genes, cystic fibrosis genes or to introduce genes that result in the expression of therapeutically beneficial proteins, such as, growth factors, lymphokines, cytokines, enzymes, etc. . For example, the gene encoding brain derived from the growth factor can be introduced into human-based or embryonic-like cells, the cells differentiated into neural cells and the cells transplanted into a Parkinson's patient to retard the loss of neural cells during such disease. . Previously, cell types transfected with BDNF varied from primary cells to immortalized cell lines, either neural or non-neural derived cells (myoblasts and fibroblasts). For example, astrocytes have been transfected with BDNF gene using vectors retrovirals, and cells grafted in a rat model of Parkinson's disease (Yoshimoto et al., Brain Research, 691: 25-36 (1995)). This ex vivo therapy reduced Parkinson's-like symptoms in rats up to 45% 32 days after the transfer. In addition, the tyrosine hydroxylase gene has been placed on astrocytes with similar results (Lundberg et al., Develop Neurol., 1 39: 39-53 (1996) and references cited therein). However, such ex vivo ems have problems. In particular, the retroviral vectors currently used are sub-regulated in vivo and the transgene is expressed only transiently (review by Mulligan, Science, 260: 926-932 (1993)). In addition, such studies used primary cells, astrocytes, which have a finite life span and replicate slowly. Such properties adversely affect the rate of transfection and prevent the selection of stably transfected cells. Moreover, it is almost impossible to propagate a large population of primary gene cells focused on being used in homologous recombination techniques. In contrast, the difficulties associated with retroviral systems should be eliminated by the use of human-based or embryonic-like cells. It has been previously demonstrated by the current transferee that the embryonic cell lines of cattle and pigs can be transfected and selected for stable integration of heterologous DNA. Such methods are described in the commonly assigned US patent serial no. 08 / 626,054, filed on April 1, 1996, incorporated herein by reference in its entirety. Accordingly, using such methods or other known methods, the desired genes can be introduced into the present human-based or embryonic-like cells, and the cells differentiated into desired cell types, for example, hematopoietic cells, neural cells, pancreatic cells, cartilage cells, etc. Genes that can be introduced into the present base-like or embryonic-like cells include, by way of example, 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, cytokine genes (interleukins, interferons, colony-stimulating factors, tumor necrosis factors (alpha and beta), etc.), genes that encode therapeutic enzymes, collagen, human serum albumin, etc.

In addition, it is also possible to use one of the negative selection systems now known in the art for removing therapeutic cells from a patient if necessary. For example, donor cells transfected with the thymidine kinase (TK) gene will lead to the production of embryonic cells containing the TK gene. The differentiation of these cells will lead to the isolation of therapeutic cells of interest, which also express the TK gene. Such cells can be selectively removed at any time from a patient on the administration of ganciclovir. Such a selection system Negative is described in U.S. Patent No. 5,698,446, and is incorporated herein by reference. The present base or embryonic similar cells, preferably human cells, can also 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. In addition, differentiated cell tissues and organs using the present base-like or embryonic cells can be used in drug studies. In addition, the present base or embryonic-like cells can be used as nuclear donors for the production of other similar base or embryonic cells and cell colonies. In order to more clearly describe the present invention, the following examples are provided.

EXAMPLE 1 MATERIALS AND METHODS Donor cells for nuclear transfer Epithelial cells were scraped lightly from the interior of an adult's mouth with consent with a standard glass slide. The cells were washed from the slide in a petri dish containing phosphate buffered saline without Ca or Mg. The cells were pipetted through a small orifice pipette to break up cell clusters into a single cell suspension. The cells were then transferred into a microdrop of TL-H EPES medium containing serum from fetal calf 10% (FCS) under oil for nuclear transfer in enucleated oocytes of cattle.

Nuclear transfer procedures Basic nuclear transfer procedures have been previously described. Briefly, after slaughterhouse oocytes matured in vitro, the oocytes were extracted from accumulated cells and enucleated with a bevelled micropipette at approximately 18 hours post maturation (hpm). Enucleation was confirmed in TL-HEPES medium plus bisbenzimide (Hoechst 33342, 3 μg / ml, Sigma). The individual donor cells were then placed in the perivitelline space of the recipient oocyte. The cytoplasm of bovine oocytes and the donor nucleus (NT unit) are fused using electrofusion techniques. A fusion pulse consisting of 90 V for 1 5 μs was applied to the NT unit. This occurred 24 hours post-initiation of the maturation (hpm) of the oocytes. NT units were placed in CR1 aa medium up to 28 hpm. The procedure used to artificially activate oocytes has been described elsewhere. NT unit activation was at 28 hpm. A brief description of! Activation procedure is as follows: the NT units were exposed for four min to ionomycin (5 μM; CalBiochem, La Jolla, CA) in TL-HEPES supplemented with 1 mg / ml of BSA and then washed for five min in TL-HEPES supplemented with 30 mg / ml of BSA. The NT units were then transferred to a microdrop of culture medium CR 1 aa containing 0.2 mM DMAP (Sigma) and were cultured at 38.5 ° C and 5% CO2 for four to five hours.

The NT units were washed and then placed in a medium CR1 aa plus 10% FCS and 6 mg / ml BSA in four cavity plates containing a confluent feeder layer of mouse embryonic fibroblasts (described below). The NT units were cultivated for three more days at 38.5 ° C and 5% CO2. The culture medium was changed every three days until day 12 after the activation time. At this time, NT units that reached the desired cell number, i.e., approximately 50 cells, were mechanically removed from the zone and used to produce embryonic cell lines. A photograph of an NT unit obtained as described above is contained in Figure 1. / Fibroblast feeder layer The primary cultures of embryonic fibroblasts were obtained from murine fetuses of 14-16 days of age. After the head, liver, heart and alimentary tract were aseptically removed, the embryos were minced and incubated for 30 minutes at 37 ° C in preheated trypsin EDTA solution (0.05% trpsin / 0.02% EDTA, GIBCO, Grand Island, NY). Fibroblast cells were platinized in tissue culture flasks and cultured in alpha-MEM medium (BioWhittaker, Walkersville, MD) supplemented with 10% fetal calf serum (FCS) (Hyclone, Logen, UT), penicillin (100 lU / ml) and streptomycin (50 ul / ml). Three to four days after the passage, embryonic fibroblasts were irradiated in Nunc 35 x 10 culture plates (Baxter Scientific, McGaw Park, IL). The irradiated fibroblasts were grown and kept in a humidified atmosphere with 5% CO2 in air at 37 ° C. Culture plates that had a uniform monolayer of cells were then used to culture embryonic cell lines.

Production of embryonic cell line The cells of NT units obtained as described above, were washed and platinized directly on cells of irradiated feeder fibroblasts. The cells were maintained in a growth medium consisting of alpha MEM supplemented with 10% FCS and 0.1 mM beta-mercaptoethanol (Sigma). The growth medium was exchanged every two to three days. The initial colony was observed by the second day of cultivation. The colony was propagated and exhibited a morphology similar to mouse embryonic base cells (ES), previously described. The individual cells within the colony are not well defined and the perimeter of the colony is refractive and smooth in appearance. The cell colony appears to have a slower cell folding time than mouse ES cells. In addition, unlike ES cells derived from bovines and swine, the colony does not have an epithelial appearance until now. Figures 2 to 5 are photographs of ES-like cell colonies obtained as described supra.

Production of differentiated human cells The obtained human embryonic cells are transferred to a differentiation medium and are cultured until differentiated human cell types are obtained.

RESULTS Table 1. Human cells as donor nuclei in production and development of NT units.

TABLE 1 An NT unit that developed a structure having more than 16 cells was platinized in a fibroblast feeder layer. This structure was attached to the feeder layer and began to propagate, forming a colony with a morphology similar to ES cells (see, for example, Fig. 2). Furthermore, although the structures of stage 4 to 16 cells were not used to try and produce a colony of ES cells, it has previously been shown that this step is capable of producing ES cell lines or ES-like cells (mouse, Esitetter et al., Devel. Growth and Differ., 31: 275-282 (1989), Bovine, Stice4 et al., 1996)). Therefore, it is expected that the NT units of stage 4 - 16 cells should also give rise to similar base cells or embryonic and cell colonies.

In addition, similar rests were obtained on the fusion of a keratinocyte cell line with an enucleated bovine oocyte, which was cultured in media comprising MCA, uridine, glucose and 1000 IU of LI F. Out of 50 reconstructed embryos, 22 cut and one developed a blastocyst at about day 1 2. This blastocyst was platinized and the production of an ES cell line is ongoing. Although the present invention has been described and illustrated herein by reference to several specific materials, methods and examples, it is understood that the invention is not restricted to the particular material, combinations of materials and methods selected for that purpose. Numerous variations of such details may be implied and will be appreciated by those skilled in the art.

Claims (50)

1. REIVI NDICATIONS 1 . A method for producing similar base or embryonic cells comprising the steps of: (i) inserting a desired human or mammalian cell or cell nucleus, into an enucleated animal oocyte, wherein said oocyte is derived from a different animal species that the human or mammalian cell under conditions suitable for the formation of a nuclear transfer unit (NT); (I) activate the resulting nuclear transfer unit; (iii) cultivating said activated nuclear transfer unit to more than the 2-cell development stage; and (iv) culturing cells obtained from said cultivated NT units to obtain similar embryonic-based cells.
2. 2. The method of claim 1, wherein the cell inserted into the enucleated animal oocyte is a human cell.
3. 3. The method of claim 2, wherein said human cell is an adult cell.
4. 4. The method of claim 2, wherein said human cell is an epithelial cell, keratinocyte, lymphocyte or fibroblast.
5. The method of claim 2, wherein the oocytes are obtained from a mammal.
6. 6. The method of claim 5, wherein the animal oocyte is obtained from an ungulate.
7. 7. The method of claim 6, wherein said unglulate is selected from the group consisting of bovine, ovine, porcine, equine, goat and buffalo.
8. 8. The method of claim 1, wherein the enucleated oocyte is matured prior to enucleation.
9. 9. The method of claim 1, wherein the fused nuclear transfer units are activated in vitro.
10. The method of claim 1, wherein the activated nuclear transfer units are cultured in a feeder layer culture. eleven .
11. The method of claim 10, wherein the feeder layer comprises fibroblasts.
12. The method of claim 1, wherein the cells of step (iv) of an NT unit having 1 6 cells or more are cultured in a feeder cell layer.
13. The method of claim 12, wherein said feeder cell layer comprises fibroblasts.
14. The method of claim 13, wherein said fibroblasts undertake mouse embryonic fibroblasts.
15. The method of claim 1, wherein the resulting base-like or embryonic cells are induced to differentiate.
16. The method of claim 2, wherein the resulting base-like or embryonic cells are induced to differentiate.
17. 17. The method of claim 1, wherein the fusion is effected by electrofusion.
18. 18. The base or embryonic similar cells obtained according to the method of claim 1.
19. 19. Human-like or embryonic-like cells obtained according to the method of claim 2.
20. 20. Human-based or embryonic-like cells obtained according to the method of claim 3.
21. 21. Human-based or embryonic-like cells obtained according to the method of claim 4.
22. 22. Human-based or embryonic-like cells obtained according to the method of claim 6.
23. 23. Human-like or embryonic-like cells obtained according to the method of claim 7.
24. 24. The differentiated human cells obtained by the method of claim 16.
25. 25. The differentiated human cells of claim 24, which are selected from the group consisting of neural cells, hematopoietic cells, pancreatic cells, muscle cells, cartilage cells, urinary cells, liver cells, spleen cells, reproductive cells, skin cells, intestinal cells and stomach cells.
26. 26. A method of therapy comprising administering to a patient in need of cell transplantation therapy, isogenic differentiated human cells according to claim 24.
27. 27. The method of claim 26, wherein said cell transplantation therapy is performed for treat a disease or condition selected from the group consisting of Parkinson's disease, Huntington's disease, Alzheimer's disease, ALS, defects or spinal cord injuries, multiple sclerosis, muscular dystrophy, cystic fibrosis, liver disease, diabetes, heart disease, defects or lesions in cartilage, burns, ulcers feet, vascular disease, Urinary tract disease, AI DS and cancer.
28. The method of claim 26, wherein the differentiated human cells are hematopoietic cells or neural cells.
29. 29. The method of claim 26, wherein the therapy is for treatment of Parkinson's disease and the differentiated cells are neural cells.
30. 30. The method of claim 26, wherein the therapy is for the treatment of cancer and the differentiated cells are hematopoietic cells.
31. 31 The differentiated human cells of claim 24, which coat and express an inserted gene.
32. 32. The method of claim 1, wherein a desired gene is inserted, removed or modified into base or embryonic-like cells.
33. 33. The method of claim 32, wherein the desired gene encodes a therapeutic enzyme, a growth factor or a cytokine.
34. 34. The method of claim 32, wherein said base or embryonic similar cells are human embryonic or base similar cells.
35. 35. The method of claim 32, wherein the desired gene is removed, modified or deleted by homologous recombination.
36. 36. The method of claim 1, wherein the donor cell is genetically modified to impair the development of at least one endoderm, ectoderm and mesoderm.
37. 37. The method of claim 1, wherein the donor cell is genetically modified to increase the differentiation efficiency.
38. 38. The method of claim 36, wherein the cultivated nuclear transfer unit is cultured in a medium containing at least one capsase inhibitor.
39. 39. The method of claim 1, wherein the donor cell expresses a detectable label that is indicative of the expression of a particular cyclin.
40. 40. The method of claim 36, wherein the donor cell has been modified to alter the expression of a gene selected from the group consisting of SRF, MESP-1, HN F-4, beta-1, integrin, MSD, GATA -6, GATA-4, RNA helicase A and H beta 58.
41. 41. The method of claim 37, wherein said donor cell has been genetically modified to introduce a DNA that provides for the expression of the Q7 and / or Q9 genes.
42. 42. The method of claim 41, wherein said gene or genes are operably linked to an adjustable promoter.
43. 43. The method of claim 1, wherein the donor cell has been genetically modified to inhibit apoptosis.
44. 44. The method of claim 43, wherein the reduced apoptosis is provided by altering the expression of one or more genes selected from the group consisting of Bad, Bok, BH3, Bik, Bik, Hrk, BN I P3, GimL, Bid , EGL-1, Bcl-XL, Bcl-w, Mcl-1, AI. Nr-13, BHRF-1, LMW5-HL, ORF1 6, Ks-Bcl-2, EIB-19K, and CED-9.
45. 45. The method of claim 44, wherein at least one of said genes is operably linked to an inducible promoter.
46. 46. A mammalian somatic cell that expresses a DNA encoding a detectable marker, the expression of which is linked to a particular cyclin.
47. 47. The cell of claim 46, wherein the cyclin is selected from the group consisting of cyclin D1, D2, D3, B1, B2, E, A, and H.
48. 48. The cell of claim 46, wherein the marker detectable is a fluorescent polypeptide. •
49. 49. The cell of claim 48, wherein the mammalian cell is selected from the group consisting of human, primate, rodent, ungulate, canine and feline cells.
50. 50. The cell of claim 48, wherein said cell is a human, bovine or primate cell. SUMMARY An improved method of nuclear transfer involving the transplantation of nuclei from differentiated donor cells into enucleated oocytes from a different species of the donor cell is provided. The resulting nuclear transfer units are useful for the production of isogenic embryonic base cells, in particular human isogenic base or embryonic cells. These base or embryonic-like cells are useful for producing desired differentiated cells and for introduction, removal or modification of desired genes, for example, at specific sites in the genome of such cells by homologous recombination. These cells, which may contain a heterologous gene, are especially useful in cell transplantation therapies and for in vitro study of cell differentiation. In addition, methods are provided to improve nuclear transfer efficiency by genetically altering donor cells to inhibit apoptosis, select a specific cell cycle and / or to enhance embryonic growth and development.