MXPA99006464A - Nuclear transfer with differentiated fetal and adult donor cells - Google Patents

Abstract

An improved method of nuclear transfer involving the transplantation of donor differentiated cell nuclei into enucleated oocytes of the same species as the donor cell is provided. The resultant nuclear transfer units are useful for multiplication of genotypes and transgenic genotypes by the production of fetuses and offspring, and for production of isogenic CICM cells, including human isogenic embryonic or stem cells. Production of genetically engineered or transgenic mammalian embryos, fetuses and offspring is facilitated by the present method since the differentiated cell source of the donor nuclei can be genetically modified and clonally propagated.

Description

NUCLEAR TRANSFER WITH FETAL AND ADULT DIFFERENTIAL DONOR CELLS 1. FIELD OF THE INVENTION The present invention relates to cloning methods in which the nuclei of the cells derived from fetal or adult differential mammalian cells are transplanted into enucleated mammalian oocytes of the same species as the donor nuclei. The nuclei are reprogrammed to direct the development of cloned embryos, which can be transferred into recipient females to produce fetuses and progenies or used to produce cells of cultured internal cell mass (MCIC). Cloned embryos can also be combined with fertilized embryos to produce chimeric embryos, fetuses and / or progeny. 2. BACKGROUND OF THE INVENTION Methods for deriving embryonic (SE) support cell lines in vitro from embryos of early preimplant mice are well known. (See, e.g., Evans et al., Nature, 29: 154-156 (1981); Martin, Proc. Natl. Acad. Sci., USA, 78: 7634-7638 (1981)). SE cells can be moved to an undifferentiated state, provided a fibroblast cell feeder layer is present (Evans et al., Id.) Or a source of inhibition of differentiation (Smith et al., Dev. Biol. 121: 1-9 (1987)).

Previously it had been reported that SE cells have numerous applications. For example, it has been reported that SE cells can be used as an in vitro model for differentiation, especially for the study of genes that have been implicated in the regulation of early development. SE cells of mice give rise to germline chimeras when introduced into embryos of preimplant mice, thus demonstrating their pluripotency (Bradley et al., Nature, 309: 255-256 (1984)). In view of their ability to transfer their genome to the next generation, SE cells have potential utility for the manipulation of germ lines of cattle animals using SE cells with or without a desired genetic modification. In addition, in the case of livestock animals, v.gr. , ungulates, cores of similar preimplant cattle embryos support the development of enucleated oocytes to term (Smith et al., Biol. Reprod., 40: 1027-1035 (1989); and Keefer et al., Biol. Reprod. , 50: 935-939 (1994)). This is in contrast to the nuclei of embryos from mice that beyond the eighth stage of cells after transfer are reported to not support the development of enucleated oocytes (Cheong et al., Biol. Reprod., 48: 958 (1993)). ). Therefore, SE cells from livestock animals are highly convenient since they can provide a potential source of totipotent donor nuclei, genetically engineered or in some other way, for nuclear transfer procedures.

Some research groups have reported the isolation of embryonic cell lines significantly pluripotent. For example, Notarianni et al., J. Reprod. Fert. Suppl., 43: 255-260 (1991), reports the establishment of significantly stable pluripotent cell lines of pig and sheep blastocysts that exhibit some morphological and growth characteristics similar to those of cells in primary cultures of isolated internal cell masses imuno-surgeries of sheep blastocysts. Also, Notarianni et al., J. Reprod. Fert. Suppl., 41: 51-56 (1990) describes the maintenance and differentiation in the culture of putative pluripotent embryonic cell lines of pig blastocysts. Gerfen et al., Anlm. Biotech, 6 (1): 1-14 (1995) describes the isolation of embryonic cells lines of porcine blastocysts. These SE cells stably maintained in embryonic fibroblast feeder layers of mice without the use of conditioned medium and are reported to differentiate into different cell types during culture. In addition, Saito et al., Roux's Arch. Dev. Biol., 201: 134-141 • (1992) reports cell lines similar to embryonic support cells of cultured cattle that survived three passages, but were lost after the fourth passage. Handyside et al., Roux's Arch. Dev. Biol., 196-185-190 (1987) describes the mass culture of internal cells immunosurgically isolated from sheep embryos under conditions that allow the isolation of SE cell lines derived from MCI of mice. . Handyside et al. Reports that under such conditions, the MCI attack of sheep, diffuses and develops into cell areas similar to SE cells and similar to the endoderm, but that after prolonged culture only cells similar to the endoderm are evident. Recently, Cherny et al., Theriogenology, 41-175 (1994) reported cell lines derived from primordial germ cells of significantly pluripotent bovines maintained in long term cultures. These cells, after approximately seven days in culture, produced SE-like colonies that stained positive for alkaline phosphatase (AP), exhibited the ability to form embryoid bodies and spontaneously differentiated into at least two cell types different It is also reported that these cells expressed mRNA for the transcription factors OCT4, OCT6 and H ES 1. A pattern of the homeotic box that is thought to be expressed by SE cells exclusively. Also recently, Campbell et al., Nature, 380: 64-68 (1996) reported the production of live lambs after nuclear transfer of embryonic disc (ED) cells cultured from nine-day-old sheep embryos cultured under conditions that promote Isolation of SE cell lines in the mouse. The authors concluded that DE cells from nine-day-old sheep embryos are totipotent by nuclear transfer and that totipotency remained in the culture.

Van Stekelenburg-Hamers et al., Mol. Reprod. Dev., 40: 444-454 (1995), reported the isolation and characterization of significantly permanent cell lines from cells of internal cell mass of bovine blastocysts. The authors isolated and cultured MCI from bovine blasts of 8 or 9 days of age under different conditions to determine which feeder cells and culture media are more efficient to support the binding and development of cattle MCI cells. They concluded that the union and development of cultured MCI cells was increased by the use of STO feeder cells (mouse fibroblasts) (instead of bovine uterine epithelial cells) and by the use of serum separated with charcoal (instead of of normal serum) to supplement the culture medium. Van Stekelenburg and others reported, however, that their cell lines resembled epithelial cells more than pluripotent MCI cells. Smith et al., WO 94/24274, published October 27, 1994, Evans and others WO 90/03432, published April 5, 1990 and Wheeler et al., WO 94/26889, published November 24, 1994, reported the isolation, selection and propagation of animal support cells which can be significantly used to obtain transgenic animals. Evans and others also reported the derivation of significantly pluripotent embryonic support cells from porcine and bovine species which are affirmatively useful for the production of transgenic animals. In addition Wheeler et al., WO 94/26884, published on November 24, 1994, described support cells that are affirmatively useful for the manufacture of chimeric and transgenic ungulates. Therefore, based on the above, it is evident that many groups have tried to produce SE cell lines, eg. , due to its potential application in the production of cloned or transgenic embryos and in nuclear transplantation. The use of ungulate internal cell mass (ICM) cells for nuclear transplantation has also been reported. For example, Collar et al., Mol. Reprod. Dev., 38: 264-267 (1994) described the nuclear transplantation of cattle MCI by microinjection of donor cells isolated in mature enucleated oocytes. Collas and others described embryo culture in vitro for seven days to produce fifteen blastocysts which, when transferring the recipients of bovines, resulted in four pregnancies and two births. As well. Keefer et al., Biol. Reprod., 50: 935-939 (1994), described the use of cattle MCI cells as donor nuclei in nuclear transfer procedures, to produce blasts which, when transplanted into bovine receptors, gave as Result several live descendants. In addition, Sims and others, Proc. Natl. Acad. Sci., USA, 90: 6143-6147 (1993), described the production of calves by core transfer of MCI cells from bovines cultured in vitro in the short term in mature enucleated oocytes.

The production of live lambs after the nuclear transfer of cultured embryonic disc cells has also been reported (Campbell et al., Nature, 380: 64-68 (1996)). Furthermore, the use of pluripotent embryonic cells of nuclear transfer bovines and the production of chimeric fetuses has been reported (Stice et al., Biol. Reprod., 54-100-1 10 (1996); Collas et al., Mol. Dev., 38: 264-267 (1994)). Coilas and others demonstrated that granulosa cells (adult cells) could be used in a bovine cloning procedure to produce embryos. However, there was no demonstration of early embryonic stages after development (blastocyst stage). Also, granulosa cells were not easily cultured and can only be obtained from females. Collas and others did not try to propagate the granulosa cells in cultures or tried to genetically modify these cells. While multiplications of genotypes are possible using embryonic cells as donors, there are also problems with current methods. For example, by current methods, embryoid cloning can only be done using a limited number of embryonic donor nuclei (less than 100), or with in vitro cell lines. It is unknown whether the embryonic genome encodes a superior genotype until the cloned animal becomes an adult. There are also problems in the production area of transgenic mammals. By current methods, heterologous DNA was introduced into early embryos or embryonic cell lines that differentiate into several types of cells in the fetus and eventually develop into a transgenic animal. However, several early embryos are required to produce a transgenic animal and, therefore, this procedure is very inefficient. Also, there is no simple and efficient method to select a transgenic embryo before it stops a transgenic embryo between the passage of time and the expense to place the embryos in replaced females. In addition, techniques that target genes can not be easily achieved with transgenic early embryo procedures. The embryonic support cells in mice have allowed researchers to select transgenic cells and direct the genes. This allows more genetic engineering than is possible with other transgenic techniques. However, embryonic support cell lines and other embryonic cell lines can be maintained in an undifferentiated state that requires feeder layers and / or the addition of cytokines to the medium. Even if these precautions are taken, these cells often suffer from spontaneous differentiation and can not be used to produce transgenic progenies by the methods currently available. Also, some embryonic cell lines have been propagated in a way that does not lead to the directing of the genes.

Therefore, no matter what was previously reported in the literature, there is a need for improved methods for cloning mammalian cells. OBJECTIVES AND COMPENDIUM OF THE INVENTION It is an object of the invention to provide novel and improved methods for producing cloned mammalian cells. It is a more specific objective of the invention to provide a novel method for cloning mammalian cells involving the transplantation of the nucleus of a mammalian cell differentiated into an enucleated oocyte of the same species. It is a further object of the invention to provide a method for multiplying adult mammals that has been proven to have genetic superiority or other convenient characteristics. It is another object of the invention to provide an improved method for producing genetically engineered or transgenic mammals (i.e., embryos, fetuses, progenies). It is a more specific objective of the invention to provide a method for producing genetically engineered or transgenic mammals by which a desired gene is inserted, removed or modified in a differentiated mammalian cell or cell nucleus before using said differentiated cells or cell nucleus for the formation of a TN unit. It is another object of the invention to provide genetically or transgenicly treated mammals (i.e., embryos, fetuses, progenies) obtained by transplantation of the nucleus of a differentiated cell into an enucleated oocyte of the same species as the differentiated cell. It is another object of the invention to provide a novel method for producing mammalian MCIC cells involving transplantation or a nucleus of a differentiated cell in an enucleated oocyte of the same species as the differentiated cell. It is another object of the invention to provide MCIC cells produced by the transplantation of the nucleus of a differentiated mammalian cell into an enucleated oocyte of the same species as the differentiated cell. It is a more specific objective of the invention to provide a method for producing human MCIC cells that involves the transplantation of the nucleus of a human cell, v. gr. , a human adult cell, in an enucleated human oocyte. It is another object of the invention to use said MCIC cells for therapy or diagnosis. It is a specific objective of the invention to use MCIC cells, including human and ungulate MCIC cells, for the treatment or diagnosis of any disease in which the transplantation of cells, tissues or organs is therapeutically or diagnostically beneficial. MCIC cells can be used within the same species or through the species. It is another objective of the invention to use tissues derived from TN embryos, fetuses or progenies, including tissues from humans and ungulates, for the treatment or diagnosis of any disease in which the transplantation of cells, tissues or organs is therapeutic or diagnostically beneficial. The tissues can be used within the same species or through the species. It is another specific object of the invention to use the MCIC cells produced according to the invention for the production of differentiated cells, tissues or organs. It is another specific objective of the invention to use MCIC cells produced according to the invention in vitro, e.g. , for the study of cell differentiation and for analysis purposes, v.gr. , for drug studies. It is another specific object of the invention to provide improved methods for transplantation therapy, comprising the use of cells, tissues or splenic or syngeneic organs produced from MCIC cells produced according to the invention. Such therapies include by way of example the treatment of disease and injuries including Parkinson's, H untington'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. It is another object of the invention to provide genetically engineered or transgenic MCIC cells produced by inserting, removing or modifying the desired gene in a differentiated mammalian cell or cell nucleus before using that of the differentiated cell or cell nucleus for the formation of a TN unit. It is another object of the invention to use the transgenic or genetically treated MCIC cells produced according to the invention for gene therapy, in particular for the treatment and / or prevention of the identified diseases and lesions, supra. It is another object of the invention to use MCIC cells produced according to the invention or transgenic or genetically treated MCIC cells produced according to the invention as nuclear donors for nuclear transplantation. Therefore, in one aspect, the present invention provides a method for cloning a mammal (e.g., embryos, fetuses, progenies). The method comprises: (i) inserting a desired cell or nucleus of differentiated mammalian cells into an enucleated mammalian oocyte of the same species as the differentiated cell or cell nucleus, under conditions suitable for the formation of a nuclear transfer unit (TN); (ii) activate the resulting nuclear transfer unit; (iii) cultivating said activated nuclear transfer unit until it is greater than stage 2 of cell development; and (iv) transferring the cultivated TN unit to a host mammal so that the TN unit develops into a fetus.

The cells, tissues and / or organs of the fetuses are advantageously used in the area of transplantation of cells, tissues and / or organs. The present invention also includes a method for cloning a genetically or genetically modified mammal, by which a desired gene is inserted, removed or modified in the cells or nucleus of differentiated mammalian cells prior to the insertion of the cell or cell nucleus. of differentiated mammals in the enucleated oocyte. Also the present invention provides mammals obtained according to the above method and progenies of these mammals. The present invention is preferably used to clone ungulates. In another aspect, the present invention provides a method for producing MCIC cells. The method comprises: (i) inserting a desired cell or nucleus of mammalian differentiated cells into an enucleated mammalian oocyte of the same species as the differentiated cells, under conditions suitable for the formation of a nuclear transfer unit (TN); (I) activate the resulting nuclear transfer unit; (iii) cultivating the activated nuclear transfer unit until it is greater than stage 2 of cell development; and (iv) culturing cells obtained from the cultured TN unit to obtain MCIC cells.

MCIC cells are advantageously used in the area of cell, tissue and organ transplants. With the above objectives and others, the advantages and aspects of the invention that will be 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. . DETAILED DESCRIPTION OF THE INVENTION The present invention provides improved methods for cloning animals by nuclear transfer or nuclear transplantation. The present application, nuclear transfer or nuclear or TN transplantation, are used interchangeably. According to the invention, cell nuclei derived from cells of fetal mammals or differentiated adults are transplanted into enucleated mammalian oocytes of the same species as the donor nuclei. The nucleus is reprogrammed to direct the development of cloned embryos, which can be transferred into recipient females to produce fetuses and progenies, or used to produce MCIC cells. Cloned embryos can also be combined with fertilized embryos to produce chimeric embryos, fetuses and / or progenies. The methods of the prior art have used embryonic cell types in cloning procedures. These include work by Campbell et al. (Nature, 380: 64-68, 1996) and Stice et al. (Biol. Reprod., 54: 100-1 10, 1996). In both of these studies, the embryonic cell lines were derived from embryos less than 10 days of gestation. In both studies, the SE cells were kept in a feeder layer to avoid the obvious differentiation of the donor cell that will be used in the cloning procedure. The present invention uses differentiated cells. It was hoped that cloned embryos with differentiated donor nuclei could develop into the advanced embryonic and fetal stages. The scientific dogma had been that only the types of embryonic or undifferentiated cells could direct this type of development. It was unexpected that a large number of cloned embryos could be produced from these differentiated cell types. Also, it was unexpected that the transgenic embryonic cell lines could be derived easily from cloned transgenic embryos. Therefore, in accordance with the present invention, multiplication of superior mammalian genotypes, including ungulates, is possible. This will allow the multiplication of adult animals with proven genetic superiority or other convenient characteristics. Progress will be accelerated, for example, in many species of important ungulates. By the present invention, potentially there are billions of fetal or adult cells that can be recovered and used in the cloning process. This will potentially result in many identical progenies in a short period.

The present invention also allows the simplification of transgenic procedures by working with a source of differentiated cells that can be propagated clonally. This eliminates the need to maintain the cells in an undifferentiated state, therefore, genetic modifications, both of random integration and directed to the gene, are more easily achieved. Also combining nuclear transfer with the ability to modify and select these cells in vitro, this procedure is more efficient than the techniques of previous transgenic embryos. In accordance with the present invention, these cells can be propagated clonally without cytokines, conditioned medium and / or feeder layers, simplifying and further facilitating the transgenic process. When transfected cells are used for the cloning procedures according to the invention, transgenic embryos are produced which can develop into fetuses and progenies. Also, these transgenic cloned embryos can be used to produce MCIC cell lines or other embryonic cell lines. Therefore, the present invention eliminates the need to derive and maintain in vitro an undifferentiated cell line that leads to genetic engineering techniques. The present invention can also be used to produce MCIC cells, fetuses or progenies that can be used, for example, in transplantation of cells, tissues and organs. By taking an adult fetal cell from an animal and using it in the cloning procedure, a variety of cells, tissues and organs can possibly be obtained from the cloned fetuses as they develop through organogenesis. Cells, tissues and organs can also be isolated from cloned progenies. This process can provide a source of "materials" for many medical and veterinary therapies including cell and gene therapy. If the cells are transferred back into the animal from which the cells were derived, then immune rejection is prevented. Also, because many types of cells can be isolated from these clones, other methodologies such as hematopoietic chimerism can be used to avoid immunological rejection between animals of the same species as well as between different species. Therefore, in one aspect, the present invention provides a method for cloning a mammal. In general, the mammal will be produced by a nuclear transfer process comprising the following steps: (i) obtaining cells from differentiated mammals desired to be used as a source of donor nuclei; (ii) obtain oocytes from a mammal of the same species as the cells which are the source of the donor nuclei; (iii) enucleating said oocytes; (iv) transferring the desired cell or desired differentiated cell nucleus into the enucleated oocyte, e.g., by fusion or injection, to form TN units; (v) activate the resulting TN unit; (vi) cultivating the activated nuclear transfer unit until it is greater than stage 2 of cell development; and (vii) transferring the cultivated TN unit to a host mammal so that the TN unit develops into a fetus. The present invention also includes a method for cloning a genetically or genetically modified mammal, by which a desired gene is inserted, removed or modified in the cell or nucleus of differentiated mammalian cells prior to the insertion of the cell or cell nucleus of cells. differentiated mammals in the enucleated oocyte. Also the present invention is provided by mammals obtained according to the above method and progenies of these mammals. The present invention is preferably used to clone ungulates. The present invention also provides the use of TN fetuses and TN and chimeric progenies in the area of cell, tissue and organ transplantation. In another aspect, the present invention provides a method for producing MCIC cells. The method comprises: (i) inserting a desired cell or nucleus of mammalian cells desired in. an oocyte of enucleated mammals of the same species as the differentiated cell or cell nucleus, under conditions suitable for the formation of a nuclear transfer unit (TN); (I) activate the resulting nuclear transfer unit; (iii) cultivating said activated nuclear transfer unit until it is greater than stage 2 of cell development; and (iv) culturing cells obtained from the cultured TN unit to obtain MCIC cells. MCIC cells are advantageously used in the area of transplantation of cells, tissues and organs, or in the production of fetuses or progenies, including fetuses or transgenic progenies. Preferably, the TN units will be grown to a size of at least 2 to 400 cells, preferably 4 to 128 cells and more preferably of a size of at least about 50 cells. 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, 43-181 (1995); Collas and others, Mol. Report Dev., 38: 264-267 (1994); Keefer et al., Biol. Reprod., 50: 935-939 (1994); Sims and others, Proc. Natl. Acad. Sci., USA, 90: 6143-6147 (1993); WO 94/26884; WO 94/24274 and WO 90/03432, which are incorporated herein by reference in their entirety. Also, the Patents of E. U.A. Nos. 4,944,384 and 5,057,420 describe procedures for bovine nuclear transplantation. The cells of differentiated mammals are those cells that pass the early embryonic stage. More particularly, the differentiated cells are those that at least pass the embryonic disc stage (day 10 of bovine embryogenesis). The differentiated cells can be derived from the ectoderm, mesoderm or endoderm. Mammalian cells, including human cells, can be obtained by well-known methods. Mammalian cells useful in the present invention include, by way of example, epithelial cells, neural cells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), erythrocytes, macrophages, monocytes, mononuclear cells. , fibroblasts, cardiac muscle cells and other muscle cells, etc. In addition, mammalian cells used for nuclear transfer can be obtained from different organs, e.g., skin, lung, pancreas, liver, stomach, intestine, heart, reproductive organs, bladder, 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. Fibroblast cells are a type of ideal cells because they can be obtained from developing fetuses and adult animals in large quantities. The fibroblast cells are somewhat different and, therefore, previously considered as a poor cell type for use in cloning procedures. More importantly, these cells can easily propagate in vitro with a rapid doubling time and can be propagated clonally for use in the procedure that targets the genes. Again, the present invention is novel since differentiated cell types are used. The present invention is advantageous because the cells can be easily propagated, genetically modified and selected in vitro. Sources of mammals suitable for oocytes include sheep, cows, pigs, horses, rabbits, guinea pigs, mice, hamsters, rats, primates, etc. Preferably, the oocytes will be obtained from ungulates and more preferably from bovines. Methods for oocyte isolation are well known in the art. Essentially, these will comprise the isolation of oocytes from the ovaries or reproductive tract of a mammal, eg. , a bovine. An easily available source of bovine oocytes are slaughterhouse materials. For the successful use of techniques such as genetic engineering, transfer and nuclear cloning, oocytes generally must be matured in vitro before they can be used as cells for nuclear transfer and before they can be fertilized by sperm cells to develop into an embryo. This process generally requires collecting immature oocytes (prophase I) from mammalian ovaries, eg. , ovaries of cattle obtained in a slaughterhouse and ripen the oocytes in a maturation medium before fertilization or enucleation until the oocyte obtains metaphase stage II, which in the case of bovine oocytes, generally occurs at around 18 -24 hours after the aspiration. For the purposes of the present invention, this time is known as the "ripening period". As used herein, for the calculation of time, "aspiration" refers to the aspiration of the immature oocyte of ovarian follicles. Additionally, oocytes in the metaphase stage I I, which matured in vivo, have been used successfully in nuclear transfer techniques. Essentially, mature metaphase II oocytes are surgically collected from non-ovulatory or ovulatory cows or heifers 35 to 48 hours after the onset of estrus or after injection of human chorionic gonadotropin (hCG) or similar hormone. . The maturation stage of the oocyte in nuclear enucleation and transfer has been reported to be significant in the success of TN methods. (See, for example, Prather et al., Differentiation, 48, 1-8, 1991). In general, successful cloning practices of successful mammalian embryos use the oocyte in the metaphase II stage as the receiving oocyte because it is thought that at this stage the oocyte can be "activated" or "activated" sufficiently to treat the introduced core as a fertilizing sperm does. In domestic animals and especially in cattle, the period of activation of the oocyte varies from approximately 16-52 hours, preferably approximately 28-42 hours after aspiration.

For example, immature oocytes can be washed in hamster embryo culture medium (MCEH) with pH regulated with H EPES 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 (MCT) 199 containing 10% fetal calf serum containing appropriate gonadotropins such as leutinizing hormone (HL) and follicle stimulation hormone (H EF) and estradiol under a layer of lightweight paraffin or silicon at 39 ° C. After a fixed period of maturation, which varies from about 10 to 40 hours and preferably from about 16-18 hours, the oocytes will be enucleated. Before enucleation the oocytes will preferably be removed and placed in MCEH containing 1 milligram per milliliter of hyaluronidase before the removal of cells in the form of clusters. This can be done by repeated pipetting through very fine orifice pipettes or by briefly stirring. The separated oocytes were screened for polar bodies and the selected metaphase I 1 oocytes, as determined by the presence of polar bodies, are used for nuclear transfer. Then follow the enucleation. The enucleation can be carried out by known methods, such as described in the Patent of E. U.A. No. 4,994,384 which is incorporated herein by reference. For example, oocytes in metaphase II are placed in MCEH, optionally containing 7.5 micrograms per milliliter of cytochalasin B, for immediate enucleation or can be placed in a suitable medium, for example an embryo culture medium such as CR1 aa, plus 10% cow serum in estrus and then enucleated, preferably no more than 24 hours later and even more preferably 16-18 hours later. Enucleation can be achieved microsurgically using a micropipette to remove the polar body and the adjacent cytoplasm. The oocytes can then be screened to identify those of which they have been successfully enucleated. This screening can be done by staining the oocytes with 1 microgram per milliliter of Hoechst 33342 dye in MCEH and then observing the oocytes under ultraviolet irradiation for less than 10 seconds. The oocysts that have been successfully enucleated can then be placed in a suitable culture medium, e.g. , CR 1 aa plus 10% serum. In the present invention, the receptor oocytes will preferably be enucleated in a time ranging from about 10 hours to about 40 hours after the onset of in vitro maturation, more preferably from about 16 hours to about 24 hours after the start of the maturation in vitro and even more preferably approximately 16-18 hours after the onset of in vitro maturation. A single mammalian cell of the same species can then be transferred as the enucleated oocyte in the perivitelline space of the enucleated oocyte used to produce the TN unit. The mammalian cell and the enucleated oocyte will be used to produce the TN units according to methods known in the art. For example, SE cells can be fused by electrofusion. Electrofusion is achieved by providing an impulse of electricity that is sufficient to cause a temporary rupture of the plasma membrane. This rupture of the plasma membrane is very high because it reforms rapidly in the membrane. Therefore, if two adjacent membranes are induced to break and by reforming the intermixed lipid layers, small channels will open between the two cells. Due to the thermodynamic instability of said small opening, it lengthens until the two SE cells become one. Reference is made to the U.S. Patent. No. 4,997,384 by Prather et al., (Incorporated herein in its entirety by preference) for further discussion of this process. A variety of electrofusion media can be used including, e.g., solution regulated with sucrose, mannitol, sorbitol and phosphate. Fusion can also be achieved using Sendai virus as a fusogenic agent (Graham, Wister Inot, Symp.Monogr., 9, 19, 1969). Also, in some cases (e.g., with small donor nuclei) it will 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), incorporated herein by reference in its entirety.

Preferably, mammalian cells and oocytes are electrofused in a 500 μm chamber by the application of an electric pulse of 90-120V for approximately 15 μsec, approximately 24 hours after the onset of oocyte maturation. After fusion, the resulting fused TN units are then placed in a suitable medium until it is activated, v. gr. , means of CR 1 aa. Normally the activation will be carried out a short time later, normally less than 24 hours later, and preferably approximately 4-9 hours later. The TN unit can be activated by known methods.

These methods include, for example, , cultivation of the TN unit at sub-physiological temperature, essentially applying a shock of cold to really cold temperature to the TN unit. This can be done more conveniently by growing the TN unit at room temperature, which is relatively cold for the physiological temperature conditions at which the embryos are normally exposed. Alternatively, activation can be achieved by the application of known activation agents. For example, it has been shown that the penetration of oocytes by sperm during fertilization activates the perfusion oocytes to give greater numbers of viable pregnancies and multiple genetically identical calves after nuclear transfer. Also, treatments such as electrical or chemical shock can be used to activate TN embryos after fusion. Suitable oocyte activation methods are the subject of the U.S. Patent. No. 5,496,720 to Susko-Parrish and others, incorporated herein by reference in its entirety. Additionally, the activation can be carried out simultaneously or sequentially: (i) the increase of levels of divalent cations in the oocyte, and (ii) reduction of the phosphorylation of cellular proteins in the oocyte. This will generally be done by introducing divalent cations into the cytoplasm of oocytes, e.g., magnesium, strontium, barium or calcium e.g., in the form of an onofero. Other methods to increase levels of divalent cations include the use of electric shock, treatment with ethanol and treatments with enclosed chelators. Phosphorylation can be reduced by known methods, e.g., by the addition of kinase inhibitors, e.g., serine-threonine kinase inhibitors such as 6-dimethyl-aminopurine, staurosporine, 2-aminopurine and sphingosine. Alternatively, the phosphorylation of cellular proteins can be inhibited by introducing a phosphatase into the oocyte, e.g., phosphatase 2A and phosphatase 2B. In one embodiment, TN activation is effected by briefly exposing the TN unit to a TL-HEPES medium containing 5 μM of ionomycin and 1 mg / ml of BSA, followed by washing in TL-H EPES containing 30 mg / ml of BSA within approximately 24 hours after the fusion and preferably approximately 2 to 9 hours after the fusion. The activated TN units can then be cultured in a suitable in vitro culture medium until the generation of MCIC cells and cell colonies. Suitable culture media for cultured and mature embryos are well known in the art. Examples of known media, which can be used for culture and maintenance of bovine embryos, include Ham F-10 + 10% fetal calf serum (FCS), Tissue Culture Medium-199 (MCT) - 199) + 105 fetal calf serum, Thyroid-Albumin-Lactate-Piruvate (TALP), Dulbecco's Phosphate Regula Saline (PBS), Eagle and Whitten medium. U not of the most common means used for the collection and maturation of oocytes is MCT-199 and 1 to 20% of serum supplementation including fetal calf serum, newborn serum or estrual cow serum, lamb serum or serum of steer. A preferred maintenance medium includes MCT-199 with Earl salts, 10% fetal calf serum, 0.2 mM Na 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. Another means of maintenance is described in the Patent of E. U.A. No. 5, 096, 822 of Rosenkrans, Jr. et al., Which is incorporated herein by reference. This embryo medium, named CR 1, contains the nutritional substances necessary to support an embryo. CR1 contains the hemicalcium L-lactate in amounts ranging from 1.0mM to 10mM, preferably 1.0mM to 5.0mM: the hemicalcium L-lactate is L-lactate with a hemicalcium salt incorporated therein. The L-lactate of hemicalcio is important since a single component satisfies the main requirements in the culture medium: (i) the requirement of calcium necessary to compact and the disposition of cytoskeleton; and (i) the requirement of lactate necessary for metabolism and transporting electrons. L-lactate from hemicalcium also serves as a valuable mineral and source of energy for the medium necessary for the viability of embryos. Advantageously, the CR1 medium does not contain serum, such as fetal calf serum and does not require the use of a co-culture of animal cells or other biological means, ie means comprising animal cells such as oviduct cells. Sometimes biological means can be disadvantageous because they can contain microorganisms or trace factors that can be harmful to embryos and that are difficult to detect, characterize and eliminate. The examples of the main components in the middle of CR! They include L-lactate of hemicalcio, sodium chloride, potassium chloride, sodium bicarbonate and a smaller amount of bovine serum albumin without fatty acids (Sigma A-6003). Additionally, a defined amount of essential and non-essential amino acids can be added to the medium. CR1 with amino acids is known by the abbreviation "CR1 aa". The CR 1 medium preferably contains the following components in the following amounts: sodium chloride -1 14.7 mM potassium chloride -3.1 mM sodium bicarbonate -26.2 mM L-lactate of hemicalcium -5 mM fatty acid-free BSA -3 mg / In one embodiment, the unit of activated TN embryos is placed in CR1 aa medium containing 1.9 mM DMAP for approximately 4 hours followed by a wash in MCEH and then cultured in BSA containing CR1 aa. For example, the activated TN units can be transferred to the culture medium of CR 1 to a containing 2.0 mM of DMAP (Sigma) and grown under ambient conditions, e.g. , approximately 38.5 ° C, 5% CO2 for a suitable time, v.gr. , approximately from 4 to 5 hours. Then, the cultivated TN unit or units are preferably washed and then placed in a suitable medium, v. gr. , CR 1 aa medium containing 10% FCS and 6 mg / ml content in well plates which preferably contain a suitable confluent feeder layer. Suitable enrichment layers include, by way of example, fibroblasts and epithelial cells, v. gr. , fibroblasts and uterine epithelial cells derived from ungulates, chicken fibroblasts, murine fibroblasts (e.g., mice or rats), STO and S! -m220 feeder cell lines and BRL cells. In one embodiment, the feeder cells comprise mouse embryonic fibroblasts. The preparation of a suitable fibroblast feeder layer is described in the following example and is within the skill of the skilled person. TN units are grown on the feeder layer until the TN units reach a suitable size to transfer it to a recipient female to obtain cells that can be used to produce MCIC cells and cell colonies. Preferably, these NT units will be cultured to at least about 2 to 400 cells, more preferably about 4 to 128 cells and even more preferably at least about 50 cells. The cultivation will be carried out under the appropriate conditions, that is, approximately 38.5 ° C and 5% CO2; with the culture medium changed in order to optimize the growth normally approximately every 2-5 days, preferably approximately every 3 days. The methods for embryo transfer and handling of the recipient animal in the present invention are normal procedures used in the embryo transfer industry. Synchronous transfers are important for the success of the present invention, that is, the TN embryo stage is in synchrony with the estrus cycle of the recipient female. This advantage and how to maintain the receptors are reviewed in Siedel, G. E., J r ("Critica! Review of embryo transfer procedures with cattle" in Fertilization and Embryonic Development in Vitro (1981) L. Mastroianni, Jr, and J. D. Biggers, ed. , Plenum Pres, New York, page 323), the content of which is incorporated herein by reference. The present invention can also be used to clone in genetically treated or transgenic mammals. As explained above, the present invention is advantageous since transgenic procedures can be simplified by working with a source of differentiated cells that can be propagated clonally. In particular, the differentiated cells used to donate nuclei have a desired gene inserted, removed or modified. These altered, genetically differentiated cells are those used for nuclear transplantation with enucleated oocytes. Any methods for inserting, deleting or modifying a desired gene of a mammalian cell can be used to alter the differentiated cell that will be used as the nuclear donor. These procedures can remove or part of a gene and the gene can be heterologous. The technique of homologous recombination is included, which allows the insertion, deletion or modification of a gene or genes at a specific site or specific sites in the genome of the cell.

The present invention, therefore, can be used to provide adult mammals with the desired genotypes. The multiplication of adult ungulates with proven genetic superiority or other convenient characteristics is particularly useful including animals, transgenic or genetically treated and chimeric animals. In addition, the cells and tissues of the TN fetus, including transgenic and / or chimeric fetuses, can be used in cell, tissue and organ transplants for the treatment of numerous diseases, as described below in connection with the use of cells from MCIC. For the production of MCIC cells and cell lines, after TN units of the desired size are obtained, SE cells are mechanically removed from the zone and then used. This is preferably done by taking the cell cluster comprising the TN unit, which will normally contain at least about 50 cells, washing said cells and plating the cells in a feeder layer, e.g. , irradiated fibroblast cells. Normally, the cells used to obtain the support cells or cell colonies will be obtained from the innermost portion of the cultivated TN unit which is preferably at least 50 cells in size. However, TN units of smaller or larger cell numbers as well as cells of other portions of the TN unit can also be used to obtain SE cells and cell colonies. The SE cells are maintained in the feed layer in a suitable growth medium, e.g. , M MS supplemented with 10% FCS and 0.1 mM β-mercaptoethanol (Sigma) and L-glutamine. The growth medium is changed as often as necessary to optimize growth, v. gr. , approximately every 2-3 days. This culture process results in the formation of MCIC cells or cell lines. One skilled in the art can vary the culture conditions as desired to optimize the growth of the particular MCIC cells. Also, MCIC cells from genetically engineered or transgenic mammals can be produced in accordance with the present invention. That is, the methods described above can be used to produce TN units in which a desired gene or genes have been introduced, or from which all or part of the endogenous gene or genes has been removed or modified. The genetically engineered or transgenic TN units can then be used to produce genetically engineered or transgenic MCIC cells, including human cells. The resulting MCIC cells and cell lines, preferably human cells and MCIC cell lines have numerous therapeutic and diagnostic applications. More especially, said MCIC cells can be used for therapies or cell transplantation. Human MCIC cells have application in the treatment of numerous disease conditions. The human TN units themselves can also be used in the treatment of disease conditions.

In this regard, it is known that embryonic (SE) support cells from mice are capable of differentiating into almost all cell types, e.g., hematopoietic support cells. Therefore, the human MCIC cells produced according to the invention should have similar differentiation capacity. The MCIC cells according to the invention will be induced to differentiate in order to obtain the desired cell types according to the known methods. For example, the human MCIC cells present can be induced to differentiate into hematopoietic support cells, muscle cells, cardiac muscle cells, liver cells, cartilage cells, epithelial cells, urinary tract cells, etc., by cultivating said cells in a differentiation medium and under conditions that provide cell differentiation. The means and methods that result in the differentiation of MCIC cells are known in the art since they are suitable for culture conditions. For example, Palacios et al., Proc. Natl. Acad. Sci., USA, 92: 7530-7537 (1995) teaches the production of hematopoietic support cells of an embryonic cell line by subjecting the support cells to an induction process which comprises initially culturing aggregates of said cells in a culture medium. suspension lacking retinoic acid followed by culture in the same medium containing retinoic acid, followed by transfer of the cellular aggregates to a substrate that provides cell binding. In addition, Pedersen, J. Reprod. Fertile. Dev., 6: 543-552 (1994) is a revised article that references numerous articles describing methods for in vitro differentiation of embryonic support cells to produce various types of differentiated cells including hematopoietic cells, muscle, cardiac muscle, nerve, among others. In addition, Bain et al., Dev. Biol., 168: 342-357 (1995) teaches the in vitro differentiation of embryonic support cells to produce neural cells having neuronal properties. These references are illustrative of reported methods for obtaining differentiated cells from embryonic or support cells. These references and in particulars the descriptions therein that refer to the methods for differentiation of embryonic support cells are hereby incorporated by reference in their entirety. Therefore, using known methods and culture medium, one skilled in the art can cultivate the MCIC cells present, including genetically treated or transgenic MCIC cells, to obtain the desired differentiated cell types, e.g. , neural cells, muscle cells, hematopoietic cells, etc. MCIC cells present can be used to obtain any type of desired differentiated cell. The therapeutic uses of said differentiated human cells are not parallel. For example, human hematopoietic support cells can be used in medical treatments that require bone marrow transplantation. These procedures are used for the treatment of many diseases, eg. , cancer in late stage such as ovarian cancer and leukemia, as well as diseases that comprise the immune system, such as SI DA. Hematopoietic support cells can be obtained, v. gr. , fusing adult somatic cells of a patient with cancer or SI DA, v.gr. , epithelial cells or lymphocytes with an enucleated oocyte, obtaining MCIC cells as described above and cultivating said cells under conditions that favor differentiation, until hematopoietic support cells are obtained. Said hematopoietic cells can be used in the treatment of diseases including cancer and SI DA. Alternatively, adult somatic cells of a patient with a neurological disorder may be fused with an enucleated oocyte, human MCIC cells obtained therefrom and said cells cultured under differentiation conditions to produce neural cell lines. Specific diseases that can be treated by transplantation of said human neural cells include, for example, Parkinson's disease, Alzheimer's disease, ALS and cerebral palsy, among others. In the specific case of Parkinson's disease, it has been shown that neural cells of the transplanted fetal brain make more appropriate connections to the surrounding cells and produce dopamine. This can result in the long-term reversal of the symptoms of Parkinson's disease. The great advantage of the present invention is that it provides an essentially unlimited supply of isogenic or syngeneic human cells suitable for transplantation. Therefore, the significant problem associated with current transplantation methods, i.e. rejection of transplanted tissue that may occur due to host rejection versus graft or graft versus host, will be obvious. Conventionally, rejection is avoided or reduced by the administration of anti-rejection drugs such as cyclosporin. However, said drugs have significant adverse side effects, e.g. , immunosuppression, carcinogenic properties, just as they are very expensive. The present invention should eliminate, or at least greatly reduce, the need for anti-rejection drugs. Other diseases and conditions that can be treated by isogenic cell therapy include, for example, spinal cord damage, multiple sclerosis, muscular dystrophy, diabetes, liver diseases, i.e., hypercholesterolemia, heart disease, cartilage replacement , b, foot ulcers, gastrointestinal diseases, vascular diseases, kidney disease, urinary tract disease and diseases and conditions related to old age. This methodology can be used to replace defective genes, eg. , genes of the defective immune system, genes of cystic fibrosis, or to introduce genes which results in the expression of therapeutically beneficial proteins such as growth factors, lymphokines, cytokines, enzymes, etc. For example, the growth factor derived from the brain encoding the gene can be introduced into human MC1C cells, cells differentiated into neural cells and cells transplanted into a patient with Parkinson's disease to delay the loss of neural cells during said disease. Previously, the cell types transfected with BDN F varied from primary cells to immortalized cell lines, either neural or non-neural derived cells (myoblast and fibroblast). For example, astrocytes have been transfected with the BDN F gene using retroviral vectors and cells grafted into a rat model of Parkinson's disease (Yoshimoto et al., Brain Research, 691: 25-36, (1995)). This ex vivo therapy reduced symptoms similar to Parkinson's in rats up to 45% 32 days after the transfer. Also, the tyrosine hydroxylase gene has been placed in the astrocytes with similar results (Lundberg et al., Develop Neurol., 139-39-53 (1996) and references cited therein). However, such ex vivo systems have problems. In particular, the retroviral vectors currently used down regulate in vivo and the transgene is only expressed temporarily (reviewed by Mulligan, Science, 260: 926-932 (1993)).

Also, these studies used primary cells, astrocytes, that have a finite life time and replicate slowly. These properties adversely affect the transfection regime and prevent the selection of stably transfected cells. In addition, it is almost impossible to propagate a large population of primary cells targeting genes that will be used in homologous recombination techniques. In contrast, difficulties associated with retroviral systems should be eliminated by the use of mammalian MCIC cells. Genes that can be introduced into the MCIC cells present include, by way of example, an epidermal growth factor, basic fibroblast growth factor, neurotrophic growth factor derived from the glia, insulin-like growth factor (I and II); neurotrophin-3, neurotrophin-4/5, ciliary neurotrophic factor, AFT-1, cytokine genes (interleukins, interferons, colony stimulation factors, tumor necrosis factor (alpha and beta), etc.), the genes they encode therapeutic enzymes, etc. In addition to the use of human MCIC cells in the transplantation of cells, tissues and organs, the present invention also includes the use of non-human cells in the treatment of human diseases. Therefore, MCIC cells, TN and TN fetuses and chimeric progenies (transgenic or non-transgenic) of any species can be used in the treatment of disease conditions in humans where cell, tissue or cell transplantation is guaranteed. organs. In general, the cells, fetuses and progenies of MCIC according to the present invention can be used within the same species (autologous, syngeneic or allografts) or through the species (xenografts). For example, brain cells from bovine NT fetuses can be used to treat Parkinson's disease. Also, the MCIC cells present, preferably human cells, can be used as an in vitro model of differentiation, in particular for the study of genes that are involved in the regulation of early development. Also, differentiated cells, tissues and organs using the MCIC cells present can be used in drug studies. In addition, the present cells of MCIC can be used as nuclear donors for the production of other MCIC cells and cell colonies. In order to more clearly describe the present invention, the following examples are provided. EXAMPLE 1 Isolation of primary cultures of fibroblast cells from embryonic and adult bovine and swine cattle. The primary cultures of bovine and porcine fibroblasts were obtained from fetuses (45 days of pregnancy for cattle and 35 days for fetuses of pigs). The head, liver, heart and alimentary tract were removed aseptically, the fetuses were crushed and incubated for 30 minutes at 37 ° C in prewarmed trypsin EDT solution (0.05% trypsin / O.02% EDTA; GI BCO, Grand Island, NY). Fibroblast cells were plated in tissue culture plates and cultured in alpha-M EM medium (BioWhittaker, Walkersville, MD) supplemented with 10% fetal calf serum (FCS) (Hyclone, Logen, UT) , penicillin (100 μL / ml) and streptomycin (50 μl / ml). The fibroblasts were developed and maintained in a humid atmosphere with 5% CO2 in air at 37 ° C. The adult fibroblast cells were isolated from the lung of a cow (approximately five years old). The ground lung tissue was incubated overnight at 10 ° C in trypsin EDTA solution (0.05% trypsin / 0.02% EDTA; G I BCO, Grand Island, NY). The next day the tissue and any dissociated cells were incubated for one hour at 37 ° C in EDTA solution of preheated trypsin (0.05% trypsin / 0.02% EDTA; GI BCO, Grand Island, NY) and processed through three consecutive washes and trypsin incubations (one hour). The fibroblast cells were seeded in tissue culture plates and cultured in alpha-M EM medium (BioWhittaker, Walkersville, M D) supplemented with 10% fetal calf serum (FCS) (Hyclone, Logen, UT), penicillin (10 lU / mL) and streptomycin (50 μl / ml). The fibroblast cells can be isolated virtually at any time during development, varying from approximately the post-embryonic disk stage to the adult life of the animal (bovines 12 to 15 days after fertilization at 10 to 15 years of age). animals) . This procedure can also be used to isolate fibroblasts from other mammals, including mice. Introduction of a marker gene (foreign heterologous DNA) in embryonic cells and adult fibroblasts. The following electroporation procedure was carried out for embryonic fibroblast cells (cattle and pigs) and adults (cattle). Normal micro-injection procedures can also be used to introduce heterologous DNA into fibroblast cells, however, in this example electroporation is used because it is an easier procedure. Culture plates containing propagating fibroblast cells were incubated in trypsin EDTA solution (0.05% trypsin / O.02% EDTA; GI BCO, Grand island, NY) until the cells were in a single cell suspension. The SE cells were rotated at 500 x g and re-suspended in 5 million cells per ml with phosphate buffered saline (PBS). The reporter gene construct contained the cytomegalovirus promoter and beta-galactosidase, a neomycin phosphotransferase (beta-G EO) fusion gene. The reporter gene and the cells at a final concentration of 50 μl / ml were added to the electroporation chamber. After the electroporation pulse, the fibroblast cells were again transferred into the growth medium (alpha-M MS medium) (BioWhittaker, Walkersville, MD) supplemented with 10% fetal calf serum (FCS) (Hyclone, Logen, UT), penicillin (100 l U / ml) and streptomycin (50 μl / ml)).

The day after electroporation, the bound fibroblast cells were selected for stable integration of the reporter gene. G418 (400 μg / ml) was added to the growth medium for 15 days (scale: 3 days until the end of the life cycle of the cultured cells). This drug kills any cell without the beta-GEO gene, since they do not express the neo-resistance gene. At the end of this time, colonies of stable transgenic cells were present. Each colony spread independently of one another. The transgenic fibroblast cells were stained with X-gal to observe beta-galactosidase expression and confirmed positive for integration using PCR amplification of the beta-GEO gene and run on the agarose gene. Use of transgenic fibroblast cells in nuclear transfer procedures to create MCIC cell lines and transgenic fetuses. A cell line (CL-1) derived from a colony of bovine embryonic fibroblast cells was used as donor nuclei in the nuclear transfer (TN) procedure. The general TN procedures are described below. The oocytes of the slaughterhouse were matured in vitro. The oocytes were separated from cells in clusters and enucleated with a level micropipette at about 18 to 20 hours after maturation (hpm). Enucleation was confirmed in TL-HEPES medium plus Hoechst 33342 (3 μg / ml, Sigma). The individual donor cells (fibroblasts) were placed in the perivitilino space of the recipient oocyte. The cytoplasm of bovine oocytes and the donor nucleus (TN unit) were fused using electrofusion techniques. A fusion pulse consisting of 120 V for 15 μsec in a chamber with a 500 μm space was applied to the TN unit. This happened at 24 hpm. The TN units were placed in CR 1 aa medium until 26 to 27 hpm. The general procedure used to artificially activate the oocytes was described above. Activation of the TN unit started between 26 and 27 hpm. In summary, TN units were exposed for four minutes to ionocimine (5 μM, CalBiochem, La Jolla, CA) in TL-H EPES medium supplemented with 1 mg / ml of BSA and then washed for five minutes in TL-H medium. H EPES supplemented with 30 mg / ml of BSA. During the ionomycin treatment, TN units were also exposed to 2 mM DMAP (Sigma). After washing, the TN units were transferred into a microdroplet of CR 1 culture medium containing 2 mM DMAP (Sigma) and cultured at 38.5 ° C, 5% CO2 for four to five hours. The embryos were washed and placed in CR1 aa plus 10% FCS and 6 mg / ml BSA in four well plates containing a confluent feeder layer of embryonic fibroblast from mice. The TN units were cultivated for three more days at 38.5 ° C and 5% CO2. The culture medium was changed every three days until day 5 to 8 after activation. At this time TN embryos in blastocyst stage can be used to produce MCIC cell lines and TN transgenic fetuses (cultured internal cell mass). The mass of internal cells of these NT units can be isolated and seeded in a feeder layer. Also, TN units were transferred into recipient females. The pregnancies were aborted at 35 days of gestation. This resulted in two cloned transgenic fetuses having the beta-GEO gene in all the tissues reviewed. Therefore, this is a quick and easy method to form MCIC cell lines and transgenic fetuses. This procedure generally led to MCIC cell lines and fetuses targeting the gene. The following table summarizes the results of these experiments. * 19 lines were positive for beta-GEO, 2 were negative and one line died before detection of CPR. t One fetus died and another was slightly retarded in the development at 35 days of gestation. Two fetuses recovered on day 38 were normal. All the fetuses were confirmed transgenic.EXAMPLE 2 Chimeric Fetuses Derived from Transgenic MCIC Cells. The transgenic MCIC cell line was originally derived from a transgenic TN unit (differentiated cell). A line of MCIC derived from transgenic TN embryos (a CL-1 cell transferred into an enucleated oocyte) was used to produce chimeric embryos and fetuses. Colonies of transgenic MCIC cells were disrupted using 1 -5 mg / ml pronase or 0.05% trypsin / EDTA combined with the mechanical disintegration methods so that clusters of five or less SE cells were produced. The trypsin or pronase activity is inactivated by passing the cells through multiple washes of 30 to 100% fetal calf serum. The disaggregated cells were placed in micromanipulation plates containing the TL-H EPES medium. The fertilized embryos were also placed on these plates and micromanipulation tools were used to produce the chimeric embryos. Eight to ten transgenic MCIC cells were injected into fertilized embryos at stage 8-16 of the cells. These embryos were cultured in vitro for the blastocyst stage and then transferred into recipient animals. A total of 6 chimeric embryos in blastocyst stage were transferred nonsurgically in two recipient females. After five weeks of gestation, 3 fetuses were recovered.

Various tissues of the three fetuses, including germ cells of the gonad (suggesting germline chimeras), were screened by PCR amplification and hybridized with Southern blot analysis of amplified product to a beta-galactosidase fragment. Of the three fetuses, two were positive for the contribution of the transgenic MCIC cells. Both fetuses had a contribution of transgenic MCIC to the gonad. Transgenic TN embryos derived from transgenic MCIC cell lines. The transgenic MCIC cell line was originally derived from a transgenic TN unit (differentiated cell). The same transgenic MCIC cell lines were used to produce TN embryos. The TN procedures described in Example 1 were used except that the MCIC cells instead of the fibroblast cells were used as the donor cell fused with the enucleated oocyte. Colonies of transgenic MCIC cells were already disintegrated using 1 -5 mg / ml pronase or 0.05% trypsin / EDTA combined with mechanical disintegration methods so that clusters of five or less cells were produced. The trypsin or pronase activity is inactivated by passing the cells through multiple washes of 30 to 100% fetal calf serum before transferring the cells into enucleated oocytes. The results are reported in Table 1 (third group). Five embryos were produced in the blastocyst stage.

Claims (77)

1. CLAIMS 1. A method for cloning a non-human mammal, comprising: (i) inserting a desired differentiated mammalian cell or cell nucleus into an enucleated mammalian oocyte of the same species as the cells or nuclei of the differentiated cells, under suitable conditions for the formation of a nuclear transfer unit (TN); (ii) activate the resulting nuclear transfer unit; (iii) cultivating said activated nuclear transfer until it is greater than stage 2 of cell development; and (iv) transferring the cultivated TN unit to a host mammal so that the TN unit develops into a fetus.
2. 2. The method according to claim 1, further comprising developing the fetus to a progeny.
3. 3. The method according to claim 1, wherein the desired DNA is inserted, removed or modified in the cell or cell nucleus of the differentiated mammal, resulting in the production of a genetically altered TN unit.
4. 4. The method according to claim 3, further comprising developing the fetus to a progeny.
5. 5. The method according to claim 1, wherein the cell or cell nucleus of the differentiated mammal is derived from mesoderm.
6. 6. The method according to claim 1, wherein the differentiated mammalian cell or cell nucleus is derived from ectoderm.
7. The method according to claim 1, wherein the differentiated mammalian cell or cell nucleus is derived from endoderm.
8. The method according to claim 1, wherein the differentiated mammalian cell or cell nucleus is a fibroblast cell or cell nucleus.
9. 9. The method according to claim 1, wherein the cell or cell nucleus of differentiated mammal comes from an ungulate.
10. The method according to claim 9, wherein the ungulate is selected from the group consisting of cattle, sheep, swine, horses, goats and buffaloes. eleven .
11. The method according to claim 1, wherein the differentiated mammalian cell or cell nucleus is an adult cell or cell nucleus.
12. The method according to claim 1, wherein the differentiated mammalian cell or cell nucleus is an embryonic or fetal cell or cell nucleus.
13. The method according to claim 1, wherein the enucleated oocyte matures before enucleation.
14. 14. The method according to claim 1, wherein the fused nuclear transfer unit is activated by the exposure of ionomycin and 6-dimethylaminopurine.
15. 15. The method according to claim 3, wherein the microinjection was used to insert a heterologous DNA.
16. 16. The method according to claim 3, wherein the electroporation was used to insert a heterologous DNA.
17. 17. A non-human mammal fetus obtained according to the method of claim 1.
18. 18. A progeny of non-human mammal obtained according to the method of claim 2.
19. 19. A non-human progeny according to the offspring of according to claim 18.
20. 20. A transgenic non-human mammal fetus obtained according to the method of claim 3.
21. 21. A progeny of transgenic non-human mammal obtained according to the method of claim 4.
22. 22. A progeny of the non-human mammal of the offspring according to claim 21.
23. 23. The method according to claim 1, further comprising combining the cloned TN unit with a fertilized embryo to produce a chimeric embryo.
24. 24. The method according to claim 23, further comprising developing the fetus to a progeny.
25. 25. A non-human mammal fetus obtained according to the method of claim 23.
26. 26. A offspring of a non-human mammal obtained according to the method of claim 24.
27. 27. A progeny of non-human mammal of the mammal according to the claim 26.
28. 28. A method for producing an MCIC cell line (cultured internal cell mass), comprising: (i) inserting a desired differentiated mammalian cell or cell nucleus into an enucleated mammalian oocyte of the same species as the cells or the nuclei of the differentiated cells, under conditions suitable for the formation of a nuclear transfer unit (TN); (ii) activate the resulting nuclear transfer unit; (iii) cultivating the activated nuclear transfer unit until it is greater than stage 2 of cell development; and (v) culturing cells obtained from the cultivated TN unit to obtain a MCIC cell line.
29. 29. An MCI cell line obtained according to the method of claim 28.
30. 30. The method according to claim 28, wherein a desired DNA is inserted, removed or modified in the differentiated mammalian cell or cell nucleus. , thus resulting in the production of a genetically altered TN unit.
31. 31 A transgenic MCIC cell line obtained according to claim 30.
32. 32. The method according to claim 28, wherein the resulting MCIC cell line is induced to differentiate.
33. 33. The differentiated mammalian cells obtained by the method of claim 32.
34. 34. The human differentiated cells obtained by the method of claim 32.
35. 35. A method of therapy comprising administering to a patient in need of transplantation therapy. of cells, isogenic differentiated cells according to claim 34.
36. 36. The method according to claim 35, wherein the SE cell transplantation therapy is performed to treat a disease or condition selected from the group consisting of Parkinson's disease, Huntington's disease, Alzheimer's disease, ALS, defects or lesions of spinal cord, multiple sclerosis, muscular dystrophy, cystic fibrosis, liver disease, diabetes, heart disease, defects or damage cartilage injuries, burns, foot ulcers, vascular disease, urinary tract disease, SI DA and cancer.
37. 37. A method of therapy comprising administering to a human patient in need of cell transplantation therapy, differentiated xenogeneic cells according to claim 33.
38. 38. The method according to claim 37, wherein the xenogeneic differentiated cells are bovine cells.
39. 39. The method according to claim 37, wherein the SE cell transplantation therapy is performed to treat a disease or condition selected from the group consisting of Parkinson's disease, H untington's disease, Alzheimer's disease, ALS, defects or spinal cord injuries, multiple sclerosis, muscular dystrophy, cystic fibrosis, liver disease, diabetes, heart disease, cartilage defects and injuries, burns, foot ulcers, vascular disease, urinary tract disease, SI DA and Cancer.
40. 40. The method according to claim 35, wherein the differentiated human cells are hematopoietic cells or neural cells.
41. 41 The method according to claim 35, wherein the therapy is for the treatment of Parkinson's disease and the differentiated cells are neural cells.
42. 42. The method according to claim 35, wherein the therapy is for the treatment of cancer and the differentiated cells are hematopoietic cells.
43. 43. A method of therapy comprising administering to a human patient in need of cell transplantation therapy, xenogeneic cells obtained from a fetus according to claim 17.
44. 44. The method according to claim 43, wherein the xenogeneic cells are bovine cells.
45. 45. The method according to claim 43, wherein the SE cell transplantation therapy is performed to treat a disease or condition selected from the group consisting of Parkinson's disease, H untington's disease, Alzheimer's disease, ALS, defects or injuries of the spinal cord, multiple sclerosis, muscular dystrophy, cystic fibrosis, liver disease, diabetes, heart disease, cartilage defects or injuries, burns, foot ulcers, vascular disease, urinary tract disease, SI DA and cancer .
46. 46. A method of therapy comprising administering to a human patient in need of cell transplantation therapy, xenogeneic cells obtained from offspring according to claim 18.
47. 47. The method according to claim 46, wherein in the xenogeneic cells are bovine cells.
48. 48. The method according to claim 46, wherein the SE cell transplantation therapy is performed to treat a disease or condition selected from the group consisting of Parkinson's disease, H untington's disease, Alzheimer's disease, ALS, defects or spinal cord injuries, multiple sclerosis, muscular dystrophy, cystic fibrosis, liver disease, diabetes, heart disease, cartilage defects or injuries, burns, foot ulcers, vascular disease, urinary tract disease, AIDS and cancer.
49. 49. A method of therapy comprising administering to a patient a human in need of cell transplantation therapy, transgenic xenogeneic cells obtained from a transgenic fetus according to claim 20.
50. 50. The method according to claim 49, in where the xenogeneic cells are bovine cells.
51. 51. The method according to claim 49, wherein the SE cell transplantation therapy is performed to 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, cartilage defects or injuries, burns, foot ulcers, vascular disease, urinary tract disease, AIDS and cancer.
52. 52. A method of therapy comprising administering to a human patient in need of cell transplantation therapy, transgenic xenogeneic cells obtained from a transgenic progeny according to claim 21.
53. 53. The method according to claim 52, wherein the xenogeneic transgenic cells are bovine cells.
54. 54. The method according to claim 52, wherein the SE cell transplantation therapy is performed to 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, cartilage defects or injuries, burns, foot ulcers, vascular disease, urinary tract disease, AIDS and cancer.
55. 55. The method according to claim 28, further comprising combining a TN unit with a fertilized embryo to produce a chimera.
56. 56. The method according to claim 55, further comprising developing the chimeric MCIC cell line to a chimeric embryo.
57. 57. A chimeric non-human mammalian embryo obtained according to claim 56.
58. 58. The method according to claim 56, further comprising developing a chimeric embryo to a chimeric fetus.
59. 59. A chimeric non-human mammal fetus obtained according to claim 58.
60. 60. The method according to claim 58, further comprising developing a chimeric fetus to a non-human mammal chimeric progeny
61. 61. A mammalian progeny does not chimeric human obtained according to claim 60.
62. 62. The method according to claim 55, wherein a. The desired DNA is inserted, removed or modified in the cell or differentiated mammalian cell nuclei thus resulting in the production of a genetically altered TN unit.
63. 63. The method according to claim 62, further comprising developing the chimeric MCIC cell line to a chimeric mammalian non-human embryo.
64. 64. A chimeric non-human mammalian embryo obtained according to claim 63.
65. 65. The method according to claim 63, further comprising developing the chimeric embryo to a chimeric fetus.
66. 66. A chimeric non-human mammal fetus obtained according to claim 65.
67. The method according to claim 65, further comprising developing the chimeric fetus to a progeny of chimeric non-human mammal.
68. 68. The progeny of chimeric nonhuman mammal obtained according to claim 67.
69. 69. A method for cloning a non-human mammal, comprising: (i) inserting a desired differentiated mammalian cell or MCIC cell nucleus into an oocyte of enucleated mammal of the same species as the differentiated MCIC cell or the cell nucleus, under conditions suitable for the formation of a nuclear transfer unit (TN); (ii) activate the resulting nuclear transfer unit; (iii) cultivating the activated nuclear transfer unit until it is greater than stage 2 of cell development; and (iv) transferring the cultivated TN unit to a host mammal so that the TN unit develops into a fetus.
70. 70. The method according to claim 69, further comprising developing the fetus to a progeny.
71. 71. A non-human mammal fetus obtained according to the method of claim 69.
72. 72. A non-human mammalian progeny obtained according to the method of claim 70.
73. 73. A non-human mammalian organ to be used as a xenograft organ, which is obtained from the progeny according to claim 18.
74. 74. A non-human mammalian organ for use as an organ xenograft, which is obtained from the progeny according to claim 21.
75. 75. A non-human mammalian organ to be used as an organ xenograft, which is obtained from the progeny according to claim 26.
76. 76. A non-human mammal organ to be used as an organ xenograft, which is obtained from the progeny according to claim 68.
77. 77. A non-human mammalian organ for use as an organ xenograft, which is obtained from the progeny according to claim 72. SUMMARY An improved method of nuclear transfer involving the transplantation of nuclei from donor differentiated cells into enucleated oocytes of the same species as the donor cells is provided. The resulting nuclear transfer units are useful for the multiplication of transgenic genotypes and genotypes by the production of fetuses and progenies, and for the production of isogenic MCIC cells, including human isogenic embryonic or support cells. The production of embryos of genetically treated or transgenic mammals, embryos, fetuses and progenies of genetically treated or transgenic mammals is facilitated by the present method since the source of differentiated cells of the donor nuclei can be genetically modified and propagated clonally.