MXPA98008103A - Cellular lines of internal cellular mass cultivated derivatives of embryos ungula - Google Patents

Abstract

Cells and novel cell lines of cultured internal cell mass (MCIC), derived from ungulates, in particular from pigs and cows, and methods for their preparation are provided. The MCICs present have similar morphology and express cell markers identical or substantially similar to MCI from embryos of undifferentiated development during prolonged culture periods. The heterologous DNAs can be introduced into the cells and cell lines present in MCIC to produce transgenic cells useful for the production of transgenic embryos, fetuses and descendants, e.g. by means of nucleation procedures

Description

CELLULAR INTERNAL CELLULAR MINE LINES CULTIVATED DERIVATIVES OF UNGULATED EMBRYOS Field of the Invention Cells, cell lines, of cultured internal cell mass (MCIC) and methods for their preparation are provided. The present MCIC has similar morphology and expresses cell markers identical or highly similar to MCI of developing embryos during prolonged culture periods. These MCICs are produced by novel culture techniques and / or by the introduction of an inhibitory gene for differentiation (ID). The MCIC cell lines present are used to produce cells, tissues, organs and / or complete differentiated animals, preferably ungulates, desirably those that have been genetically modified to contain within their genome a desired heterologous DNA or that have been selected to contain traits genetically convenient. This is achieved by in vitro culture techniques or producing embryos, fetuses and / or chimeric descendants or nuclear transfer. In addition, MCIC cells can also be used to clone (nuclear transfer procedures) to produce embryos, fetuses and / or genetically identical offspring. BACKGROUND OF THE INVENTION Methods for deriving embryonic (SE) cell support lines from early embryonic mouse embryos are well known. (See, for example, Eva ns et al., Nature, 29: 154-156 (1981), Martin, Proc. Nati, Acad. Sci .. USA, 78: 7634-7638 (1981)). SE cells can be passaged in an undifferentiated state, as long as a fibroblast cell feeder layer is present (Evans et al., L) or a source of inhibition of differentiation (Smith et al., Dev. Biol. 121: 1-9 (1987) Previously it has 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 are involved in the regulation of early development Se cells from mice can originate germline chimeras when introduced into preimplantation mouse embryos, thus demonstrating their pluripotency (Bradley et al., Nature, 309: 255-256 (1988) )). In view of their ability to transfer their genomes to the next generation, SE cells have potential utility for the manipulation of germ lines of livestock animals using SE with or without a desired genetic modification. In addition, in the case of livestock animals, v.gr. , ungulates, the nuclei of similar embryos of cattle for pre-implants support the development of enucleated oocytes at term (Smith et al., Biol. Reprod., 40: 1027-1035 (1989); and Keefer et al., Biol. Reprod. : 935-939 (1994)). This is in contrast to the embryonic nuclei of mice that beyond their eighth cellular stage after the transfer have been reported to not support e | development of enucleated oocytes (Cheong et al., Biol. Reprod 48: 958 (1993)). Therefore, SE cells from livestock animals are highly convenient as they can provide a potential source of totipotent, genetically engineered donor nuclei or in some other way for nuclear transfer procedures. Many research groups have reported the isolation of interestingly pluripotent embryonic cell lines. For example, Nortarianni and others, J. Reprod. Fert. Suppl .. 43: 255-260 (1991)), reports the establishment of interestingly 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 internal cell masses isolated immunosurgically from sheep blastocysts. (Id) Also, Nortarianni and others, J. Reprod. Fert. Suppl. 41: 51-56 (1990) describe the maintenance and differentiation in culture of putative pluripotent embryonic cell lines of pig blasts. further, Gerfen et al., Anim. Biotech 6 (1): 1-14 (1994) describes the isolation of embryonic cell lines from porcine blasts. These cells are stably maintained in the layers of the embryonic fibroblast feeder of mice without the use of conditioned medium. It is reported that these cells differentiate into several different cell types during culture (Gerfen et al., Id.) - In addition, Saito et al., Roux's Arch. Dev. Biol., 201: 134-141 (1992), reports lines similar to cultured bovine embryonic support cells which survived three passages, but were lost after the fourth passage. In addition, Handyside et al., Roux's Arch. Dev. Biol. 196: 185-190 (1987), describe the mass culture of internal cells immunosurgically isolated from sheep embryos under conditions that allow the isolation of SE cell lines. mice derived from MCI of mice. Handyside et al., (1987) (Id.), Report that under these conditions, the binding of sheep MCI diffuses and develops areas of cells similar to SE cells, cells similar to the endoderm, but that after prolonged culture only Cells similar to the endoderm are evident. (Id) Recently, Cherny et al., Therioqenology, 41: 175 (1994) reported cell lines derived from primordial germ cells of interestingly pluripotent bovines maintained in long-term cultures. These cells, after about seven days in culture, produce SE-like colonies that stain positive for alkaline phosphatase (FA), exhibit the ability to form embryoid bodies and spontaneously differentiate into at least two different cell types. . It is reported that these cells can express mRNA for the transcription factors OCT4, OCT6 and H ES 1, a pattern of homeotic box genes that are thought to be expressed exclusively by SE cells.

Also recently, Campbell et al., Theriogenology, 43: 181 (1995) in a summary report the production of live lambs after nuclear transfer of embryonic disk (ED) cells from nine-day-old sheep embryos cultured under conditions that promote the isolation of SE cell lines in the mouse. The authors concluded, based on their results, that DE cells from nine-day-old sheep embryos are totipotent by nuclear transfer and that totipotency is maintained in the culture for up to three passages. Even more recently, Campbell et al., Nature, 380: 64-68 (1996) reported the cloning of sheep by nucleic transfer of a cultured cell line. The cells used are not the same as the MCIC of the present invention. Unlike the MCIC cells present, the Campbell and other cells formed a monolayer in tissue culture. The authors refer to these cells as being "flattened" or exhibiting an "epithelial" appearance.In contrast, the MCIC cells of the present invention can be continuously maintained in a colony of multiple layers or portions of the colony as they develop. Also, the Campbell cells and others are cytokeratins and are positive for laminin A / C. In contrast, the MCIC cells of the present invention are cytokeratin negative, and there is no suggestion that the cells Instead, the reference only indicates that the cells are useful in nuclear transfer procedures.These cells are also not cultured under conditions where they maintain constant contact with a fibroblast feeder layer. Instead, the cultured cells (from Campbell et al. (1996)) apparently push the fibroblasts to the side in the culture and become active. They develop on the top of the culture plate. Van Stekelenburg-Hamers et al., Mol. Reprod. Dev. 40: 444-454 (1995), reported the isolation and healing of interestingly permanent cell lines from the internal cell mass cells of bovine blastocysts. The authors isolated and cultured MCIC from bovine blastocysts of 8 or 9 days under different conditions to determine which feeder cells and culture media are more efficient to support the binding and development of cattle MCI cells. They concluded, based on their results, that the binding and growth of cultured MC I cells is increased by the use of STO feeder cells (mouse fibroblast) (instead of epithelial cells of the bovine uterus) and by the use of whey prepared with charcoal (instead 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 (Id). In addition, 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. of 1994, reported the isolation, selection and propagation of animal support cells that can apparently be used to obtain transgenic animals. Also, Evans others, WO 90/03432, published April 5, 1990, reports the derivation of apparently pluripotent embryonic support cells derived from porcine and bovine species that are surely useful for the production of transgenic animals. In addition, Wheeler et al., WO 94/26884, published on November 24, 1994, describes embryonic support cells that are certainly useful for the manufacture of chimeric and transgenic ungulates. Therefore, based on the above, it is evident that one can try to produce many groups of 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 MCI cells for nuclear transplantation has also been reported. For example, Collas et al., Mol. Reprod. Dev .. 37; 264,267 (1994) describe the nuclear transplantation of cattle MCI by microinjection of lysed donor cells in mature enucleated oocytes. The reference describes embryo culture in vitro for seven days to produce fifteen blastocysts that, when transferred to bovine recipients, resulted in four pregnancies and two births. Also Keefer et al., Biol. Reprod. , 50: 935-939 (1994), describes the use of cattle MC I cells as donor nuclei in nuclear transfer procedures, to produce blastocysts, which upon transplantation into bovine receptors, resulted in several live offspring. In addition, Sims and others, Proc. Natl. Acad. Sci .. USA. 90: 6143-6147 (1993), describe the production of calves by transfer of nuclei from MCI cells of bovines cultured in vitro in the short term in enucleated mature oocysts. Also, the production of live lambs after the nuclear transfer of embryonic disc cells cultured in the short term (up to three passages) has been reported (Campbell et al., Theriogenology, 43: 181 (1995)). In addition, the use of bovine pluripotent embryonic cells in nuclear transfer and the production of chimeric fetuses has also been reported (Stice et al., Theriogenology, 41: 301 (1994)). However, regardless of what has been previously reported in the literature, there is still a significant need for cultured MCI cells and cell lines that have improved properties, e.g. , which have morphological properties and express identical cell markers substantially similar to MCI cells to develop embryos, in particular, ungulate embryos. In addition, there is a significant need in the art for methods to produce said cultured MC I cells and cell lines. OBJECTIVES OF THE INVENTION Therefore, it is an object of the invention to provide novel and improved cells or cell lines of internal or cultured cell mass (MCI). It is a more specific objective of the invention to provide improved and novel cultured and cultured MCI cell lines and cells that exhibit morphological characteristics and express cellular markers identical or substantially similar to those of embryonic MCIs in development. It is still a more specific objective of the invention to provide novel and improved ungulate cultured MC I cells from cell lines that exhibit morphological characteristics and express cellular markers identical or substantially similar to MC I of ungulate embryos in development. It is yet another specific objective of the invention to provide improved cell lines of cultured MC I, preferably derived from ungulates, which exhibit morphological characteristics and which express cell markers identical or substantially similar to MC I to develop ungulate embryos, during prolonged culture times. It is still another specific objective of the invention to provide novel methods for the isolation and / or production of said cells and cultured MCI cell lines or improved nguladas. It is still a specific object of the invention to provide novel methods for the isolation and / or production of cells from cultured ungulate MC I cell lines exhibiting morphological characteristics and expressing cellular markers identical or substantially similar to embryo MCIs. ungulates in development, preferably during long periods of cultivation. It is a specific object of the invention to provide a novel method for culturing cells or cell lines of MCI that exhibit morphological characteristics and express cell markers identical or substantially similar to the MCI of ungulate embryos in development, which method comprises: (i) obtaining an MCI of a blastocyst or MCI progenitor cells of embryos in the preblastocyst stage by mechanical and / or enzymatic means; (ii) culturing said MCI in a feeder layer culture, preferably fibroblasts; and (iii) identifying from among other cultured cells those that exhibit the following properties: (a) small cytoplasmic / nuclear volume ratio; (b) cytoplasmic vesicles; and (c) small individual cells; (iv) separating cells having said properties from the remaining cultured cells; and (v) passing the separated cells in a feeder layer, preferably fibroblasts, under conditions such that the separated cells are in direct physical contact with the feeder layer.

It is a more specific objective of the invention to provide MC! cultivated improved by the following steps: (i) obtain an ICM of blastocyst stage embryos, preferably ungulates, by suitable mechanical and / or enzymatic means; (ii) culturing said MCI obtained in a monolayer of feeder cells, preferably coarse, confluent, preferably comprised of fibroblast cells; (iii) culturing said MCI under conditions that result in a multilayer cell colony that includes a substantially internal population of flattened epithelium-like cells and a second population in multiple layers of cells substantially surrounding said epithelial-like cells and include relatively small cells that have cytoplasmic vesicles and small relationships of cytoplasmic / nuclear volume; (iv) separating said second population into multiple layers of cells that are substantially comprised in the perimeter of the multilayer cell colony by suitable non-degradable means, i.e., mechanical and / or enzymatic means; and (v) passing the separated cells in a new feeder layer preferably comprised of fibroblasts under conditions such that the separated cells are in direct physical contact with the aliquot layer.

It is another object of the invention to provide cultured MCIs that exhibit morphological characteristics and that express cellular markers identical or substantially similar to the MCI of developing ungulate embryos by a method comprising: (i) obtaining ungulated MCI cells or an MCI cell line established ungulate; (ii) introducing into the core of the MCI cells or the cultured MCI cell line established in one or more genes that inhibit differentiation, (ID genes) whose DI gene or DI genes are preferably expressed under the control of a inducible promoter; (iii) culturing the resulting transgenic MCI cells or cultured MCI cell line on a feeder layer. preferably a confluent fibroblast layer, under conditions that provide for the expression of said gene or genes that inhibit differentiation. It is also an object of the invention to use MCI cells or cell lines that express one or more ID genes in the culture method described above. It is another specific objective of the invention to use the improved cultured MCIs present that exhibit morphological characteristics and express cellular markers identical or substantially similar to MCI of ungulate embryos in development for any use where the cultured MCI or MCI have applicability. Such uses include, eg, the production of differentiated cells, tissues, organs and / or whole animals by in vitro cell culture techniques or the production of chimeric or nuclear transfer embryos that may or may not be transgenic. It is a specific objective of the invention to provide cells of Cultured MCIs that can be used in cloning (nuclear transfer procedures) to produce genetically identical embryos, fetuses and / or descending ungulates, or produce chimeric ungulate embryos, fetuses or descendants. It is another specific objective of the invention to use the present cultured MCIs or cell lines derived therefrom for the integration of desired heterologous DNA and the use of resulting transgenic cultured MCI to produce transgenic ungulates, embryos, fetuses and / or descendants or to produce embryos and / or transgenic chimeric ungulate descendants. BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a photograph of cultured MCIC cells grown without contact, feeder layer. Embryoid bodies can be observed. Figure 2 is a photograph of MCIC cells cultured positive for cytokeratin. Figure 3 is a photograph of MCIC cells on a fibroblast feeder layer. Multilayer colonies are visible only after 2 days of culture.

Figures 4 and 5 are photographs showing colonies of MCIC cells positive for FA and negative for cytokeratin. Figures 6 and 7 are photographs showing cells similar to epithelia that are obtained during the culture of MCIC cells. These cells are negative for FA and positive for cytokeratin. Figure 8 is a photograph of colonies of MCIC cells-this photograph shows that multilayer colonies begin to flatten on epithelial-like cell laminas. The cells in the middle of the colony are negative for FA and exhibit a flattened epithelial-like appearance. In contrast, cells on the perimeter are smaller, exhibit a multilayer morphology and have cytoplasmic vesicles. Figure 9 is a photograph showing MCIC cells from cultured pigs expressing a beta-galactosidase DNA construct five days after the microinjection. DETAILED DESCRIPTION OF THE INVENTION Before discussing the invention in more detail, the following definitions are provided. Internal cell mass cells (MCI cells): This is one of the two different cell types that occur during embryonic development, that is, the blasts, and eventually form part of the feeder. These cells have known application in nuclear transfer techniques and production of chimeric and cloned descendants.

Trofectoderm (TE cells): This refers to the second of two different types of cells that occur during embryonic development, that is, the blastocyst stage and eventually part of the placenta. MCI progenitor cells: These are cells comprised in preblastocyte stage embryos that develop in MCI cells. Cultured internal cell mass cells: This refers to the cells of internal cell mass that have been cultured in vitro for a long time. Internal cultured cell mass (MCIC or cultured MCI) are cells that exhibit morphological characteristics and that express cell markers identical or substantially similar to cells of internal cell mass to develop embryos: In the present invention, this will refer to MC cells I cultures that exhibit a morphology identical or highly similar to ICM from developing embryos, v. gr. , ungulate embryos. In general, said cells will grow as small, multi-layered colonies; however, some cells can differentiate if the size exceeds approximately 50 to 100 cells. MC I C expressing cell markers identical to MC I from developing ungulate embryos refers to MC I C cells that express or do not express cellular markers in a manner that is characteristic of undifferentiated MCI from developing ungulate embryos. Suitable cell markers that can be used to identify suitable MCIC include, by way of example, cytokines, in particular cytokeratin 8 and cytokeratin 18, enzymes such as alkaline phosphatase and other similar markers expressed in MCI such as rex-1, 1 amin ac , and oct4. Ideally, the expression levels (if any) of these cell markers will be the same as in undifferentiated MCI obtained from ungulate embryos. MCICs expressing cell markers substantially similar to MCIs of developing ungulate embryos refer to MCICs that express a majority of cell markers that are characteristic of undifferentiated developing ungulated MCI embryos, e.g. , cytokeratins such as cytokeratin 18 and enzymes, such as alkaline phosphatase and other cellular markers such as rex-1 ac and oct4. Substantially similar, it refers to the fact that the amount of expression of some cell markers may vary and some cell markers may be expressed differently in the MCIC present than in the undifferentiated MCI of ungulate embryos as long as this does not adversely affect the capacity of the resulting IC ICs that will be grown and maintained in accordance with the invention. In general, MCIC cells expressing cell markers identical and substantially similar to MC I of developing ungulate embryos will not express cytokeratin 18 and will express alkaline phosphatase. Methods for detecting the expression of said cell markers and others are known in the art (some of which are mentioned below) and include, by way of example, immunodetection methods. For example, such methods detect the expression or lack of expression of a particular cellular marker based on the reactivity of cells with a suitable immuno-probe, e.g., a labeled antibody which provides specific detection. However, as discussed below, there may be species differences in cell marker expression. (For example, where the MCIC obtained from pigs are positive for FA, the MCIC obtained from cows are predominantly negative for FA). In addition, the cultured MCIs of the present invention may also contain genes that are not normally contained in the MCI, v. gr. , genes that encode a desired product and / or one or more genes that inhibit differentiation. Gene that inhibits differentiation: In the present invention this will normally refer to any nucleic acid sequence that inhibits MCI differentiation. This includes, by way of example, tsA58 as well as other genes encoding other T antigens and products of oncogenes, cytokines and transcription factors, e.g. , OCT3, LI F and LI receptor. Said genes that inhibit differentiation are known in the art and are described in WO 91/13150; Okamoto and others, Cell. 60: 461 (1990); Rosner et al., Nature, 345: 686 (1990); and Smith et al., Nature, 336: 688 (1988), all of which are incorporated herein by reference in their entirety. Other suitable genes include R EX-1 (Rodgers et al.

Develop. 113: 815-824 (1991)), and FGF-5 (Herbert et al., DevelQP.1 12: 407-415 (1991)). Promoter that can be regulated or regulated: This refers to any promoter that, when it is operably linked to a structural gene, v.gr. , gene that inhibits differentiation, is "turned on", that is, it promotes transcription under specific conditions. Normally, this requires the presence or absence of one or more substituents in the culture medium, e.g. , metal ions, or other specific culture conditions, e.g. , particular temperature conditions, etc. Examples of well-known inducible or regulatable promoters include, by way of example, the response elements such as tetracycline (WO 94/29442), interferon (Kimura et al., Cell, 44: 261 (1986)), steroids and promoters. metallothionine (Yarranton, GT, Curr Opin. Biotech., 3: 506 (1992)), inducible temperature promoters, etc. These references are incorporated herein in their entirety by reference. Ceiling the aliquots: This refers to any cells that can be used to obtain and / or propagate cultured, undifferentiated MCI cell lines. Preferably, said feeder cells will be fibroblasts and more preferably murine embryonic fibroblasts, v. gr. , of fetuses of murines of 12-16 days of age. Other suitable feeder cells include, e.g. , fibroblasts and ungulate uterine epithelial cells, chicken fibroblasts, rat fibroblasts, STO and Sl-m220 feeder cell lines, and BRL cells.

MCI Multilayer Colony: This refers to a multilayered colony of MCI grown on the feeder layer that exhibits a multilayer structure that has two different cell populations. The first cell population substantially constitutes the perimeter of the multilayer cell colony and has multiple layers. Cells in it include cells that are relatively small, have a cytoplasmic vesicle and stain strongly positive for FA activity. The other cell population is comprised substantially of half of the cell colony and consists substantially of a flattened epithelial-like population of cells exhibiting little or no activity for AF. As discussed, the present invention is generally directed to cells and cultured MC I cell lines that exhibit improved properties relative to the previously reported cultured MCI. In particular, these MC I and cultured cells exhibit morphological characteristics and express cellular markers identical or substantially similar to the MCI of developing embryos. The present invention provides cultured MCIs having a novel combination of properties that are identical or substantially similar to MCI from developing ungulate embryos, i.e., having a morphology of the multilayer cell colonies defined above and expressing identical or identical cell markers. substantially similar to MC I of developing ungulate embryos, e.g., do not express cytokeratin 18 and may or may not express alkaline phosphatase (depending on the species of origin in particular). Both the alkaline phosphatase marker and the cytokeratin marker 18 have been used independently by previous investigators to determine whether the cultured cells are putatively similar to developing ungulate MCI (Piedrahita et al., Theriogenology, 34: 876 (1990), Wheeler et al. others, Reprod. Fert. Dev .. 6: 463 (1994) and Talbot et al., Mol. Reprod Dev. 36: 139 (1993)). Therefore, the expression of these cell markers by the MCICs produced herein provides strong evidence that the culture techniques present can be used to obtain MCI that are identical or substantially similar to the MCI found in developing ungulate embryos. This is in contrast to the cultured MCIs previously described, such as cultured pork MCI from Talbot et al. (Id.), which after being cultured for only two weeks in vitro, differentiate and lose FA activity. The cultured MCIs present are also different from the MCI cells cultured from cattle of Sims et al. (Id.), Which were cultured for short periods of time but which were disaggregated and grown as individual cells in a cell suspension system. The MC I cultures present in addition are different from SE-like cells that have been previously described in the literature which grow as an epithelial monolayer and which are negative for AF and positive for cytokeratin. In addition, the cultured MCI present are different from those of Wheeler, Reprod. Fert. Dev .. 6: 563 (1994) and WO 94/26884 while, growing in multilayer colonies and being negative for cytokeratin (similar to the MCIC cell lines of the present invention) are independent of the feeder layer. Therefore, the present invention provides novel MCIC cells and cell lines which, given their morphological characteristics and cell markers, should be well suited for chimeric and nuclear transfer studies to produce differentiated cells, fetuses and descendants. In general, the novel MCIC cell lines present are produced by any of the following two methods. The first method comprises obtaining MCI from blasts or MCI progenitor cells from embryos in the pre-blastocyst stage, preferably those derived from ungulates. Ungulates include many important livestock animals, eg. , pigs, cattle, sheep, horses and goats. Therefore, cultured ungulate cells potentially suitable for the production of ungulate animals are highly convenient. Also, ungulates give significant advantages over species known to be useful for production of cultured MCI and transgenic or cloned animals, e.g. , rodents, since they are immunologically and physiologically more similar to humans. thick confluent monolayer of cells on the culture plate-For example, this can be done by keeping the fibroblasts in a humid atmosphere, e.g. , one containing 5% CO2 in air at 37 ° C. However, it is expected that specific culture conditions may vary depending on the factors that they include, eg. , the type of feeder cells, age, species, among other factors. As discussed, culture plates containing the desired feeder layer, preferably in the form of a monolayer of thick confluent cells, are then used to obtain and culture the cells and MCIC cell lines present. Preferably, these will comprise plating partially intact or intact ungulate ICMs containing at least a portion of the trofectoderm directly on the confluent feeder layer treated with mitomycin C. This is effected by any means that provides direct physical contact between the cultured ICM. and the feeder cells. This can be achieved by different methods. For example, this can be done using a glass pipette to initiate contact between the MCI and the fibroblast feeder layer. Alternatively, the physical contact between the feeder cell layer and the cultured MCIs can be effected by placing the MCI cells under the feeder layer and the lower part of the culture plate, or by centrifuging the MCI cells in such a way as to force them on the cell layer. feeder Embryos ungulate in the blastocyst stage or preblastocytes can be obtained by well-known methods. For example, embryos of blastocysts or preblastocysts can be surgically recovered from the reproductive tract of ungulates, eg, pigs or cows, after dying during surgical paraparatomy, or non-surgically. Generally, these embryos will vary in age from 2 to 15 days and preferably 8 days. After recovery, the ICMs of ungulate embryos in the blastocyst stage or preblastocytes will be partially separated (eg, MCI separated from the trophoblast cells) or left intact. If partial separation is carried out, it is normally carried out by mechanical and / or enzymatic methods, eg. , by using a culture glass needle and / or by incubation with trypsin or pronase. Partially separated or intact MCIs of blastocysts containing MCI and at least a portion of the trofectoderm or MCI progenitor cells derived from embryos in the preblastocyst stage are then introduced into a culture medium of suitable feeder cell layers. All cells derived from embryos in the preblastocyst stage are introduced in a suitable feeder layer. As discussed above, the feeder cell layer will comprise any cell layer that allows the selection and / or propagation of undifferentiated MC I. Preferably, the feeder cell layer will comprise fibroblasts and more preferably those derived from primary cultures of embryonic murine fibroblasts. However, it is expected that fibroblast cell lines or other types of fibroblasts can be substituted in this way. It has been found by the present inventors that the morphological characteristics of the feeder layer is an important factor in obtaining and propagating undifferentiated MCIC cell lines in culture. More particularly, it has been found that the culture plate used to culture the MCI should preferably comprise a thick confluent monolayer of feeder cells, more preferably a thick confluent monolayer of murine fibroblast cells. As discussed in greater detail in the Examples, the feeder layer is preferably obtained from primary cultures of embryonic fibroblasts, e.g. , cells derived from murine fetuses of 12-16 days. Methods for the isolation of fibroblasts are well known in the art. For example, Fibroblasts can be recovered by aseptic removal of the head, liver, heart and alimentary tract from suitable murine fetuses, which are then cut and incubated under suitable conditions to provide cell dissociation, eg , incubation with a composition containing trypsin and the dissociated fibroblasts are seeded onto plates on tissue culture plates containing suitable culture medium. Any suitable means can be used to maintain the feeder cells cultured, e.g. , murine fibroblasts. In particular, ios present inventors choose plating the fibroblast cells on culture plates and tissue culture medium alfa-MEM (BioWhittaker, Walkersville, MD) supplemented with 10% fetal calf serum (FBS) ( Hyclone, Logen, UT), penicillin (100 μL / ml) and streptomycin (50 μg / ml). However, it is expected that, therefore, other means may be substituted, including, e.g. , DMEM supplemented with glutamine, glucose, 2-mercapto, ethanol, MEM non-essential amino acids, 5-20% serum, antibodies, nucleosides, glutamine (See Strojek et al, Theriogenology 33: 981 (1990); Notarianni and others, J. Reprod. Fert. 43 (Suppl): 255 (1990)); and beta CM and conditioned medium with BRL (See Handyside et al., Roux's Arch. Dev. Biol. 196: 185 (1987)). After the fibroblasts were cultured for confluence, they are then passed over the tissue culture plates or used directly to culture cells and MCI cell lines. Preferably, some time after the passage, and before the introduction of an MCI, the feeder cells, e.g. , fibroblasts, are also treated with an amount of antibiotic, e.g. , mitomycin C, preferably 5 to 100 μg / ml, and more preferably about 10 μg / ml of mitomycin C, contained in a suitable culture medium, e.g. , MEM-alpha or exposed to irradiation to stop or prevent the growth of fibroblasts. The feeder cells, v.gr. , fibroblasts, are preferably grown under conditions that allow the production of an MCI are grown on the feeder layer using any culture medium that allows the growth and maintenance of the MCI and the desired multilayer colony morphology. Preferably, the MCIC cells or cell lines will be maintained in a growth medium consisting of alpha-MEM supplemented with SBF and 0.1 mM beta-mercaptoethanol (Sigma). However, another culture medium can be substituted, including, e.g. , the cultivated medium described above. The growth medium is exchanged for fresh medium as necessary during culture in order to optimize cell growth. This is usually done approximately every 2-3 days. However, it can be varied depending on the specific feeder cells and the selected culture medium. After cultivation is carried out for several days, usually about 4 days, the first colonies of cultured MCI or MCIC will be observed some time later, for at least about 1 day after, the cultured MC I will pass on another fibroblast feeder layer contains culture plates. It has also been shown that the passage efficiency of MCIC cells is improved when they are passed along with some associated feeder cells on a new feeder layer. Therefore, the new step contains some of the feeder cells from the previous step. By passing the MCIC cells together with associated feeder cells (fibroblasts), it has now been found that step efficiency (percentage of MCIC cell groups resulting in new colonies) is significantly improved. As discussed above, an important part of the invention comprises the discovery that MCIC cells are preferred which comprise a particular combination of morphological properties to be passed and the production of cultured MCI cells and cell lines having the desired properties. Specifically, cells having the following morphological characteristics are preferred: (i) small cytoplasmic / nuclear volume ratios (ranging from about 10/90 to about 50/50, more preferably from about 10/90 to about 30/70 , and more preferably about 25/75); (ii) observable cytoplasmic vesicles; and (iii) small individual cells, varying from about 10-20 μm in diameter, and preferably less than about 15 μm in diameter. The calculation of cytoplasmic / nuclear volume ratios can be easily determined by observing the cells grown under the microscope, taking appropriate measurements and making appropriate volumetric calculations. Similarly, cytoplasmic vesicles can be easily observed in cultured cells. Finally, the size of the cells can be easily determined by measuring diameters of MCIC cells comprised in the culture of the feeder layer. It has surprisingly been discovered by the inventors of the present, that these morphological properties are important for the isolation and propagation of MCI cells and the production of cell lines that have the desired morphological and cell marker characteristics and that maintain said properties during prolonged periods in tissue culture, that is, after of the repeated step. More specifically, the present invention was based on the observation that when the passed MCI or MCIC cell lines were initially attached to the feeder layer, the multilayer colonies became visible in a short time, usually after 2 days. However, these multilayer colonies generally begin to flatten in the epithelial sheets of the cells as they propagate in vitro. Related to this observation, it was found that while the cells contained in the multilayer section of the colony are positive for AF and negative for cytokeratin 18, the flattened epithelium-like cells are negative for AF and positive for cytokeratin 18. Therefore, , epithelium-like cells express cell markers differently than ICM from developing fetuses. Consequently, it was discovered that MC I grown on feeder cells over time gradually exhibit a morphology and express cell markers that do not agree with the MCI of undifferentiated developing embryos. This is undesirable since MCICs exhibiting properties identical or substantially similar to MCI undifferentiated from developing embryos will potentially be totipotent and therefore should be useful in chimeric and nuclear transfer (TN) techniques. Therefore, the goal of the present invention was to develop culture methods that maintain or reverse these colonies of MCI cells so that they comprise the morphology of the desired multilayer colonies. It is theorized, based on the described morphology of the MCI colony, that specific cells could be separated and used to pass and these separate cells can result in the potential production of cultured MCIs having the desired multilayer morphology. As noted, it is observed that the growing MCI cell colony, while initially in multiple layers as a whole, flattens very rapidly to produce an epithelial sheet of cells having two distinct cell ratios within the colony. The first population is comprised of the outer perimeter of the cell colony and has a multilayer structure and includes cells having the following morphological characteristics: (i) small cell size (cells varying from about 10-20 μm in diameter, preferably smaller at 15 μm in diameter); (ii) observable cytoplasmic vesicles; and (iii) small cytoplasmic / nuclear volume ratio (ranges from about 10/90 to about 50/50, preferably from about 10/90 to about 30/70, and more preferably about 25/75). This external section of the colony was also found to stain strongly positive for FA activity. In contrast, cells in the middle of the colony tend to be comprised of flattened epithelium-like cells that exhibit little or no AF activity. Based on the activity of observed FA and multilayer structure, the present inventors choose to selectively pass only or substantially only the cells on the outer perimeter of the cell colony and specifically the cells that have a small cytoplasmic volume / volume ratio, vesicles observable cytoplasm and small cell size (defined above) in the hope that these cultured cells can produce colonies of MCIC cells and multiple additional layers that have the desired morphology. However, this result was not insured in its entirety. On the contrary, it was possible that such a step could have resulted in colonies consisting entirely of flattened epithelium-like cells, particularly if the observed epithelial-like appearance and altered cell marker expression were a consequence of in vitro culture of MCI for a prolonged time or a consequence of the passage of MCI cells. Very surprisingly, the inventors of the present invention found that the selective passage of cells comprised in the perimeter of the multilayer cell colony, have the morphological characteristics listed above to produce multilayer colonies of MCI. Also, it was surprisingly found that these multi-layered colonies contain cells expressing cellular markers identical or substantially similar to MCI from developing embryos. In addition, it was found that the present method provides for the sustained production of MCIC that exhibits a morphology and expresses identical or substantially similar markers to MCI from theoretically undefined developing ungulate embryos. The MCICs produced in accordance with the invention maintain said properties for prolonged times in culture, ie, at least one pass, after about 5 to 10 passes, and more preferably after about 10 to 50 passes. The selective passage of cells having desired morphological characteristics can be achieved by known cell separation methods. For example, the multi-layered portion of the colony constituting the perimeter of the colony may be separated from the middle portion of the colony by physical means, e.g. , using a glass pipette or a needle. The resulting large cell groups can also be separated by physical and / or chemical and / or enzymatic means. For example, cell dissociation can be effected by the use of enzymes, e.g. , trypsin or pronase. Alternatively, mechanical cell separation can be effected by repeated pelletizing of large groups of cells or groups of cells through a cell pipette or by the use of a shaving needle or knife to cut large groups of cells into small groups. Preferably, said chemical and / or mechanical cell separation will produce groups of MCIC suitable for the passage, ie, containing approximately 5 to 10 cells. The separate cells used for passing preferably will consist of cells contained in the multilayer peripheral section of the cell colony, preferably having the morphological characteristics cited above. However, in some cases, even the cells of the inner portion of the cell colony, when treated according to the invention, are found to produce multilayer colonies after passing over a feeder layer. It is thought to occur because the cells are reverted back to the desired multilayer colonies either due to the step procedure or to the reestablishment of cell-to-cell contact with the new feeder layer. Nevertheless, the inventors of the present do not wish to be united to this belief. During passage, it is essential that small MCI cells or cell groups are placed in direct contact with the feeder layer to avoid differentiation of MCIC colonies. It has been found that if the cells or groups of cells develop without sufficient contact with the feeder layers, instead, this results in embryoid bodies. Said embryoid bodies can be observed in Figure 1 which is a photograph showing cultured MCI cells developed without sufficient contact with the feeder layer. To initiate direct contact, any method can be used that provides direct cell contact between past MCIC cells and the layer of feeder cells that are not degradable for MCIC and do not adversely affect colony production, the least efficient means they are simply allowing small groups of cells to settle on top of the feeder layer. It is more preferable to use methods that provide more efficient cell-to-cell contact between the MCIC cells and the feeder layer. This has been found to result in a higher number of multilayer MCIC cell colonies. Any physical means providing improved physical contact between the MCIC cells and the feeder cells, but which are not unduly degradable, i.e., which do not adversely affect the production of the desired multilayer type cells and the cell lines, can be used. of MC IC. The methods for providing the increased direct contact of past MCI C and the feeder layer include, by way of example, the use of a pipette to press the individual groups of cells in the feeder layer.; placing the groups of MCIC cells under the feeder layer so that the cells are sandwiched between the feeder layer and the bottom of the layer; and centrifuging the cell groups on top of the feeder layer, e.g., between 100 and 5000 g for about 10 minutes to 5 hours to force the groups of cells in the feeder layer. These methods are merely illustrative of methods that can be used to force groups of MC IC cells into close contact with the feeder layer. Other methods may be substituted as long as they do not adversely affect the formation of the desired multilayer MCIC cell colonies. As discussed above, it has also been found that step efficiency can also be increased by passing the MCIC cells together with some associated feeder cells on the new feeder colon. Apparently the presence of some cells fed the previous step increases the percentage of groups of MCIC cells that result in new colonies. In general, the above culture procedure for cultivating MCIC cells is applied to any ungulated derived MCI C cell. For example, the procedures that were initially found to be suitable for cuing the pig MC I C cells have been found to be applicable for cattle MCIC cells. However, an observed difference is that the majority of cells derived from bovine embryos are negative for AF whereas those derived from embryos of pigs are positive for FA. It is hypothesized that there may be several species differences in the expression of FA in ICM of goats and pigs. However, this is not clear since the inventors were able to produce a bovine cell line that was weakly positive for FA and that grows in small groups. (See Figure 2). Also, bovine cells differed from pig cells on those edges of the colony were not well defined. However, the cells derived from pigs, the cells comprised in the perimeter of the colony are positive for FA where the cells in the center of the colony have to lose the activity of FA. This can also be seen by reviewing Figure 2. Similar to MCIC derived from pigs, these cells are negative for cytokeratin 18. As shown in the Examples, and described in more detail above, the MCICs present are useful for the introduction of Heterologous DNA. In particular, the transgenic MCIC cell lines have been produced, which contain within their genome a heterologous DNA (construction of beta-galactosidase DNA). Also, recently the inventors obtained cells using the culture methods described above that are somewhat different from the MC IC cells described in their morphology, ability to differentiate, endogenous beta-galactosidase (higher) activity level, and ability to express constructs. of beta-galactosidase DNA. In addition, these cells were negative for AF from the start of culture while previous MCIC cells of pigs tended to lose FA activity over time during culture. Similar cells have been observed and propagated in MCIC derived from cows. Although these cells lack FA activity, they exhibit some characteristics in common with MCI cells. Therefore, they can also be useful for the production of transgenic cloned embryos, as well as the other applications described in MCIC. The present invention also provides another method for providing cultured MCI cells and cell lines that have the morphological characteristics and markers of desired cells, e.g. , MCIC that are positive for FA and negative for cytokeratin 18. This second method will also involve maintaining and cultivating MCI, preferably embryos in the blastocyst stage or preglastocytes of ungulates on the cell-feeder cell cultures. Preferably, the cultivation techniques and methods of passages will be carried out as described above. However, the second method differs in the fact that the cultured MCI cells were expressed, preferably only under specific conditions, a differentiation inhibition (ID) gene. This is preferably achieved by introducing into the MCI cells, at some point during the culture or passage procedures, a sequence of nucleic acids including a differentiation inhibition gene, which is preferably expressed under the control of an inducible or regulatable promoter. As noted above, the ID genes differ from any gene or genes that can inhibit cell differentiation in colonies of MCIC cells that do not adversely affect the isolation of MCIC cell lines that have the desired morphological characteristics and expression of cell markers. . The ID gene preferably operably linked in the appropriate reading frame to an adjustable or inducible promoter can be introduced into the nucleus of embryonic cells from which MCIC cells are derived during passage, or alternatively introduced into a MCIC cell line established This is done in a way that results in the DI gene, or transgene as can be accurately described, being integrated into the embryonic MCI genome or cells or cultured MCI cell line. Methods for introducing the desired DNA into mammalian cells and embryonic cells in particular are known in the art and include, by way of example, microinjection, electroporation, lipofection, retroviral insertion, Ca precipitation and liposome insertion. To date, microinjection seems to be the most efficient means to introduce DNA into the MCIC cell lines. However, other methods with appropriate optimization are also expected to be effective. Genes that inhibit differentiation suitable for use in the invention, include, e.g. , tsA58 (See WO 91/13150), other antigens and oncogene products known to inhibit differentiation (see WO 91/13150 for examples thereof), OCT3 (Okamoto et al., Cell.60: 461 (1990), Rosner and others, Nature 345: 686, (1990), LIF and the LIF receptor (Smith et al., Nature 336: 688 (1988).) These ID genes are only illustrative of those that can be used herein. invention The ID gene is preferably placed under the control of an inducible or regulatable promoter As noted, examples of inducible promoters are well known in the art and include by way of example the metallothionine (inducible metal ion) promoter, as well as the response elements for tetracycline, interferon and steroid (See WO 94/29442; Kimura et al., Cell, 44: 261 (1986); Yarranton, Curr. Qpm Biotech 3: 506 (1992) After the transgene integrates into embryonic or cultured MCI cells or cell lines and the resulting transgenic cells are established in the feeder cell cultures, the ID gene or genes are ignited by inducing the particular inducible promoter. . This is usually done by adjusting culture conditions. For example, if the promoter is the metallothionine promoter, the induction is effected by the introduction of a culture medium containing appropriate metal ions that induce ("turn on") the promoter. Therefore, when the cells are cultured under induction conditions, the cultured MCI cells must maintain continuously or for prolonged periods in the tissue culture, the morphology characteristics of MCIC cells and expression of desired genes - more specifically, the cells should display the morphology of desired multilayer cell colonies and express cellular markers identical or substantially similar to MCI of developing embryos, ie, the cells will generally be AF positive and cytokeratin negative (cytokeratin 18). Therefore, minimize or even eliminate problems such as colonies of MCI cells that differentiate into flattened epithelial sheets that are negative for FA and positive for cytokeratin 18 (which are observed when MCI are cultured under conventional conditions). In addition this should also prevent differentiation of MCI cells during passage and during sustained culture periods. The resulting cultured MCI cells and cell lines that are obtained by the methods described above have numerous uses. More especially, MCIC cell lines can be used to produce offspring that have the genetic formation of MCIC in whole or in part. The chimeric descendants can be obtained by injecting the MCIC cells directly into the blastocel cavity of recipient embryos or combined in embryos in the preblastocyte stage. The resulting chimeric embryos are then placed in a recipient female. The resulting offspring should have a genetic contribution of MCIC to all organ systems including the germ cells of the reproductive organ. Therefore, the chimeric animal can pass the genetics of MCIC in subsequent generations of the offspring. Also, the introduced MCIC may have introduced a desired gene or genes into its genome. Therefore, the chimeric descendant can be obtained which expresses a desired gene or genes. For example, genes can be introduced to MCICs that provide improved livestock properties, e.g. , which encode hormones, which provide resistance to the disease, (e.g., lymphokines, viral resistance genes, bacterial resistance genes), improved milk production, altered fat percentages, improved body weight, among other properties. Also, genes encoding the desired gene products can be introduced, e.g. , genes that encode products useful as human therapy or as xenotransplantation. Also, the MCIC cells present can be used in nuclear transfer procedures to obtain embryos, fetuses and descendants of nuclear transfer. Nuclear transfer techniques are known in the literature and are described in many of the references discussed in the background of the invention. See in particular, Campbell et al., Theriogenology, 43: 181 (1995); Collas and others, Mol. Reprod. Dev. 38: 264-267 (1994); Keefer et al., Biol. Reprod. , 50: 935-939 (1994); Sims and others, Proc Nati. Acad. Sci. USA. 91: 6143 (1994); Stice et al., Therioqenology, 41: 301 (1994); Sims and others, Proc. Nati Acad. Sci. USA, 90: 6143-6147 (1 993); WO 94/26884; WO 94/24274; and WO 90/03432 which are incorporated herein by reference in their entirety. Again, if MCIC contributes to fetal germ cells, the genetics of MCIC can be passed on to subsequent generations of animals. Similarly, MCIC cells can be genetically treated so that a desired gene or genes are integrated into their genome, eg, genes that provide improved properties for cattle, or that encode the desired gene products, v.gr ., human therapeutic or other polypeptides. Still further, differentiated cells, tissues or organs produced by nuclear transfer or obtained from fetuses or chimeric descendants can be used in transplant therapies. For example, nuclear or chimeric transfer fetuses derived from MCIC cells contain an anti-rejection gene or can provide a source of useful hematopoietic cells to supplement or replace human hematopoietic support cells. This is potentially useful in immunocompromised patients v.gr. , patients with SI DA or other diseases that affect hematopoietic support cells. Also, support cells may be useful in the treatment of Huntington's disease, Parkinson's disease and Alzheimer's disease. In addition, pancreatic cells may be useful in diabetes treatments. The transplanted liver cells can also be useful for the treatment of liver diseases. Alternatively, whole soft organs can potentially be transplanted from ungulates derived from MCIC genetically altered in humans (See Durling et al., Curr.Omp. Immunol., 6: 765 (1994) incorporated herein by reference in its entirety. The present MCIC cell lines can be used as an in vitro model of differentiation, particularly for the study of genes that are involved in the regulation of early development.This is only illustrative of potential applications of MCIC cell lines obtained according to the present invention The invention will now be described in greater detail in the following Examples: EXAMPLE 1 The production of MCIC cell lines from embryos of pigs in the stage of preblastocytes and blast cells was carried out using the following general protocol. primary embryonic fibroblasts were obtained from murine fetuses of 12-16 days After the head, liver, heart and alimentary tract were removed aseptically, the embryos were cut and incubated for 30 minutes at 37 ° C in prewarmed trypsin EDTA solution (p 0.05% trypsin / 0.02% ETA; GIBO, Grand Island, NY). The fibroblast cells were placed in tissue culture plates and cultured in 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). Three to four days after! Step, embryonic fibroblasts, in 35 x 10 culture plates (Baxter Scientific, McGaw Park, IL), were treated with mitomycin C (10 μg / ml; Sigma) in MEM alpha supplemented for a minimum of three hours. The fibroblasts were developed and kept in humid atmosphere with 5% CO2 in air at 37 ° C. Only culture plates that had a thick confluent monolayer of cells were used to culture the MCIC cell lines. The characteristics of the feeder layer is an important factor in obtaining and propagating undifferentiated MCIC cell lines. Porcine embryos were surgically recovered from the reproductive tract after samples or during surgical laparotomy. The MCI embryos in blastocyst stage were partially separated from the trophoblast cells using a cutting needle, incubated in trypsin or pronase, or left intact. MCIs and at least a portion of the trofectoderm were plated directly onto the blocked fibrombotic cells of mitomycin C by frequently using a glass pipette to initiate contact between the MCI and the fibroblast feeder layer. The MCIC cell lines were maintained in a growth medium consisting of alpha MEM supplemented with 10% FBS and 0.1 mM beta-mercaptoethanol (Sigma). The growth medium was exchanged every two to three days. The initial colonies were observed by the fourth day of cultivation and could happen any time after the fifth day. Only the cells having the following three morphological characteristics were isolated for passage: a small cytoplasmic / nuclear volume ratio, cytoplasmic vesicle and generally small individual cells (less than 15 μm in diameter). Often these cells were isolated from the multilayer portions of the colony that maintained direct contact with the feeder layer. Portions of the colony that meet this criterion were frequently positive for AF and negative for cytokeratin when moving to a new feeder layer. When the MCI or the passed MCIC cells were initially bound to the feeder layer, the multilayered colonies consisting of small cells are visible after two days in culture. This can be seen in Figure 3. The multilayered colonies are positive for AF and negative for cytokeratin (See Figures 4 and 5). However, some colonies begin to form a lamina similar to cell epithelium. Epithelium-like cells are negative for AF and positive for cytokeratin (Figures 6 and 7). In addition, multilayered colonies often begin to flatten in the epithelial sheets of cells as they propagate in vitro (Figure 8). It was observed by the inventors that a multilayer colony in development begins to flatten, forming an epithelial sheet of cells having two different populations of cells within the colony. The first population resides in an area around the perimeter of the colony. This section of the colony is in multiple layers and the individual cells are small and have cytoplasmic vesicles (Figure 8). This area also dyes positive for FA activity. The other area of the colony contains the population similar to flattened epithelium of the cells. These cells tend to be in the middle of the colony. In this population of cells, individual cells and cell borders can be observed when the colony is observed under a microscope (Figure 8). Again, these cells have very little or no FA activity. It is theorized that to maintain the desired multilayer type cells, preferably, only the cells around the perimeter could selectively pass to produce additional multilayer colonies. This is probably accomplished using a glass pipette, razor blade, or a needle to cut the multilayer portions of the colony (perimeter cells). Large groups of cells can be separated either by mechanical separation or by using trypsin (0.05% trypsin / 0.2% EDTA) together with mechanical separation. Mechanical separation was carried out by repeatedly pipetting large groups of overhead cells already low through a small orifice pipette. Alternatively, a shaving needle or blade can be used to cut the large group cells into small groups. Surprisingly it was found that the MCIC groups obtained by these methods (from 5 to 100 cells) can be passed on new feeder layers to produce cultures having the desired multilayer morphology. In some cases, the cells of the inner portion of the colony, when treated in this same manner, produced multilayer colonies after passage in new feeder layers. It is hypothesized that these cells were reverted back to the multilayer colonies as a result of the step procedure or the reestablishment of cell-to-cell contact with the new feeder layer. It was observed that the small groups of cells should be put back in direct contact with the feeder layer to avoid the differentiation of MCIC colonies. In contrast, cells developed without contact with the feeder layers form embryoid bodies (Figure 1). There are several methods used to restart the feeder layer contact. The least efficient means is to allow small groups of cells to settle on top of the feeder layer after the cell culture plates are placed back in the incubator. A preferred method involves forcing the cell-to-cell contact between the MCIC cells and the feeder layer. This results in a higher number of colonies of newly established MCIC cells. One way to force cell-to-cell contact is to use a pipette to press the individual groups of cells down on top of the feeder layer. Another method is to place the group of MCIC cells under the feeder layer so that the cells are forced between the feeder layer and the bottom of the culture plate. Alternatively, the groups of cells on top of the feeder layer can be centrifuged between (100 and 5000 g) for (10 minutes to 5 hours) to force the cells from below on top of the feeder layer. Essentially, any method that forces the last groups of MCIC cells in close contact with the feeder layer results in the production of clones of MCIC cells having the desired multilayer morphology. Also, as previously discussed, step efficiency can be further improved by passing MCIC cells together with some associated feeder cells on the new feeder layer. EXAMPLE 2 The MCIC cells obtained according to Example 1 were used for heterologous DNA insertion. Specifically, these cells were microinjected with both linear and supercoiled DNA constructs containing different promoters placed on the front of the beta-galactosidase gene and / or the neomycin phosphotransferase gene. The specific promoters used were the cytomegalovirus promoter (CMV promoter), phosphoglycerate kinase promoter (PGK promoter), mammary promoter (MAM promoter), reCMV promoter and beta-actin promoter of chickens. These gene constructs were diluted in a regulated solution (containing 80mM KCl and 70mM HEPES). However, other regulators can be easily replaced for the same, such as Tris EDTA. The concentration of DNA construction in solution ranged from 5 to 10 μg / ml. However, concentrations ranging from 0.1 to 100 μg / ml would be effective. These DNA preparations were microinjected into cultured MCIC cells obtained according to Example 1. In this procedure, the individual MC IC cells within the colonies were localized by observing the colonies through an inverted microscope. Then, the cell membrane alone or additionally the nuclear membrane, was stung using a small injection needle containing the DNA preparation. The opening in the needle was approximately 1 μm. Said needle of a diameter was selected to help prevent lysis of MCIC cells. In addition, the microinjection procedure was facilitated by the use of a micromanipulader that was connected to the inverted microscope.

After the injection pipette was introduced into the nucleus, approximately 700 copies of the DNA in the nucleus were released. The micropipette was then removed from the cell. This process was repeated for other cells in the MCI colony C. Ideally, 1000 cells per hour could be microinjected. The results obtained using the different promoters are summarized in the following table.

Expression of Heterologous Genes in Microinjected IC IC Cells The above results demonstrate that the vectors containing each of the tested promoters result in cells expressing the inserted heterologous DNA. It was also observed that microinjection with several constructs does not provide any additive effect on gene expression. Figure 9 is a photograph of microinjected pig MCIC cells containing a CMV beta-galactosidase construct detected by X-gal tinsion. The nests of cells expressing beta-galactosidase can be observed. This indicates that the β-galactosidase gene has been effectively incorporated into the cell genome and is being transmitted and expressed to the daughter cells. The above results were obtained using MC IC cells from pigs. In addition, using similar methods, bovine MCI C cells were also injected with constructs of CMV and PGK-beta-galactosidase. Both DNA constructs resulted in the recovery of cells expressing beta-galactosidase. Therefore, these results demonstrate that the MC IC cultured according to the invention can be used successfully for the integration and expression of desired heterologous DNA. Also, these gene expression characteristics can be passed on to daughter cells. EXAMPLE 3 In this culture method, an I D gene was conditionally expressed in colonies of MCIC cells to prevent cell differentiation. The passage of cells and culture techniques are the same as in Example 1 with the difference of the genetic formation of the MCIC cells. Specifically, an ID gene is introduced into the nucleus of embryonic cells from which the MCIC cell lines are derived or in a MCIC cell line. The transgene is then integrated into the genome of the MCIC cells. Any known methods for introducing transgenes into embryonic cells can be used. including, by way of example, microinjection, electroporation, retroviral insertion, Ca precipitation, and liposome insertion. The inserted transgene is expressed under the control of an inducible promoter. Inducible promoters include, e.g. , response elements such as tetracycline (WO 94/29442), interferon (Kimura et al. 1986), steroid and metallothionein (reviewed by Yarranton, 1992). Consequently, the I D gene is inserted so that it is operably linked, in the appropriate reading frame, with an inducible promoter. After the construction of chimeric genes is integrated into the embryonic cell genome and the colony of multilayer MC I cells is established, the I D gene is expressed by inducing the implantable promoter. This expression provides that the MCI cell colony maintains continuously, or for a prolonged time in tissue culture, the desired multilayer morphology and that it expresses genes that match MCI of developing embryos. Therefore, problems such as differentiation of cells into flattened epithelial sheets that lose their FA expression and express cytokeratin 18 and are minimized or avoided. This method is also useful to prevent cell differentiation during long-term culture periods. While the invention has been described with respect to certain specific embodiments, it will be appreciated that many modifications and changes may be made thereto by those skilled in the art without departing from the spirit of the invention. It is intended, therefore, by the appended claims to cover all modifications and changes that fall within the spirit and actual scope of the invention.

Claims (41)

1. REVIVAL DICTION IS 1. A method for producing a cultured cell mass cell (MCIC) comprising the following steps; (i) obtaining an MCI of a blastocyst or MC I progenitor cells from embryos in the preblastocyst stage; (ii) culturing said MCI or MCI progenitor cells on a feeder layer culture under conditions that provide for the formation of multilayer cell colonies; (iii) identify among other cells contained in the colony of cultured MCI cells, those that exhibit the following properties: (a) small cytoplasmic / nuclear volume ratio; (b) you see cytoplasmic particles; and (c) small individual cells; (iv) separating one or a group of said identified cells from the rest of the cell colony; and (v) passing the separated MCI cells or MCI progenitor cells into another feeder layer culture, under conditions such that the separated cells have at least some physical contact between the feeder cell layer and the cells or group of cells separated. The method of claim 1, wherein the MC I obtained in step (i) includes at least a portion of the trofectoderm. 3. The method of claim 1, wherein the feeder cell layer comprises fibroblasts. 4. The method of claim 3, wherein the fibroblasts are embryonic murine fibroblasts. 5. The method of claim 4, wherein the embryonic fibroblasts of m urins are primary cells. 6. The method of claim 3, wherein the fibroblasts comprise a coarse confluent monolayer. The method of claim 1, wherein the method for producing physical contact of the separated cultured MCI cells and the feeder cell layer is selected from the group consisting of (i) placing the separated MC I cells on the part of the cell layer feeders, (ii) pressing the MC IC cell groups onto the feeder layer, (ii) placing the MCIC cell groups between the feeder cell layer and the culture plate, and (iv) centrifuging the MC IC cells or groups of cells on the layer of feeder cells. The method of claim 1, wherein the separate identified cells are comprised in the outer portion of the multilayer MCIC cell colony. The method of claim 8, wherein the further separated cells include some cells comprised in the inner epithelial-like portion of the cell colony. The method of claim 1, wherein the identified cells are separated from the colony by at least one of the following chemical or physical or enzymatic means. 11. The method of claim 10, wherein the physical means comprises the use of a glass pipette, hypodermic needle or shaving blade. The method of claim 11, further comprising treating cells separated with trypsin or pronase. The method of claim 1, wherein the separated cells comprise a group of about 5 to 100 cells. The method of claim 1, wherein steps (ii) to (v) are repeated. The method of claim 1, wherein the cytoplasmic / nuclear ratio scales range from about 10/90 to about 50/50. 16. The method of claim 15, wherein the ratio is from about 25 to 75. 17. The method of claim 1, wherein the cells vary in size from about 10 to 20 microns in diameter. 18. The method of claim 2, wherein the cells are less than about 15 microns in diameter. 19. Cultured internal cell mass cells (MCIC cells) derived from a blastocyst MCI or MCI progenitor cell derivative obtained from ungulate preblastocyte embryos where the cultured internal cell mass maintains the morphological characteristics in culture and expresses cell markers identical or substantially similar to MC I of ungulate embryos in development. 20. The IC IC of claim 19, which express alkaline phosphatase. 21, The MCICs of claim 19, which do not express cytokeratin 18. 22. The MCICs of claim 19, wherein the cells exhibit a multilayer colony morphology in tissue culture. 23. The MCIC of claim 21, wherein the morphology of multilayer colonies comprises an inner epithelial type portion and a multilayer potion substantially surrounding the epithelium-like portion. 24. The MCIC of claim 19, wherein the ungulate embryos are obtained from a ungulate selected from the group consisting of pigs, cows, sheep, horses and goats. 25. The MCIC of claim 24, wherein the ungulate is a pig. 26. The IC IC of claim 24, wherein the ungulate is a cow. 27. A cultured internal cell mass cell line obtained from the MC ICs of claim 19. 28. The MCICs of claim 19, comprising one or more genes that inhibit MCIC differentiation. 29. The MCIC of claim 28, wherein the gene is selected from the group consisting of tsA58, OCT3, LIF, LI F receptor, FGF-5, Rex 1 and other oncogene products, T antigens, cytokines and transcription factors . 30. The IC ICs of claim 10, which comprise a heterologous DNA integrated within its genome. 31 The MCICs of claim 30, wherein the heterologous DNA encodes a selectable marker. 32. The IC ICs of claim 19, wherein the heterologous DNA encodes a desired polypeptide. 33. A method for inner cell mass cells which exhibit morphological characteristics cultivated and express cell markers that match or are substantially similar to, the MCI of ungulate embryos for prolonged times differentiation in tissue culture comprising: (i ) obtain ungulate MCI cells or a LACTED, stablished MCI cell line, established; (ii) introducing into the core of the MCI cells or the established MC I cell established one or more genes that inhibit differentiation of said cells or MCI cell line (I D genes); (iii) culturing the resulting transgenic ungulated transgenic MC I cells or cell line on a suitable feeder layer under conditions that inhibit differentiation and provide for the production of a multilayer cell colony. 34. The method of claim 33, wherein the genes are selected from the ID group with sists of tsA58, OCT3, LI F, F LI receptor and other oncogene products or antigens T. 35. The method of claim 33, wherein said ID gene is expressed under the control of an inducible promoter and the culture conditions comprise those that induce the promoter. 36. The method of claim 33, wherein the cells are passed. 37. The method of claim 36, wherein the step method comprises: (i) culturing said MCIs in a feeder layer culture, under conditions that provide colonies of multilayer cells; (ii) identifying from the colony of cultured MC I cells the cells exhibiting the following properties: (a) small cytoplasmic / nuclear volume ratio; (b) cytoplasmic vesicles; and (c) small individual cells relative to the rest of the cell colony; (iii) separating one or a group of said identified cells from the rest of the cell colony by suitable means; and (iv) passing the separated MCI cells into another feeder layer, preferably fibroblasts, under conditions such that the separated cells have at least some physical contact between the feeder cell layer and the separated cells or group of cells. 38. The method of claim 33, wherein step (iv) of the separated passed MC I cells includes some of the associated feeder cells. 39. The method of claim 33, wherein the ungulate embryo is obtained from a ungulate selected from the group consisting of pigs, cows, sheep, goats and horses. 40. The method of claim 39, wherein the ungulate is a pig. 41 The method of claim 39, wherein the ungulate is a cow.