MXPA00000201A

MXPA00000201A - Cloning using donor nuclei from non-serum starved, differentiated cells

Info

Publication number: MXPA00000201A
Application number: MXPA/A/2000/000201A
Authority: MX
Inventors: Steven L Stice; James M Robl; Jose Cibelli; Paul Golueke; De Leon F Abel Ponce; D Joseph Jerry
Original assignee: University Of Massachusetts
Priority date: 1997-07-03
Filing date: 2000-01-03
Publication date: 2000-09-08

Abstract

An improved method of nuclear transfer involving the transplantation of donor differentiated cell nuclei from non-serum starved cells 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

CLONING USING DONOR CELLS OF DIFFERENTIATED CELLS THAT SUFFER FROM THE LACK OF SERUM 1. FIELD OF THE INVENTION The present invention relates to cloning methods in which the nuclei of cells derived from differentiated mammalian cells that suffer from the lack of serum are transplanted into oocytes of enucleated mammals of the same species as the nuclei. donors The nuclei are reprogrammed to direct the development of cloned embryos, which can then be transferred into female donors to produce fetuses and progenies, or used to produce cells of cultured internal cell mass (CICM). Cloned embryos can also be combined with embryos fertilized to produce chimeric embryos, fetuses and / or progenies. 2. BACKGROUND OF THE INVENTION The use of cells with internal cell mass (ICM, for its acronym in English) ungulate for nuclear transplantation has been reported. For example, Collas et al., Mol Reprod. Dev., 38: 264-267 (1994), describe nuclear transplantation of cattle ICM by microinjection of donor cells Used in mature enucleated oocytes. Collas and others describe embryo culture in vitro for seven days to produce fifteen blastocysts which, when transferred to bovine recipients, resulted in four pregnancies and two births. Also, Keefer et al., Biol.

Reprod., 50: 935-939 (1994), describe the use of cattle ICM cells as donor nuclei in nuclear transfer procedures, to produce blastocysts which, when transplanted into bovine donors, resulted in several live progenies. In addition, Sims and others, Proc. Nati Acad. Sci., E.U.A., 90: 6143-6147 (1993), describe the production of calves by in vitro short-term nuclei transfer of ICM cells from bovines cultured in enucleated mature oocytes. The production of live sheep following the nuclear transfer of cultured embryonic disc cells has also been reported (Campbell et al., Nature, 380: 64-68 (1996)). Still further, the use of pluripotent embryonic cells in nuclear transfer and the production of chimeric fetuses has been reported (Stice et al., Biol. Reprod., 54: 100-110 (1996); Collas et al., Mol Reprod. Dev., 38: 264-267 (1994)). Collas and others demonstrated that granulosa cells (adult cells) can be used in a bovine cloning procedure to produce embryos. However, there was no demonstration of development after the early embryonic stages (blastocyst stage). Also, granular cells are not easily cultured and are only obtained from females. Collas and others did not try to propagate the granular cells in culture or try to genetically modify these cells. Wilmut et al. (Nature, 355: 810-813 (1997)), produced progenies of sheep by nuclear transfer derived from fetal fibroblast cells, and a progeny of a cell derived from an adult sheep.

There are also problems in the area of transgenic production in mammals. By current methods, heterologous DNA is introduced into either early embryos or embryonic cell lines that differentiate into various cell types in the fetus and eventually develop into a transgenic animal. However, many early embryos are required to produce a transgenic animal and, therefore, this procedure is very inefficient. Also, there is no simple and efficient method of selection for a transgenic embryo before going through the time and cost of placing the embryos in surrogate females. In addition, gene targeting techniques can not be easily completed with transgenic early embryo procedures. Embryonic support cells in mice have allowed researchers to select transgenic cells and perform gene targeting. This allows more genetic engineering than is possible with other transgenic techniques. Nevertheless, embryonic support cells and other embryonic cell lines must be maintained in an undifferentiated state that requires feeder layers and / or the addition of cytokines to the medium. Although these precautions are followed, these cells frequently undergo spontaneous differentiation and can not be used to produce transgenic progenies by currently available methods. Also some embryonic cell lines have to be propagated in a way that does not lead to gene targeting procedures. Methods for deriving embryonic (ES) support cell lines in vitro from fresh preimplant mouse embryos are well known. (See, for example, Evans et al., Nature, 29: 154-156 (1981); Martin, Proc. Nati Acad. Sci., E.U.A., 78: 7634-7638 (1981)). ES cells can be passaged in an undifferentiated state, as long as 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) ES cells have been previously reported for numerous applications, for example, it has been reported that ES cells can be used as an in vitro model for differentiation, especially for the study of genes which they are involved in the regulation of recent development.The mouse ES cells can originate germline chimeras when introduced into preimplant mouse embryos, 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, ES cells have potential utility to generate the manipulation of cattle animals using ES cells with or without n a desired genetic modification. In addition, in the case of cattle animals, e.g., ungulates, the nuclei of similar preimplant 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: 935-939 (1994)). This is in contrast to the nuclei of mouse embryos which beyond the eight-cell stage after the transfer, did not report support for the development of the enucleated oocytes (Cheong et al., Biol. Reprod., 48: 958 (1993 )). Therefore, ES cells from livestock animals are highly desirable because they provide a potential source of totipotent donor nuclei, genetically engineered or in some form, by nuclear transfer methods. Some research groups have reported the apparent isolation of pluripotent embryonic cell lines. For example, Notarianni et al., J. Reprod. Fert. Suppl., 43: 255-260 (1991), report the apparent establishment of pluripotent, stable blastocyte cell lines of pigs and sheep which exhibit some morphological and growth characteristics similar to those of cells in primary cultures of internal cell masses isolated immunosurgically from 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 swine blasts. Gerfen et al., Anim. Biotech, 6 (1): 1-14 (1995) describe the isolation of embryonic cell lines from porcine blasts. These cells are stably maintained in feeder layers of mouse embryonic fibroblasts using conditioned medium, and it is reported that they differentiate into several different cell types during culture. In addition, Saito et al., Roux's Arch. Dev. Bíol., 201: 134-141 (1992) report cell lines similar to embryonic bovine support cells, cultured which survived three steps, but were lost after the fourth step. Handyside et al., Roux's Arch. Dev. Biol., 196: 185-190 (1987) describe the culture of internal cell masses isolated immunosurgically from embryos of sheep under conditions which allow the isolation of mouse ES cell lines derived from Mouse ICMs. Handyside et al. Report that under such conditions sheep ICM bind, disseminate and develop areas of both cells similar to ES cells and similar to endoderms, but after prolonged culture only cells similar to the endoderm are evident. Recently, Cherny et al., Theriogenology, 41: 175 (1994) reportedly reported cell lines derived from primordial germ cells of pluripotent bovines maintained in a long-term culture. These cells, after approximately seven days in culture, produced similar colonies of ES which were stained positive for alkaline phosphatase (AP), exhibited the ability to form embryo bodies, and spontaneously differentiated into at least two different cell types. . These cells reportedly also expressed mRNA for the transcription factors of OCT4, OCT6 and HES1, a pattern of homeocasilla genes which are thought to be expressed by ES cells only. Also recently, Campbell et al., Nature, 380: 64-68 (1996) reported the production of live lambs after the nuclear transfer of embryonic disc cells (ED) cultured from ovine embryos on day nine cultured under conditions which promote the isolation of ES cell lines in the mouse. The authors concluded that the ES cells of sheep embryos on day nine are totipotent by nuclear transfer and the totipotency is maintained in the culture. Van Stekelenburg-Hamers et al., Mol. Reprod. Dev., 40: 444-454 (1995), reported the isolation and characterization apparently of permanent cell lines of cells of internal cell mass of bovine blastocysts. The authors isolated and cultured BCCs from bovine blastocysts of 8 or 9 days under different conditions to determine which feeder cells and culture medium are more efficient in supporting the binding and growth of cattle ICM cells. They concluded that the binding and growth of ICM cell cultures is increased by the use of feeder cells (mouse fibroblasts) STO (instead of bovine uterine epithelial cells) and by the use of serum separated with charcoal vegetable (instead of normal serum) to the culture medium supplement. Van Stekelenburg and others reported, however, that their cell lines resemble epithelial cells more than pluripotent ICM cells. 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 November 24, 1994, report the isolation, selection and propagation of animal support cells which apparently can be used to obtain transgenic animals. Evans and others also reported the apparent derivation of pluripotent embryonic support cells from porcine and bovine species which are surely useful for the production of transgenic animals. In addition, Wheeler et al., WO 94/26884, published on November 24, 1994, describe embryonic support cells which are surely useful for the production of chimeric and transgenic ungulates. Therefore, based on the above, it is evident that many groups have tried to produce ES cell lines, for example, due to their potential application in the production of cloned or transgenic embryos and in nuclear transplantation. Without considering what has been previously reported in the literature, there is a need for improved methods to clone mammalian cells.

OBJECTIVES AND COMPONENTS OF THE I NVENTION It is an objective of the invention to provide novel and improved methods for producing cloned mammals (e.g., embryos, fetuses and progenies). It is a more specific objective of the invention to provide a novel method of cloned mammals which involves the transplantation of nuclei from a differentiated mammalian cell that suffers from the lack of serum in an enucleated oocyte of the same species. It is another object of the invention to provide a method for multiplying adult mammals that have proven genetic superiority or other desirable traits. It is another object of the invention to provide an improved method for producing genetically treated mammals or transgenic mammals (ie, embryos, fetuses, progenies). The invention also provides genetically treated or transgenic mammals produced by said method. It is a more specific objective of the invention to provide a method for producing genetically treated mammals or transgenic mammals by which a desired DNA sequence is removed or modified in a differentiated mammalian cell or cell nucleus prior to the use of the cell. differentiated or cellular nucleus for the formation of a TN unit. The invention also provides genetically treated or transgenic mammals made by said method.

It is another object of the invention to provide a method for producing genetically treated or transgenic mammals by transplantation of the nuclei of a differentiated, transgenic cell, which suffers from the lack of serum in an enucleated oocyte of the same species as the differentiated cells. The invention also provides genetically engineered or transgenic mammals produced by said method. It is another object of the invention to provide a novel method for producing mammalian CICM cells which involves the transplantation of a nucleus of a differentiated cell, which suffers from the lack of serum in an enucleated oocyte of the same species as the differentiated cell. It is another object of the invention to provide CICM cells produced by the transplantation of the nuclei of a differentiated mammalian cell, which suffers from the lack of serum in an enucleated oocyte of the same species as the differentiated cells. A more specific objective of the invention is a method for producing human CICM cells which involves the transplantation of the nucleus of a human cell, which suffers from the lack of serum, for example, a human adult cell, in a human oocyte enucleated It is another object of the invention to use such CICM cells for therapy or diagnosis. It is a specific objective of the invention to use such CICM cells, including ungulate and human CICM cells, for the treatment of the diagnosis of any disease wherein the transplantation of cells, tissues or organs is beneficial therapeutically or for diagnosis. CICM cells can be used within the same species or through the same species. It is another object of the invention to use cells or tissues derived from TN embryos, fetuses or progenies, including human and ungulate tissues, for the treatment or diagnosis of any disease or injury where the transplantation of cells, tissues or organs is beneficial therapeutically or for diagnosis. Such diseases and injuries include Parkinson's, Huntington's, Alzheimer's, ALS, spinal cord injuries, multiple sclerosis, muscular dystrophy, diabetes, liver diseases, heart disease, cartilage replacement, burns, vascular diseases, diseases of the tract urinary, as well as for the treatment of immune defects, bone marrow transplantation, cancer, among other diseases. The tissues can be used within the same species or through the species. It is another specific objective of the invention to use the CICM cells produced according to the invention for the production of differentiated cells, tissues or organs. It is a more specific objective of the invention to use the human CICM cells produced according to the invention for the production of cells, tissues or human organs differentiated. It is another specific object of the invention to use CICM cells produced according to the invention in vitro, for example for the study of cell differentiation and for analysis purposes, for example for drug studies. It is another object of the invention to provide improved methods of transplantation therapy, which comprises the use of ¡hegenic or syngeneic cells, tissues or organs produced from CICM cells produced according to the invention. Such therapies include, by way of example, the treatment of diseases and injuries including Parkinson's, Hunttington's, Alzheimer's, ALS, spine 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 CICM cells produced by inserting, removing or modifying a desired DNA sequence in a differentiated mammalian cell or cell nuclei before the use of differentiated cells or cell nuclei for the formation of a unit of TN. It is another object of the invention to use the genetically engineered or transgenic CICM cells produced according to the invention for gene therapy, in particular for the treatment and / or prevention of the diseases and lesions identified, supra.

It is another object of the invention to use the genetically treated or transgenic CICM cells produced according to the invention as nuclear donors for nuclear transplantation. Therefore, in one aspect, the present invention provides a method for the cloning of a mammal (e.g., embryos, fetuses, progenies). The method comprises: (i) inserting a differentiated mammalian cell, suffering from lack of serum or cell nuclei in an enucleated mammalian oocyte of the same species as differentiated cells or cell nuclei, under conditions suitable for formation of a nuclear transfer TN unit; (ii) activate the resulting nuclear transfer unit; and (iii) transferring said cultivated TN unit to a mammalian host such that the TN unit develops into a fetus. Preferably, the activated nuclear transfer unit is cultured until it is greater than stage 2 of cell development. 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 of cloning a genetically or genetically modified mammal, by which a desired DNA sequence is inserted, removed or modified, in the cell of the differentiated mammal or cell nucleus prior to cell insertion. of differentiated mammal or cell nucleus in an enucleated oocyte.

Mammals obtained according to the above method, and the progeny of these mammals are also provided in the present invention. The present invention is preferably used for cloning ungulates. In another aspect, the present invention provides a method for producing CICM cells. The method comprises: (i) inserting a differentiated mammalian cell, which suffers from the lack of serum or cell nucleus in an enucleated mammalian oocyte of the same species as the differentiated cells or cell nuclei, under conditions suitable for the formation of a nuclear transfer unit (TN); (ii) activate the resulting nuclear transfer unit; and (iii) culturing cells obtained from said cultivated TN unit to obtain CICM cells. Preferably, the activated nuclear transfer unit is cultured to be greater than stage 2 of cell development. CICM cells are advantageously used in the area of cell, tissue and organ transplantation. With the foregoing and other objects, advantages and aspects of the invention which will be apparent hereafter, the nature of the invention will be more clearly understood 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 mammals by nuclear transfer or nuclear transplantation. In the present application, nuclear transfer or nuclear or TN transplantation are used interchangeably. According to the invention, cell nuclei derived from differentiated mammalian cells, suffering from lack of serum, are transplanted into enucleated mammalian oocytes of the same species as the donor nuclei. The nuclei are programmed to direct the development of the cloned embryos, which can be transferred into female receptors to produce fetuses and progenies, or are used to produce CICM cells. Cloned embryos can also be combined with fertilized embryos to produce embryos, fetuses and / or chimeric progenies. The methods of the prior art have used embryonic cell types in cloning procedures. This includes work by Campbell et al. (Nature, 380: 64-68, 1996) and Stice et al. (Biol. Reprod., 54: 100-110, 1996). In both studies, embryonic cell lines were derived from embryos less than 10 days of gestation. In both studies, the cells were maintained on a feeder layer to prevent premeditated differentiation of the donor cell for use in the cloning procedure. It has been found that the present is effective using both fetal and adult cells.

It was unexpected that embryos cloned with fetal or adult donor nuclei could develop into advanced embryonic and fetal stages. The scientific dogma has been that only recent types of embryonic cells could direct this type of development. It was unexpected that a large number of cloned embryos could be produced from fetal or adult cells. Also, it was unexpected that the new transgenic embryonic cell lines could be derived easily from transgenic cloned embryos. The adult cells and fetal fibroblast cells of a sheep have apparently been used to produce a progeny of sheep (Wilmut et al., 1997). In that study, however, it was emphasized that the use of a core donor cell, which does not suffer from lack of serum in the resting state, was important for the success of the Wilmut cloning method. Said requirement for lack of serum or rest does not exist for the present invention. In contrast, cloning is carried out using differentiated mammalian cells, which suffer from lack of serum. In addition, the cloning efficiency according to the present invention may be the same regardless of whether or not fetal or adult donor cells are used, while Wilmut et al. (1997) reported that the cloning efficiency was carried out with adult donor cells. . Therefore, in accordance with the present invention, the multiplication of higher genotypes of mammals, including ungulates, is possible. This will allow the multiplication of adult animals with genetic superiority provided or other desirable characteristics. Progress will accelerate, for example, in many important ungulate species. By the present invention, there are potentially billions of fetal or adult cells that can be harvested and used in the cloning process. This will potentially result in many identical progenies in a short period. There was also speculation that the Wilmut and others method will allow the generation of transgenic animals (see MacQuitty, Nature Biotech., 15: 294 (1997)). However, there is no reason to assume, for example, that the nuclei of adult cells that have been transfected with exogenous DNA will be able to survive the process of nuclear transfer. In this regard, it is known that the properties of mouse embryonic (ES) support cells are altered by in vitro manipulation in such a way that their ability to form viable chimeric embryos is impaired. Therefore, prior to the present invention, the cloning of transgenic animals could not be predicted. The present invention allows the simplification of transgenic procedures by working with a source of cells that can be clonally propagated. This eliminates the need to maintain the cells in an undifferentiated state, therefore, genetic modifications, both the random integration and the direction of genes, are more easily completed. 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, further simplifying and facilitating the transgenic process. When the transfected cells are used in 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 CICM cell lines or other embryonic cell lines. Therefore, the present invention eliminates the need to derive and maintain a line of undifferentiated cells in vitro which leads to genetically treated techniques. The present invention can also be used to produce cells, fetuses or progenies of CICM, which can be used, for example, in the transplantation of cells, tissues and organs. Taking a fetal or adult cell from an animal and using it in the cloning procedure a variety of cells, tissues and possibly organs can be obtained from cloned fetuses since they are developed by organogenesis. Cells, tissues, and organs can be isolated from also cloned progenies. This process can provide a source of "materials" for many veterinary and medical therapies that include cell and gene therapy. If the cells are transferred back into the animal in 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 evade immunological rejection between animals of the same species as well as between 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, which suffer from lack of serum 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 donor nuclei; (iii) enucleating said oocytes; (V) transferring the differentiated cell or cell nuclei in the enucleated oocyte, for example, by fusion or injection, to form TN units; (v) activate the resulting TN unit; and (vi) transferring said unit of cultured TN to a mammalian host in such a way that the TN unit develops into a fetus.

Preferably, the activated nuclear transfer unit is cultured until it is greater than the stage of development 2 of the cells. The present invention also includes a method of cloning a genetically or genetically modified mammal, by which a desired DNA sequence is inserted, removed or modified in the differentiated mammalian cell or cell nuclei prior to insertion into the oocyte. enucleated Also the present invention provides mammals obtained according to the above method, and the progeny of these animals. The present invention is preferably used to clone ungulates.

The present invention also provides the use of TN and TN fetuses and chimeric progeny in the transplantation area of cells, tissues and organs. In another aspect, the present invention provides a method for producing CICM cells. The method comprises: (i) inserting a differentiated mammalian cell, which suffers from the lack of serum or cell nuclei in an enucleated mammalian oocyte of the same species as the differentiated cells or cell nuclei, under conditions suitable for formation of a TN transfer unit; (ii) activation of the resulting nuclear transfer unit; and (iii) cultivating cells obtained from said cultivated TN unit to obtain CICM cells. Preferably, the activated nuclear transfer unit is cultured to be greater than the stage of development 2 of the cells. CICM cells are advantageously linked in the area of transplantation of cells, tissues and organs, or in the production of fetuses or progenies, including fetuses or transgenic progenies.

As used herein, a fetus is a young product not born of a viviparous animal after it has taken form in the uterus. Therefore, in cows, the fetal stage occurs 35 days after conception until birth. In pigs, the fetal stage occurs from 30 days after conception until birth. A mammal is an adult from birth to death. Preferably, the NT units will be grown to a size of at least 2 to 400 cells, preferably 4 to 128 cells, and more preferably to 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., Theryiogenology, 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. Nati Acad. Sci., E. U.A, 90: 6143-6147 (1993); WO 94/26884; WO 94/24274, and WO 90/03432, which are hereby incorporated by reference in their entirety. Also, the patents of US Pat. Nos. 4,944, 384 and 5,057,420 describe procedures for nuclear transplantation of bovines. Differentiated, refers to cells that have a different character or function from the surrounding structures or from the cell of origin. Differentiated mammalian cells are those cells which passed the early embryonic stage. More particularly, the differentiated cells are those that passed at least 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 others muscle cells, etc. In addition, mammalian 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. The fibroblast cells are an ideal cell type because they can be obtained from developing fetuses and adult animals in large quantities. The fibroblast cells differ in some way and, therefore, were previously considered as a poor cell type for use in cloning procedures. Importantly, these cells can be easily propagated in vitro with a rapid doubling time and can be propagated clonally for use in gene targeting procedures. Again, the present invention is novel because differentiated cell types are used. The present invention is advantageous because the cells can be easily propagated, genetically modified and selected in vitro. Other reported cloning methods (eg, Wilmut et al., 1997) have relied on the use of cells that do not suffer from lack of serum. In the present invention, however, the donor cells are not in a state of suffering from lack of serum. According to Wilmut et al. (1997), cells that do not suffer from lack of serum are at rest, that is, they leave the growth phase. Other methods (chemical, temperature, etc.) are also capable of producing cells at rest. The donor cells used in the present invention are not at rest. The sources of mammals suitable for oocytes include sheep, cows, pigs, goats, horses, rabbits, guinea pigs, mice, hamsters, rats, primates, etc. Preferably, the oocytes will be obtained from ungulates, and more preferably from bovines. Methods for the proper isolation of oocytes are well known in the art. Essentially, these will comprise oocytes for isolation of the ovaries or reproductive tracts of a mammal, for example, a bovine. An easily available source of bovine oocytes are meat materials. For the successful use of techniques such as genetic engineering, transfer and nuclear cloning, oocytes must be generally matured in vitro before these cells can be used as donor cells for nuclear transfer, and before they can be fertilized by the cell. sperm to develop into an embryo. This process generally requires the collection of immature oocytes (prophase I) from ovaries of mammals, for example, ovaries from bovines obtained in a slaughterhouse, and ripening the oocytes in a medium maturation prior to fertilization or enucleation until the oocyte completes the stage metaphase II, which in the case of bovine oocytes, usually occurs around 18-24 hours after aspiration. For the purposes of the present invention, this period is known as the "ripening period". As used herein to calculate times, "aspiration" refers to the aspiration of the immature oocyte of ovarian follicles. Additionally, oocytes from the metaphase stage I I, which have been matured in vivo, have been used successfully in n uclear transfer techniques. Essentially, mature metaphase I I oocytes are surgically collected from either non-superovulated or superovulated cows or heifers from 35 to 48 hours after the onset of estrus or after injection of human cryonic ganadotropin (hCG) or similar hormones.

It has been reported that the maturation stage of the oocyte in nuclear enucleation and transfer is important for the success of TN methods. (see for example, Prather et al., Differentiation, 48, 1-8, 1991). In general, successful mammalian embryo cloning practices use the metaphase II stage of oocytes since it is thought that the donor oocyte at this stage may be, or is, sufficiently "activated" to treat the introduced core as It is formed in a fertilization sperm. In domestic animals, and especially cattle, the period of activation of the oocyte generally varies from about 16-52 hours, preferably around 28-42 hours after aspiration. For example, immature oocytes can be washed in hamster embryo culture media equilibrated in pH with H EPES (H ECM) 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 10% fetal calf serum in which it contains appropriate ganadotropins such as luteinizing hormone (LH) and follicle-stimulating hormone (FSH), an estradiol under a paraffin or low-weight silicon layer at 39 ° C. After a fixed period of maturation, which varies from about 10 to 40 hours, and preferably around 16-18 hours, the oocytes will be enucleated. Prior to oocyte enucleation it will preferably be removed and placed in HEMC containing 1 milligram per milliliter of hyaluronanidase prior to removal of the cell cluster. This can be done by repeated pipetting through very fine orifice pipettes or by briefly stirring. The separated oocytes are then screened by polar bodies, and the selected metaphase II oocytes, as determined by the presence of polar bodies, are then used for nuclear transfer. The enucleation continues. Enucleation can be affected by known methods, such as are described in U.S. Patent No. 4,994,384 which is incorporated by reference herein. For example, metaphase II oocytes are placed in some way in HECM, optionally containing 7.5 micrograms per milliliter of cytochalasin B, by immediate enucleation, or they can be placed in a suitable medium, for example an embryo culture medium such as CR1aa. , plus 10% cow serum in estrus, and subsequently enucleated, preferably no more than 24 hours later, and more preferably 16-18 hours later. Enucleation can be completed microsurgically using a micropipette to remove the adjacent polar body and cytoplasm. The oocytes can then be screened to identify those of which they have been successfully enucleated. The screening can be done by staining the oocytes with a microgram per milliliter 33342 Hoechst inked in HECM, and then observing the oocytes under ultraviolet irradiation for less than 10 seconds. Oocytes that have been successfully enucleated can then be placed in a suitable culture medium, eg, CR1 aa plus 10% serum. In the present invention, the donor 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 24 hours after the start of the maturation in vitro, and more preferably around 16-18 hours after the initiation of in vitro maturation. A single mammalian cell of the same species as the enucleated oocyte will then be transferred into a perivitelline space of the enucleated oocyte used to produce the TN unit. The mammalian cell and the enucleated oocytes will be used to produce TN units according to methods known in the art. For example, the cells can be fused by electrofusion. The electrofusion is completed by providing an impulse of electricity that is sufficient to cause a temporary breakdown of the plasma membrane. This breaking of the plasma membrane is very short because the membrane is easily reformed. Therefore, if two adjacent membranes are induced to break and as the intermixed lipid bilayers reform, small channels will open between the two cells. Due to the thermodynamic instability of said small opening, it lengthens until the two cells become one. Reference is made to the patent of US Pat. No. 4,997,384 to Prather et al. (Incorporated herein by reference in its entirety) by further discussion of this process. A variety of average electrofusion can be used including, for example, sucrose, mannitol, sorbitol and phosphate buffer. Fusion can also be completed using Sendai virus as a fusogenic agent (Graham, Wister Inot, Symp, Monogr., 9, 19, 1969). Also, in some cases (for example with small donor nucleus) it might 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 by reference completely herein. Preferably, the mammalian cell and the oocyte are electrofused in a 500 μm chamber containing fusion medium (o.25 M D-sorbitol, 100 μm calcium acetate, 0.5 mM magnesium acetate, 1.0 g / l BSA (free fatty acid), pH 7.2) by the application of an electric impulse of 90-120V for about 15 μsec, about 24 hours after the initiation of oocyte maturation. After fusion, the resulting fused TN units are then placed in a suitable medium until activation, for example, usually less than 24 hours later, and preferably about 2-9 hours later.

The TN unit can be activated by known methods. Such methods include, for example, culture of the TN unit at sub-physiological temperature, essentially by applying a cold or really cooled temperature shock to the TN unit. This can be done more conveniently by the TN unit culture 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 the application of known activation agents. For example, the penetration of oocytes by sperm during fertilization has been shown to activate oocyte pre-fusion to produce larger numbers of viable pregnancies and multiple calves identical genetically after nuclear transfer. Also, treatments such as electrical and chemical shock can be used to activate TN embryos after fusion. Suitable oocyte activation methods are the subject of U.S. Patent No. 5,496,720, to Susko-Parrish et al., Incorporated herein by reference in its entirety. Additionally, the activation can be carried out simultaneously or sequentially by: (i) the increase of the levels of divalent cations in the oocyte, and (ii) the reduction of phosphorylation of cellular proteins in the oocyte.

This will generally be accomplished by introducing divalent cations into the cytoplasm of the oocyte, for example, magnesium, strontium, barium or calcium, for example, in the form of an ionophore. Other methods of increasing divalent cation levels include the use of electric shocks, treatment with ethanol and treatment with enclosed 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-dimethylaminopurine, staurophorin, 2-aminopurine, and sphingosine. Alternatively, phosphorylation of cellular proteins can be inhibited by the introduction of a phosphatase into an oocyte, for example, phosphatase 2A and phosphatase 2B. In one embodiment, TN activation is effected by brief exposure of the TN unit fused to a TL-HEPES medium containing 5 μM of ionomycin and 1 mg / ml of BSA, followed by washing in TL-HEPES containing 30 mg / ml of BSA within about 24 hours after the fusion, and preferably about 2 to 9 hours after the fusion. Activated TN units can then be cultured in a suitable in vitro culture medium until the generation of CICM cells and cell colonies. The culture medium suitable for the culture and maturation of embryos are well known in the art. Examples of known media, which can be used for bovine embryo cultures and maintenance, include Hams's F-10 + 10% fetal calf serum (FCS), culture tissue medium-199 ( MCT-199) + 10% fetal calf serum, thyroid-albumin-lactate-pyruvate (TALP), Dulbecco's phosphate pH-regulating saline (PBS), Eagle's and Whitten's medium. One of the most common means used by the collection and maturation of oocytes is TCM-199, and 1 to 20% of serum supplement that includes fetal calf serum, newborn serum, estrual cow serum, sheep serum or whey. A preferred maintenance medium includes TCM-199 with Earl salts, 10% fetal calf serum, 0.2 mN Na pyruvate and 50 μg / ml gentamicin sulfate. Any of the foregoing may 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. 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 1.0 mM to 10 mM, preferably from 1.0 mM to 5.0 mM. The L-lactate of hemicalcio is L-lactate with a salt of hemicalcio incorporated in it. Hemicalcium L-lactate is important in that a single component satisfies two main requirements in the culture medium: (i) the calcium requirement necessary for the compaction and disposition of the cytoskeleton; and (ii) the requirement of lactate necessary for the metabolism and transport of electrons. The L-lactate of hemicalcio also serves as a valuable mineral and source of energy for the necessary medium for the embryos' viability. 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, i.e., which comprises animal cells such as oviduct cells. The biological environment can sometimes be of disadvantage in that they may contain microorganisms or trace factors which may be harmful to the embryos and which are difficult to detect, characterize and eliminate. Examples of the main components in the CR1 medium include hemicastium L-lactate, sodium chloride, potassium chloride, sodium bicarbonate and a lower amount of bovine serum albumin-free fatty acid (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 CR1 medium preferably contains the following components in the following amounts: Sodium chloride -114.7 mM Potassium Chloride -3.1 mM Sodium Bicarbonate -26.2 mM Hemicathium L-lactate -5 mM Free fatty acid BSA -3 mg / ml In one embodiment, the activated TN embryo units are placed in the CR1 aa medium containing 1.9 mM DMAP for about 4 hours followed by a wash in HECM and then cultured in CR1 aa containing BSA For example, the activated TN units can be transferred to a CR1 aa culture medium containing 2.0 mM DMAP (Sigma) and cultivated under ambient conditions, for example, at about 38.5 ° C, 5% CO2 for a suitable time, for example, about 4 to 5 hours.Then, the unit or units of TN grown are preferably washed and discarded. They are placed in a suitable medium, for example, CR1 aa medium containing 10% FCS and 6 mg / ml contained in well plates which preferably contain a suitable confluent feeder layer. Suitable feeder layers, for example, fibroblast and epithelial cells, e.g., fibroblast and uterine epithelial cells derived from ungulates, chicken fibroblasts, murine fibroblasts (e.g., mouse or rat), STO feeder cell lines and Sl-m220, 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 experience of ordinary experts.

TN units are grown on the feeder layer (5 x 105 cells / ml) until the TN units reach a suitable size to be transferred to a female donor, or to obtain cells which can be used to produce CICM cells or colonies of cells. Preferably, these NT units will be cultured to at least about 2 to 40 cells, more preferably about 40 to 128 cells, and even more preferably at least about 50 cells. The culture will be carried out under suitable conditions, that is, around 38.5 ° C and 5% CO2, with the culture medium changed in order to optimize the growth around every 2-5 days, preferably around every 3 days. The methods of embryo transfer and handling of donor animals in the present invention are standard procedures used in the embryo transfer industry. Synchronous transfers are important for the success of the present invention, ie the stage of the TN embryo is in synchrony with the estrous cycle of the female donor. This advantage and the way to maintain the donors was reviewed in Siedel, GE, Jr. (Critical review of embryo transfer procedures with cattie "in Fertilization and Embryonic Development in Vitro (1981) L. Mastroianni, Jr. and JD Biggers, ed. , Plenum Press, New York, NY, page 323), the contents of which are incorporated herein by reference.

By the present invention, the cloning efficiency using donor nuclei of adult cells can be the same as when using fetal cellulars nucleus. For example, the development efficiency for embryos of the morula and blastocyst stage is the same as whether cell nuclei of fetal or adult cows are used or not. The present invention can also be used to clone genetically treated genetically or transgenic mammals. As explained above, the present invention is advantageous in such transgenic procedures can be simplified by working with a differentiated cell source that can be propagated clonally. In particular, the differentiated cells used by donor nuclei have a desired DNA sequence inserted, removed or modified. These differentiated cells, genetically altered, are then used for nuclear transplantation with enucleated oocytes. Any known method for inserting, deleting or modifying a DNA sequence of a mammalian cell can be used to alter the differentiated cell in order to be used as the nuclear donor. These procedures can remove all or parts of a DNA sequence, and the DNA sequence can be heterologous. The technique of homologous recombination is included, which allows the insertion, deletion or modification of a DNA sequence or sequences at a specific site or sites in the genome of the cell.

The present invention can therefore be used to provide adult mammals with desired genotypes. The multiplication of adult ungulates with provided genetic superiority or other desirable characteristics is particularly useful, including transgenic or genetically treated animals and chimeric animals. In addition, the cells and tissues of TN fetuses, including chimeric and / or transgenic fetuses, can be used in the transplantation of cells, tissues and organs for the treatment of numerous diseases as described below in connection with the use of cells of CICM. For the production of cell lines and CICM cells, after TN units of the desired size are obtained, the cells are mechanically removed from the area and then used. This is preferably done by taking the group of cells which comprises the TN unit, which will usually contain at least about 50 cells, washing such cells, and plating the cells on a feeder layer, eg, fibroblast cells. irradiated Usually, the cells used to obtain the cells or colonies of stagnant cells will be obtained from the innermost portion of the cultivated TN unit which is preferably at least 50 cells in size, however, the TN units of the cell numbers more small or larger as well as cells from other portions of the TN unit can also be used to obtain ES cells and cell colonies. Cells are maintained in the feeder layer in a suitable growth medium, eg, alpha MEM supplement 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, for example, about every 2-3 days. This culture process results in the formation of CICM cells or cell lines. One skilled in the art can vary the culture conditions as desired to optimize the growth of CICM cells in particular. Also, CICM cells of genetically treated 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 DNA sequence or sequences have been introduced, or from which all or part of an endogenous DNA sequence or sequences have been removed or modified. These genetically treated or transgenic TN units can then be used to produce genetically or transgenic CICM cells, including human cells.

The resulting CICM cells and cell lines, preferably cells and human CICM cell lines, have numerous therapeutic and diagnostic applications. More especially, such CICM cells can be used for cell transplantation therapies. The human CICM cells have application in the treatment of numerous disease conditions. The human TN units per se can also be used in the treatment of disease conditions. In this regard, it is known that mouse embryonic (ES) support cells are capable of differentiating into almost any cell type, e.g., hematopoietic support cells. Therefore, the human CICM cells produced according to the invention should possess similar differentiation capacity. The CICM cells according to the invention will be induced to differentiate in order to obtain the desired cell types according to known methods. For example, individual human CICM cells can be induced to differentiate into hematopoietic support cells, nerve cells, muscle cells, cardiac muscle cells, liver cells, cartilage cells, epithelial cells, urinary tract cells, etc., cultivating such cells in a differentiation medium and under conditions which are provided for cell differentiation. The means and methods which result in the differentiation of CICM cells are known in the art as such under suitable culture conditions. For example, palaces and others, Proc. Nati Acad. Sci., E.U.A., 92: 7530-7537 (1995) teaches the production of hematopoietic support cells from an embryonic cell line by subjecting the support cells to an induction procedure initially comprising the aggregate culture of such cells in a suspension culture medium that it lacks retinoic acid followed by culture in the same medium containing retinoic acid, followed by the transfer of aggregated cells to a substrate which is provided for cell attachment. In addition, Pedersen, J. Reprod. Fertile. Dev., 6: 543-552 (1994) is a review of article which refers to numerous articles describing methods by in vitro differentiation of embryonic support cells to produce various types of differentiated cells including hematopoietic, muscle cells, cardiac muscle, nerve cells, among others. In addition, Brain et al., Dev. Biol., 168: 342-357 (1995) teaches the in vitro differentiation of embryonic support cells to produce neural cells which possess neuronal properties. These references are examples of reported methods to obtain differentiated cells of embryonic or support cells. These references, and in particular the descriptions herein, refer to methods for differentiating embryonic support cells are hereby incorporated by reference in their entirety. Therefore, using known methods and culture media, one skilled in the art can cultivate the CICM cells present, which include genetically treated or transgenic cells, to obtain desired differentiated cell types, e.g., neural cells, muscle cells, hematopoietic cells, etc. The CICM cells present can be used to obtain any type of differentiated cell. The therapeutic uses of such differentiated human cells are not parallel. For example, human hematopoietic support cells can be used in medical treatments that require bone marrow transplantation. Such methods are used to treat many diseases, for example, advanced stage cancers such as ovarian cancer and leukemia, as well as diseases comprising the immune system, such as AIDS. Hematopoietic support cells can be obtained, for example, by fusing adult somatic cells from a cancer or AIDS patient, for example, epithelial cells or lymphocytes with an enucleated oocyte, which obtain CICM cells as described above, and culturing such cells under conditions which taste differentiation, to hematopoietic support cells are obtained. Such hematopoietic cells can be used in the treatment of diseases including cancer and AIDS. Alternatively, the adult somatic cells of a patient with a neurological disorder can be fused with an enucleated oocyte, the human CICM 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, cerebral palsy and ALS, among others. In the specific case of Parkinson's disease, it has been shown that transplanted fetal brain neural cells make their own connections with surrounding cells and produce dopamine. This can result in long-term reversal of the symptoms of Parkinson's disease. The great advantage of the subject invention is that it provides an essentially limited supply of isogenic or syngeneic human cells suitable for transplantation. Therefore, it will be obvious that the significant problem associated with common transplant methods, that is, the rejection of the transplanted tissue which may occur due to host rejection against graft or anti-host graft. Conventionally, rejection is prevented or reduced by the administration of anti-rejection drugs such as cyclosporin. However, such drugs have adverse side effects, that is, immunosuppression, carcinogenic properties, as well as being very expensive. The present invention should eliminate, or at least reduce to a greater extent, the need for drugs against rejection. Other diseases and conditions treatable by isogenic cell therapy, for example, spinal column lesions, multiple sclerosis, muscular dystrophy, diabetes, liver diseases, i.e. hypercholesterolemia, heart disease, cartilage replacement, burns, ulcers foot, gastrointestinal diseases, vascular diseases, kidney diseases, urinary tract diseases, and diseases and conditions related to aging.

This methodology can be used to replace defective genes, for example, genes of the defective immune system, genes of cystic fibrosis, or to introduce genes which result in the expression of therapeutically beneficial proteins such as, growth factors, lymphokines, cytichines, enzymes , etc. For example, the DNA sequence encoding the brain derived from the growth factor in neural cells and the cells transplanted in a Parkinson's patient to retard the loss of neural cells during such a disease. Previously, the cell types transfected with BDNF varied from primary cells to immortalized cell lines, either neural or non-neural derived cells (myoplasts and fibroblasts). For example, astrocytes have been transfected with BDNF genes using retroviral vectors, and cells grafted into a rat model of Parkinson's disease (Yoshimoto et al., Brain Research, 691: 25-36, (1995)): reduced live of similar symptoms of Parkinson's in rats above 45% 32 days after transfer. Also, the tyrosine hydroxylase gene has been located in astrocytes with similar results (Lunderberg et al., Develop Neurol., 139: 39-53 (1996) and references cited therein). However, such ex vivo systems have problems. In particular, the commonly used retroviral vectors are down regulated in vivo and the transgene is only expressed temporarily (reviewed by Mulligan, Science, 260: 926-932 (1993)). Also, such studies used cells, astrocytes, which have a finite life extension and replicate slowly. Such properties adversely affect the transfection regime and prevent the selection of stably transfected cells. In addition, it is almost impossible to propagate a long population of bleached primary cells of genes for use in homologous recombination techniques. In contrast, the difficulties associated with retroviral systems could be eliminated by the use of cloning of mammals using differentiated cells and CICM cells. The DNA sequences which can be introduced into the differentiated or CICM present cells include, as an example, those which encode the epidermal growth factor, insulin-like growth factor (I and II), neurotrofin-3, neurotrophin. -4/5, ciliary neurotrophic factor, AFT-1, cytokines (interleukins, interferons, colony stimulation factors, tumor necrosis factors (alpha, and beta), etc.), therapeutic enzymes, etc. In addition to the use of TN units and CICM cells in cell, tissue and organ transplants, the present invention also includes the use of non-human cells in the treatment of human diseases. Therefore, CICM cells, fetuses and progeny of chimeric TN and TN (transgenic or non-transgenic) of any species can be used in the treatment of conditions of human diseases where the transplantation of cells, tissues and organs is guaranteed. In general, the CICM cell, fetuses and progeny according to the present invention can be used within the same species (autologous, syngeneic or allografts) or crossed species (xenografts). For example, brain cells from bovine TN fetuses can be used to treat Parkinson's diseases. Also the CICM cells present, preferably human cells, can be used as an in vitro model of differentiation, in particular for the study of genes which are involved in the regulation of near development. Also, the differentiated cells, tissues and organs using the CICM cells present can be used in drug studies. In addition, the CICM cells present can be used as nuclear donors for the production of other CICM cells and cell colonies. In order to more clearly describe the purpose of the invention, the following examples are provided. EXAMPLE 1 Isolation of primary cultures of adult and bovine embryonic bovine and porcine fibroblast cells The primary cultures of bovine and porcine fibroblasts were obtained from fetuses (45 days of pregnancy for cattle and 35 days for pig fetuses). The head, liver, heart and feeding tract were removed aseptically, the fetuses were crushed and incubated for 30 minutes at 37 ° C in an EDTA solution of trypsin (0.05% trypsin / 0.02% EDTA; GIBCO, Grand Island, NY). The fibroblast cells were plated in tissue culture dishes and grown in alpha-MEM medium (BioWhittaker, Walkersville, MD) supplemented with 10% fetal calf serum (CBF) (Hiclon, Logen, UT), penicillin. (100 lU / ml) and streptomycin (50 μl / ml). The fibroblasts were developed and maintained in a humidified atmosphere with 5% CO2 in air at 37 ° C. The cells were passed regularly upon reaching the confluence. The adult fibroblast cells were isolated from the lung and skin of a cow (approximately five years old). The crushed lung tissue was incubated overnight at 10 ° C in trypsin ADTA solution (0.05% trypsin / 0.02% EDTA, GIBCO, Grand Island, NY). The next day the tissue and any disassociated cells were incubated for one hour at 37 ° C in prewarmed trypsin EDTA solution (0.05% trypsin / 0.02% EDTA, GIBCO, Grand Island, NY) and processes through three washes consecutive and trypsin incubations (one hour). The fibroblast cells were plated in tissue culture dishes and cultured in alpha-MEM medium (BioWhittaker, Walkersville, MD) supplemented with developing time, which varies from approximately post-embryonic disk stage through to adult life. animal (bovine day 12 to 15 after fertilization up to 10 to 15 years of animal age). This method can also be used to isolate fibroblasts from other mammals, including mice.

Introduction of a marker gene (foreign heterologous DNA) into embryonic and adult fibroblast cells The following electroporation procedure was carried out for both embryonic (cattle and pigs) and adult (cattle) fibroblast cells. Normal microinjection procedures can also be used to introduce heterologous DNA into fibroblast cells, however, in this example electroporation was used because it is an easier procedure. Culture plates containing propagating fibroblast cells were incubated in trypsin EDTA solution (0.05% / 0.02% EDTA; GIBCO, Grand Island, NY) until the cells were in a single cell suspension. Cells were turned down at 500 x g and re-suspended at 5 million cells per ml with phosphate buffer (SRpHF). The reporter gene constructs the cytomegalovirus promoter and beta-galactosidase, the neomycin phosphoransferase fusion gene (beta-GEO). The reporter gene and the cells at 50 μm / ml final concentration were added to the electroporation chamber. After the electroporation pulse, the fibroblast cells were transferred back into the growth medium (alpha-MEM medium (BioWhittaker, Walkersville, MD) supplemented with 10% fetal calf serum (SBF) (Hyclone, logen, UT) , penicillin (100 lU / 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 extension of life of the cultured cells). This drug kills any cell without the beta-GEO gene, while 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 fibroblast cells were stained with X-gal to observe the expression of beta-galactosidase, and confirmed positive to integrate using PCR amplification of the beta-GEO gene and run on an agarose gel. Use of transgenic fibroblast cells in nuclear transfer procedures to create CICM cell lines and transgenic fetuses. A cell line (CL-1) derived from a colony of bovine embryonic fibroblast cells was used as the donor nucleus in the nuclear transfer (TN) process. The general TN procedures are described above. The slaughterhouse oocytes were matured in vitro. The oocytes were dislodged from cell clusters and enucleated with a bevelled micropipette in approximately 18 to 20 hours post-maturation (hpm). Enucleation was confirmed in the medium of TL-HEPES plus Hoechst 33342 (3 μg / ml, Sigma). The individual donor cells (fibroblasts) were then placed in the perivitelline space of the donor oocyte. The cytoplasm of the bovine oocyte and the donor nucleus (TN unit) were fused together using electrofusion techniques. A fusion pulse containing 120 V per 15 μsec in a 500 μm aperture chamber with melting medium was applied to the TN unit. This happened at 24 hpm. The TN units were placed in CR1 aa medium until 26 to 27 hpm. The general procedure used to artificially activate oocytes has been described above. Activation of the TN unit started between 26 and 27 hpm. Briefly, TN units were exposed for four min. to onomycin (5 μM; CalBiochem, La Jolla, CA) in TL-HEPES supplemented with 1 mg / ml BSA and then washed for five min. in TL-HEPES supplemented with 30 mg / ml BSA. Through the ionomycin treatment, the TN units were also exposed to 2mM of DMAP (sigma). Following washing, the units were then transferred to a microdrop of CR1 aa culture medium containing 2 mM of DMAP (Sigma) and cultivated at 38.5 ° C 5% CO2 by four to five hours. The embryos were washed and then crushed in CR1 medium to plus 10% FCS and 6 mg / ml BSA in four well plates containing a confluent feeder layer of mouse embryonic fibroblasts. 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 days 5 and 8 min. after activation. At this time TN embryos of blastocyst stage can be used to produce transgenic CICM (cultured internal cell mass) cell lines or fetuses. The internal cell mass of these TN units can be isolated and plated on a feeder layer. Also the N units were transferred in female donors. The pregnancies were aborted between 35 and 48 days of gestation. This resulted in seven cloned transgenic fetuses having the beta-GEO gene in all the tissues reviewed. Six of the seven embryos had a normal heartbeat detected via ultrasound observation. As well, histological sections of fetuses shown without anomalies. Therefore, this is a quick and easy method to make transgenic CICM and fetus cell lines. This procedure is generally conductive for bleached CICM cell line genes and fetuses. The following table summarizes the results of these experiments. or * 19 lines were positive for beta-G EO, 2 were negative and one line died before PCR detection. One fetus died and another was slightly retarded in development in 35 days of gestation. Five fetuses recovered between 38 to 45 days were normal. All the fetuses were confirmed as transgenic. f First progeny to be born in October, 1997.

EXAMPLE 2 Fetuses and chimeric progeny derived from transgenic CICM cells. The transgenic CICM cell line was originally derived from a transgenic TN unit (differentiated cell). A CICM line 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 cells were either disaggregated using 1-5 mg / ml pronase or 0.05% trypsin / EDTA combined with mechanical disaggregation methods such that groups of five or less cells were produced. The activity of trypsin or pronase was deactivated by passing the cells through multiple washes of 30 to 100% fetal calf serum. The disaggregated cells were placed in micromanipulation plates containing TL-HEPES media. Fertilized embryos were also placed on these plates and micromanipulation tools were used to produce the chimeric embryos, Eight to ten transgenic CICM cells were injected into 8-16 stage cells of fertilized embryos. These embryos were cultured in vitro at the blastocyst stage and then transferred into animal donors. A total of 6 chimeric blastocyst stage embryos were not surgically transferred into female donors. After 5 weeks of gestation 3 fetuses recovered. Various tissues of the three fetuses, including germ cells of the gonad (suggesting chimeras of germination lines), where they were screened by PCR amplification and southern blot hybridization of the amplified product to a fragment of beta-galactosidase. Of the three fetuses, two were positive for the contribution of the transgenic CICM cells. Both fetuses had a contribution of transgenic CICM to the gonad. Ten chimeric embryos were allowed to go to term, and 7 of the 10 were developed to produce a progeny. Ear cuts were taken and DNA was isolated from each calf. When amplifying PCR, one of the seven was confirmed as a transgenic chimeric progeny. Transgenic TN embryos derived from transgenic CICM cell lines. The transgenic CIC cell line was originally derived from a transgenic TN unit (differentiated cell). Transgenic CICM cell lines were used to produce TN embryos. The TN procedures described in Example 1 were used except that the CICM cells instead of fibroblast cells were used as a donor cell fused with the enucleated oocyte. Colonies of transgenic CICM cells were separated in any way using 1-5 mg / ml pronase or 0.05% trypsin / EDTA combined with mechanical separation methods such that groups of five or a few fewer cells were produced. The trypsin or pronase activity was deactivated by passing the cells through multiple 30 to 1055 washes of fetal calf serum before transferring the cells into enucleated oocytes. The results are reported in Table 1 (third group). Five embryos were produced in blastocyst stage.

Claims

CLAIMS 1. A method of cloning a mammal, comprising: (i) inserting a cell or cell nucleus of differentiated mammals, suffering from the lack of serum in an enucleated mammalian oocyte of the same species as the cell or cell nucleus Differentiated or cellular nucleus, under conditions suitable for the formation of a nuclear transfer unit (TN); (I) activate the resulting nuclear transfer unit; and (iii) transferring said cultivated transfer unit to a host mammal in such a way that the TN unit develops into a fetus.
2. The method according to claim 1, which further comprises the development of a fetus to a progeny.
3. The method according to claim 1, wherein the desired DNA is inserted, removed or modified in said differentiated mammalian cell or cell nucleus, thereby resulting in the production of a genetically altered TN unit.
4. The method according to claim 3, which further comprises the development of the fetus in a progeny.
The method according to claim 1, wherein the cell or nucleus of the differentiated mammalian cell is derived from a cover of the mesoderm.
6. The method according to claim 1, wherein the differentiated mammalian cell or cell nucleus is derived from a cover of the ectoderm.
The method according to claim 1, wherein the differentiated mammalian cell or cell nucleus is derived from the endoderm envelope.
The method according to claim 1, wherein the differentiated mammalian cell or cell nucleus is a fibroblast cell or cell nucleus.
The method according to claim 1, wherein the mammalian cell or cell nucleus is an ungulate.
The method according to claim 9, wherein the ungulate is selected from the group consisting of bovine, ovine, porcine, equine, goat and buffalo.
11. The method according to claim 1, wherein the differentiated mammalian cell or cell nucleus is an adult cell or cell nucleus.
The method of claim 1, wherein the differentiated mammalian cell or cell nucleus is an embryonic or fetal cell or cell nucleus.
The method according to claim 1, wherein the enucleated oocyte matures prior to enucleation.
The method according to claim 1, wherein the fused nuclear transfer unit is activated by exposure to ionomycin and 6-dimethylaminopurine.
15. The method according to claim 3, wherein the microinjection is used to insert a heterologous DNA.
16. The method according to claim 3, wherein the electroporation is used to insert a heterologous DNA.
17. A fetus obtained according to the method of claim 1.
18. A progeny obtained according to the method of claim 2.
19. Progeny of the progeny according to claim 18.
20. A transgenic fetus obtained from according to the method of claim 3.
21. A transgenic progeny obtained according to the method of claim 4.
22. A progeny of the progeny according to claim 21.
23. The method according to claim 1, which also comprises the combination of the cloned TN unit with a fertilized embryo to produce a chimeric embryo.
24. The method according to claim 23, which further comprises the development of the fetus to a progeny.
25. A fetus obtained according to the method of claim 23.
26. A progeny obtained according to the method of claim 24.
27. Progeny of the mammal according to claim 26.
28. The method according to claim 1, wherein said activated nuclear transfer unit is grown to become greater than 2 cells in the development stage.
29. A method of producing CICM cell lines, comprising: (i) inserting a differentiated mammalian cell or cell nucleus, which suffers from the lack of serum in an enucleated mammalian oocyte of the same species as the cell or cell nucleus differentiated, under conditions suitable for the formation of a nuclear transfer unit (TN); (ii) activate the resulting nuclear transfer unit; and (ii) cultivating cells obtained from said cultivated TN unit to obtain a CICM cell line.
30. The method according to claim 20, wherein said activated nuclear transfer unit is grown to be greater than 2 cells in the developmental stage.
31. A line of CICM cells obtained according to the method of claim 29.
32. The method according to claim 29, wherein a desired DNA is inserted, removed or modified in said mammalian cell or cell nucleus. differentiated, which results in the production of a genetically altered TN unit.
33. A transgenic CICM cell line obtained according to claim 32.
34. The method of claim 29, wherein the resulting CICM cell line is induced to differentiate.
35. The differentiated cells obtained by the method of claim 34.
36. The human differentiated cells obtained according to claim 34.
37. A method of therapy which comprises administration to a patient in need of a transplant therapy. isogenic differentiated cell cells according to claim 36.
38. The method of claim 37, wherein said 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 injuries. , multiple sclerosis, muscular dystrophy, cystic fibrosis, liver disease, diabetes, heart disease, cartilage defects or injuries, burns, foot ulcers, vascular diseases, urinary tract diseases, AIDS and cancer.
39. A method of therapy which comprises administering to a human patient the need for xenogeneic differentiated cell cell transplantation therapy according to claim 35.
40. The method according to claim 39 wherein the xenogeneic differentiated cells are bovine cells.
41. The method of claim 39, wherein said 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 the spine, multiple sclerosis, muscular dystrophy, cystic fibrosis, liver disease, diabetes, heart disease, cartilage defects or injuries, burns, foot ulcers, vascular diseases, urinary tract diseases, AIDS and cancer.
42. The method of claim 37, wherein the differentiated human cells are hematopoietic cells or neural cells.
43. The method of claim 37, wherein the therapy is for treatment of Parkinson's disease and the differentiated cells are neural cells.
44. A method of claim 37 wherein the therapy is for the treatment of cancer and the differentiated cells are hematopoietic cells.
45. A method of therapy which comprises administering to a human patient the need for cell transplantation therapy of xenogeneic cells obtained from a fetus according to claim 17.
46. The method according to claim 45 wherein the xenogeneic cells are bovine cells.
47. The method of claim 45, wherein said 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 the spine, multiple sclerosis, muscular dystrophy, cystic fibrosis, liver disease, diabetes, heart disease, cartilage defects or injuries, burns, foot ulcers, vascular diseases, urinary tract diseases, AIDS and cancer.
48. The method of claim 46 wherein said cell transplant therapy is performed to treat Parkinson's disease.
49. A method of therapy which comprises the administration to a human patient of cell transplantation therapy of xenogeneic cells obtained from a progeny according to claim 18.
50. The method of claim 49 wherein the xenogeneic cells are cells of cattle.
51. The method of claim 49, wherein said 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 injuries of the spine, multiple sclerosis, muscular dystrophy, cystic fibrosis, liver disease, diabetes, heart disease, cartilage defects or injuries, burns, foot ulcers, vascular diseases, urinary tract diseases, AIDS and cancer .
52. a method of therapy which comprises administering to a human patient the need for cell transplantation therapy of transgenic xenogeneic cells obtained from a transgenic fetus according to claim 20.
53. The method according to claim 52 wherein the xenogeneic transgenic cells are bovine cells.
54. The method of claim 52, wherein said 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 the spine, multiple sclerosis, muscular dystrophy, cystic fibrosis, liver disease, diabetes, heart disease, cartilage defects or injuries, burns, foot ulcers, vascular diseases, urinary tract diseases, AIDS and cancer.
55. The method of claim 53 wherein said cell transplantation therapy is performed to treat Parkinson's disease.
56. A method of therapy which comprises administering to a human patient the need for cell transplantation therapy of transgenic xenogeneic cells obtained from a transgenic progeny according to claim 21.
57. The method according to claim 56 wherein the xenogeneic transgenic cells are bovine cells.
58. The method of claim 56, wherein said 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 the spine, multiple sclerosis, muscular dystrophy, cystic fibrosis, liver disease, diabetes, heart disease, cartilage defects or injuries, burns, foot ulcers, vascular diseases, urinary tract diseases, AIDS and cancer.
59. The method of claim 29, which further comprises the combination of the cloned TN unit with a fertilized embryo to produce a chimera.
60. The method of claim 59, which further comprises the development of chimeric CICM cell lines to a chimeric embryo.
61. A chimeric embryo obtained according to claim 60.
62. The method of claim 60, which further comprises the development of the chimeric embryo to a chimeric fetus.
63. A chimeric fetus obtained according to claim 62.
64. The method according to claim 62, which further comprises the development of chimeric fetus to a chimeric progeny.
65. A chimeric progeny obtained according to claim 64.
66. The method according to claim 59, wherein a desired DNA is inserted, removed or modified in said cell or mammalian cell nucleus differentiated, so that results in the production of a genetically altered TN unit.
67. The method according to claim 66, which further comprises the development of chimeric CICM cell line to a chimeric embryo.,
68. An embryo obtained according to claim 67.
69. The method according to claim 67, which also includes the development of the chimeric embryo to a chimeric fetus.
70. A chimeric fetus obtained in accordance with the claim 69.
71. The method according to claim 69, which further comprises the development of a chimeric fetus to a chimeric progeny.
72. a chimeric progeny obtained according to claim 71.
73. A method of cloning a mammal, comprising: (i) inserting a different cell or cell nucleus CICM, which suffers from the lack of serum in an enucleated mammalian oocyte. of the same species as the cell or cell nucleus differentiated CICM, under conditions suitable for the formation of a nuclear transfer unit (TN); (ii) activate the resulting nuclear transfer unit; and (iii) transferring said TN unit grown in a host mammal in such a way that the TN unit develops into a fetus.
74. The method according to claim 73, wherein said nuclear transfer unit is grown to be greater than 2- cells in the development stage.
75. The method according to claim 73, which also includes the development of the fetus to a progeny.
76. A fetus obtained according to the method of claim 73.
77. A progeny obtained according to the method of claim 75.
78. An organ for use as an organ xenograft, which is obtained from the progeny of according to claim 18.
79. An organ for use as an organ xenograft, which is obtained from the progeny according to claim 21.
80. an organ to be used as an organ xenograft, which is obtained from the progeny according to claim 26.
81. An organ for use as an organ xenograft, which is obtained from the progeny according to claim 72.
82. An organ for use as an organ xenograft, which obtains from the progeny according to claim 77.
83. The method according to claim 13, wherein the enucleated oocyte is matured in vitro.
84. The method according to claim 1, wherein the activation occurs after the formation of the TN unit.
85. The method according to claim 84, wherein the activation occurs 4 hours or later after the formation of the TN unit.
86. The method according to claim 1, wherein the TN unit is co-cultivated in vitro with helper cells in indefinite medium.
87. A method of producing a pharmaceutically active protein, comprising the isolation of a pharmaceutically active protein which is expressed by a transgenic progeny according to claim 21.