JPH09508020A - Isolation of embryonic stem cells - Google Patents

Abstract

  (57) [Summary] Embryonic stem (ES) cells are isolated from cultured embryos containing both ES cells and differentiated cells by selectively killing the differentiated cells in situ. This improves the efficiency of known (manual) techniques and allows the induction of ES cell lines in previously difficult to perform species. Killing is performed by the drug selectable marker system or by expression of a lethal gene. In the former case, undifferentiated ES cells are protected against toxic effects of drugs by selective expression of drug resistance markers (neo etc.). In the latter case, a lethal gene (diphtheria toxin, etc.) is selectively expressed in differentiated cells. The expression cassette is then removed by the site-specific recombination system.

Description

DETAILED DESCRIPTION OF THE INVENTION Isolation of embryonic stem cells The present invention relates to a method for isolating embryonic stem cells. Mouse embryonic stem (ES) cells are rapidly proliferating, undifferentiated, totipotent cells derived from early embryos. These cells can grow in large numbers in vitro and then reintroduced into early embryos to become both somatic and germ cells of chimeric animals. ES cells have been widely used as a procedure by which gene conversion performed in vitro can be introduced into mice and stably inherited. A particularly powerful method is to obtain a given genetic modification with gene targeting in ES cells. This makes it possible to inactivate, convert or replace a beneficial gene (Bradley et al., Bio / Technology, 10: 534-539 (1992)). . Applications include: detailed analysis of the regulation of gene expression, analysis of protein structure and function, development of animal models of human disease and precise placement of heterologous genes in transgenic animals. The induction of ES cell lines from mouse embryos is essentially the release of a generally short-lived population of undifferentiated cells from the regulatory actions of the embryonic environment. ES cells inherently have the ability to grow permanently without any form of oncogenic transformation. The standard method for obtaining mouse ES cell lines is by Evans et al. (Nature, 292, pp. 154-156 (1981)) and separately by Martin (Proc National Academia USA. Proc. Natl'l. Acad. Sci. USA. ), 78, p. 7634-7638 (1981)), and Robertson, E. J. ), “Embryo-derived stem cells” in “Teratocarcinomas and Embryonic Stem Cells, A Practical Approach”, Eger, Robertson (E. J. Robertson), published by IRL Press Ltd, Oxford, 1987. Briefly, blastocyst stage embryos are drained from the uterus and then cultured in culture on a layer of mitotically incapable fibroblasts, or "supporting" cells. After a few days, the blastocysts attach and spread on the feeder layer and are exposed to the cells of the inner cell population. Depending on the explant, the inner cell population proliferates to form an undifferentiated cell mass. Portions of each such explant are manually isolated, separated and seeded again on a new feeder layer. Colonies of cells form, which are usually composed of various cell types. Colonies with ES morphology are further isolated, separated and replated. By repeating such operations by various routes, a homogeneous ES cell population can be obtained. Physical selection of ES cells from differentiated tissues such as trophectoderm and endoderm is an important part of the induction process. These differentiated tissues arise from the differentiated cells of the explant embryo as well as by the natural differentiation of ES cells. The endoderm is generally the first product of ES cell differentiation, and cells of these types tend to be located in close proximity to undifferentiated ES cells. The close proximity of differentiated cells induces further differentiation of ES cells. The presence of feeder cells is required during the first step of ES cell induction in order to suppress the natural differentiation of undifferentiated cells. However, the mechanism is only partially defined. Established ES cell lines include cytokines, leukaemia inhibitory factor, LIF (Smith et al., Nature, 336, 688-689 (1988)) and Williams (Williams) in culture medium. Willi ams) et al., Nature, vol. 336, pages 684-687 (1988)), generally they can grow well in the absence of a feeder layer (Magin). Nuc Acids Less (Nuc. Acids. Res. ), Vol. 14, p. 3795 (1992)). However, soluble LIF alone cannot necessarily be substituted for support during the induction of ES cells. The frequency with which ES cell lines are derived from the embryo varies widely. According to experts, ES cell lines are obtained from 5-10% of mouse blastocysts with strain 129Sv (Robertson, E .; J. ), M Etch Kaufman (M. H. Kaufman, Bradley, A. ) And MJ Evans (M. J. Evans), 1983, "Teratocarcinoma Stem Cells", Cold Spring Harbor Conferences on Cell Proliferation, Vol. 10, pp. 647-663, Martin. , G (Matin, G. R. ), El Silver (L. Silver) and S-Strick Cand (S. Strickland) "Isolation, Properties and Karyotype Analysis of Pluripotential (EK) Cells from Normal and Parthenogenetic Embryos)" . It would be highly desirable to extend the use of gene targeting in ES cells to more commercially available animals than mice, especially rats and livestock. Various researchers have been trying to obtain ES cells from farm animals (Handyside et al., Roux's Arch. Dev. Biol. 196, pp. 185-190 (1987), Notorianni et al., J. Leprod Fertill (J. Reprod. Fertil. ) (Supplement), Volume 41, pages 51-6 (1990), Piedrahita et al., Theriogenology, Volume 34, pages 865-877 (1990), Piedrahita et al., Serioger. Theriogenology, Vol. 34, pages 879-901 (1990) and Salto et al., Roux's Arch. Dev. Biol. ), 201, pp. 134-141 (1992)) had limited success. Although ES-like cells were induced, there is no report that chimeric animals can be formed from developed embryos. Progressive differentiation of undifferentiated cells that ultimately kills the cell line is a common difficulty in the induction and maintenance of ruminant ES cells. It has been reported that ES cell differentiation is largely induced by adjacent differentiated cells. Traditionally, as mentioned above, differentiated cells have been manually separated from ES cells, if possible. The present invention is based on the fact that the elimination of differentiated cells, ie the specific destruction of differentiated cells in situ, improves the efficiency of inducing ES cells from the embryo. According to a first aspect of the invention, there is provided a method of selecting embryonic stem (ES) cells from an embryo in culture, which comprises selectively killing the differentiated cells of the embryo. Differentiated cells are derived from the embryo in the same way as they are explanted (usually in vitro) in culture or arise from the differentiation of ES cells within the embryo. The purpose of the present invention will usually be to selectively kill all the differentiated cells. The present invention is applicable not only to mouse stem cells but also to other animals, particularly rat (Ratus spp. )) And other placental mammals, other rodents, rabbits and livestock animals, in particular cows, pigs, farm animals such as sheep and goats, for the production of stem cells. To be done. However, the present invention is not limited to a particular species and has been found to have very broad applicability to the animal kingdom. Explanted embryos usually contain undifferentiated (ES) cells as well as differentiated cells such as trophectoderm and endoderm cells. Explanted embryos were prepared in the same manner as described above (eg, at the blastocyst stage), as outlined by Robertson (1897) (see citation above). It is obtained by extracting an embryo from the uterus and culturing it in a suitable culture medium on a feeder layer which is a fibroblast that has lost mitotic ability. Explanted embryos that undergo the selective extinction step of the invention are subjected to one or more pathways to isolate a mass of mostly undifferentiated cells, separated and placed on a new feeder layer. Seed again. According to a preferred embodiment of the invention, the differentiated cells are killed by a drug selection method. Such methods are used to kill the differentiated cells during the early stages of explanted embryos. Since undifferentiated ES cells express markers that are resistant to drugs that are toxic to both differentiated and undifferentiated cells, adding drugs to colonies kills only the differentiated cells. For the embodiments described above, it is important to use undifferentiated cell-specific or substantially specific promoters, and in embryonic cells such promoters encode DNA sequences encoding drug resistance markers. To transform a construct ligated into or to allow expression of such a construct. Two reports have been published regarding genes that strongly down-regulate ES cell differentiation. These are transcription factors Oct-3 / 4 (Okazawa et al., Embo J. ), 10: 2997-3005 (1991)) and growth factor FGF-4 (Ma) et al., Dev. Biol. ), 154, pp. 45-54 (1992)). Although the examples shown below use the Oct-3 / 4 promoter, the FGF-4 promoter can also be used and other suitable ES-specific promoters. The transcription factor Oct-3 / 4 was first identified to be present in undifferentiated mouse embryonic carcinoma (EC) cells (closely related to ES cells), but not a differentiated derivative (Okamoto et al., Cell, Volume 60, pages 461-472 (1990)). Analysis of Oct-3 / 4 mRNA expression in mice revealed that expression was undifferentiated in ES and EC cells (Ben-Shusan et al., Mol Cell Biol (Mol. Cell. Biol. ), 13: 891-901 (1993)), embryonic inner cell populations and primitive germ cells (Rosner et al., Nature, 345, 686-92 (1990). )). The Oct-3 / 4 promoter is capable of directing Oct-3 / 4 specific expression of a heterologous reporter gene in embryos of transgenic mice (Okazawa et al., Embo J. ), 10: 2997-3005 (1991)). Various genes have been used to confer drug resistance to non-mutated cells, eg, selection by G418 for the neo gene (Colbere-Garapin et al., J. Morbiol (J. Mol. Biol. ), 150, pp. 1-14 (1981)), selection by hygromycin for hygro gene (Santerrer et al., Gene, 30, 147-156 (19784)), Selection with histidinol for the his gene (Hartman et al., Proc National Academia (Proc. Natl'l. Acad. Sci. ), 85, pp. 8047-8051 (1988)), selection by methotrexate for the dh fr gene (Wigler et al., Proc National Academia USA (Proc. Natl'l. Acad. Sci. US A. ), 77, pp. 3567-3570 (1980)), aminopterin / mycophenolic acid selection for the gpt gene (Mulligan et al., Proc National Academia USA (Proc. Natl'l. Acad. Sci. USA. ), 78, pp. 2072-2076 (1981)), selection by methionine sulphoximine for the glutamine synthetase (gs) gene (Hayward et al., Nucl Acidsless (Nucl. Acids. R es. ), 14, pp. 999-1008 (1986)) and selection by deoxycoformicin for the adenosine deaminase (ada) gene (Kaufman et al., Proc. National Acad. Natl'l. Acad. Sci. USA. ), 83, pp. 3136-3140 (1986)). The examples below use the neo (aminoglycoside phosphotransferase) gene, but the hygro, his, dhfr, gpt, gs or ada genes can also be used, and other suitable drug resistance genes can also be used. It is noted that the use of the expression "gene" as used herein does not mean that natural genomic DNA has to be used, although it is preferred to use natural genomic DNA. Should. A "minigene" can be used, in which the cDNA is at least preferred, and which contains some, but not all, of the introns naturally present in the gene. According to another important embodiment, DNA sequences whose expression results in cell death (eg toxin genes) are selectively expressed in differentiated cells. For the above embodiments, it is important to use a promoter that is specific or substantially specific to the differentiated cell, such that in embryonic cells such a promoter encodes a lethal DNA sequence (eg, toxin). ), Which is operatively linked to, or is capable of expressing such a construct. One of the preferred promoters is that of the transforming growth factor β-2 gene, which is activated during ES cell differentiation regardless of the type of cell formed (Mummery et al. Dev Biol Biol. ), 137, pp. 161-170 (1990)). The genes encoding various toxins are placed under the control of promoters that are active in differentiated cells. For example, the diphtheria toxin subunit-A gene (Maxwell et al., Mol. Cell. Biol. ), 7, pp. 1576-1589 (1987)) or subunit of ricin toxin-A gene (Lande1 et al., Jeans and Dev. ), 2 pp. 1168-1178 (1988)) under the control of a promoter expressed only in differentiated cells. Again, either the native gene encoding the toxin or the non-native sequence can be used. Either (a) a selective marker gene under the control of a promoter specifically expressed in undifferentiated ES cells and / or (b) a toxin gene under the control of a promoter specifically expressed in differentiated cells Embryos capable of expressing either are obtained using standard methods. Such embryos may be transformed, in which case the heterologous DNA construct will be stably integrated into the embryo's genome, or non-integrated expressiable DNA. May be simply included. ES cells are then derived from embryos that have been transformed, injected or transfected by culturing under selective conditions. The embryos preferably used in the above method are also included in the concept of the present invention. According to a second concept of the invention, (a) a drug resistance gene (or other DNA sequence conferring drug resistance) under the control of a promoter which is specifically expressed in undifferentiated ES cells and / or (b) From expressible DNA consisting of a DNA sequence (such as a toxin gene or other DNA sequence encoding a toxin) whose expression results in cell death under the control of a promoter that is specifically expressed in differentiated cells To provide an embryo. The embryo may be transformed, in which case a transgene construct consisting of expressible DNA as described in (a) or (b) above will be integrated into the genome of the embryo. The transformed embryo in case (a) above is derived from a parent prepared or transformed de novo. The transformed embryo in case (b) above is prepared de novo without any special means so that the expression of the toxin construct in the differentiated cells causes the death of the whole animal. Therefore, transformed animals also form part of the present invention. According to a third concept of the invention, a transgene consisting of a drug resistance gene (or other DNA sequence conferring drug resistance) under the control of a promoter that is specifically expressed in undifferentiated ES cells. The present invention provides a transgenic non-human animal in which the construct has integrated into the genome. Embryos containing the selective DNA construct in case (a) above may be prepared by mating adult animals, at least one of which has been transformed, and then removing the embryos from the reproductive tract or by de novo It is obtained by carrying out transgenesis. Transgenesis is performed, for example, by microinjection or transfection of DNA. The microinjection method is the microinjection of DNA into zygotes or embryos at early division stages. The transformed mouse method is described in Hogan et al., “Manipulating the Mouse Embryo: A Laboratory Mannual”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory Press. (Cold Spring Harbor), New York (1986). Microinjection using substantially similar techniques, although less efficient, has been successfully applied to other species such as farm animals (Wall et al., Theriogenology, 38, p. 337-). 357 (1992) and Wilmut et al., Reprod Fert Supple (Reprod. Fert. Suppl. ), 43, pp.265-275 (1991)). Transformed animals are identified by DNA analysis of living tissues and are bred appropriately to produce transformed embryos. In most cases, as described below, such a transgene construct does not have to be a transgene construct integrated into the genome of the animal. Not all embryos according to the second concept of the invention are transformed. Alternatively, the expression construct may simply be introduced into the embryonic cell by a suitable means such as microinjection (eg, Burdon et al., Molleprod & Dev (Mol. Reprod. and Dev. ), 33, pp. 436-442 (1992)). Such a construct does not remain in the absence of the replicative sequence (and unless integrated) for a long period of time, but is maintained for a period sufficient to perform the method of the first concept of the invention on the embryo. Non-integer rating, non-replicating constructs of relatively short duration may be used when long-term undesired or when it is essential to remove them. It is actually preferred as it does not require the special step of removing the construct. The latter case applies to ES cells containing a toxin gene under the control of a promoter that is specifically expressed in differentiated cells. Such constructs are generally removed before ES cells can participate in embryonic development. A third possibility is that there is an expression construct in embryonic cells linked to a sequence that causes episomal replication. Vectors of this type of episome are described in bovine papillomavirus (Mathias et al., EMBO J. 2), pp. 1487-1492 (1983)) and adenovirus (Quantin et al., Proc National Academia (Proc. Natl'l. Acad. Sci. ), 89, pp. 2581-2584 (1992)). The advantage of using episomal vectors is that most embryos and their cultured derivatives contain long-term selection cassettes after microinjection. The selection method for the isolation of ES cells depends on the expression of ES-specific salvage transgenes or ES-specific expression of toxin transgenes that retain the ES lineage under general lethal selection conditions. Integration and maintenance of the transgene after isolation of ES is considered unimportant and is undesirable. Therefore, a short-term transfection system such as lipofection (Fergner et al., Nature, 337, p. 387-388 (1989)) is used for the above-mentioned methods, especially for large animals. Transient infection occurs under physiologically close conditions (Brunette et al., Nucl Acidsless. Acids. Res. ), Volume 20, page 1151 (1992)). In lipofection, DNA is spontaneously associated with a liposome containing a cationic lipid and then fused with a cell membrane to internalize the DNA. Suitable liposome preparations are commercially available. According to the present invention, there are many advantages to using lipofection as a transgenesis means. First, the method is considerably less laborious and time consuming compared to microinjection; thus, large numbers of embryos can be processed. Second, lipofection is a relatively mild treatment that can be used repeatedly without toxicity. Therefore, repeated lipofections aid in the presence of DNA in a high proportion of explanted embryos. Third, the selective DNA exists mainly as a non-integrated non-replicating molecule; therefore, unselected ES cells can be easily isolated when selection is removed. . DNA constructs that can be used for the preparation of embryos as described above are also included in the concept of the invention. According to a fourth concept of the invention, (a) a drug resistance gene (or other DNA sequence conferring drug resistance) under the control of a promoter which is specifically expressed in undifferentiated ES cells and / or (b) Provided is a DNA construct comprising a DNA sequence (such as a toxin gene or another DNA sequence encoding a toxin) that is expressed so as to cause cell death under the control of a promoter that is specifically expressed in differentiated cells. is there. Once the ES cell line is established, the selectable marker or toxin gene construct can be removed if necessary or desired. In the case of non-integrated non-replicating DNA (non-integrated non-replicating DNA), the removal is, in fact, automatic. More careful steps must be taken for transformed embryos and embryos in which the expression construct replicates episomally. Recently, the use of site-specific recombination systems has been demonstrated to accurately effect certain deletions in cultured mammalian cells. Gu et al. (Cell, Vol. 73, pp. 1155 to 1164 (1993)) show how deletion in the switch region of immunoglobulin of mouse ES cells is Cre site-specific. It has been described whether transient expression of recombinase occurs between two copies of the bacteriophage P1 loxP site, leaving a single loxP site. Similarly, yeast FLP recombinase was used to precisely eliminate the selectable marker defined by the recombinase target site in mouse erythrole ukemia cells (Fiering et al., Proc National. Academia USA (Proc. Natl'l. Acad. Sci. USA. ), 90, pp. 8469-8473 (1993)). The Cre lox system is specifically shown below, but other site-specific recombinase systems may be used. Constructs used in the Cre lox system generally have three functional elements: Expression cassette; A negative selectable marker expressed under the control of a ubiquitously expressed promoter (eg, thymidine kinase (TK) gene of Herpes si mplex virus) (eg, phosphoglycerate kinase (Soriano et al. , Cell, Volume 64, pages 693-702 (1991)); and 3. Bacteriophage P1 site-specific recombination site loxP (Baubonis et al., Nucl. Acids. Res. ), 21 volumes, pages 2025-2029 (1993)). The constructs described above are Cre recombinase proteins that can be introduced into cells by lipofection (Baubonis et al., Nucl. A cids. Res. ), 21, 2025-2029 (1993)), can be excreted from established ES cell lines containing the construct by means of site-specific recombination between the two loxP sites. Cells deficient in the DNA between the two loxP sites are deleted for the TK gene (or other negative selectable marker) by growing in medium containing the appropriate agent (ganciclovir in the case of TK). Selected by. Embryonic stem cells isolated by such selection methods can be tested for totipotency by assessing their ability to form adult tissues, and most importantly germ cells. Totipotent embryonic stem cells can be used as a means of manipulating the genome of an animal by introducing the transgene once or by the broader modifications possible using the targeting technology of the gene. Preferred aspects of each concept of the invention are the same as each other concept, mutatis mutandis. The present invention will be described in detail with reference to the following examples. This example refers to the drawings, which are as follows: Figures 1A to 1F are representative examples of embryoid bodies obtained in the same manner as described in Example 2. FIG. 2 shows the structure of the OctneoTL construct. Example 1 Preparation of Octneo DNA Construct A 1.94 kb fragment containing the 5'flank and promoter region of the Oct-3 / 4 gene was amplified from mouse genomic DNA by the polymerase chain reaction. The primer sequence used was the known sequence of Oct-3 / 4 (Okazawa et al., Embo J., vol. 10) with additional restriction enzyme cleavage sites added to facilitate cloning and construction. , Pages 2997-3005 (1991)). This Oct-3 / 4 fragment was used to obtain a construct, designated Octneo, designed to confer resistance to the drug G418 on undifferentiated ES cells. Octneo was constructed from the following three DNA fragments: 1.94 kb Oct-3 / 4 5'funking fragment with 5'SalI and 3'HindIII sites from PCR primers (1940 bp up to 34 bp upstream of translation initiation site); Modified aminoglycoside phosphotransferase (neo) gene and polyadenylation signal of human growth hormone (Selfridge et al., Som. Cell and Mol. Gen.), 18, 325-336. (1992)) and a 1.7 kb HindIII / EcoRI fragment; and PUC18 cut with EcoRI and SalI. Example 2 Expression of Octneo gene in ES cells The tissue specificity of Oct3 / 4-related neo expression was examined in the following two experiments: 2.1 Expression of Octneo in ES cells and fibroblasts Octneo DNA was expressed in NIH 3T3 (differentiated). Fibroblasts) and HM-1 (undifferentiated ES cells) (Magin et al., Nucl. Acids. Res., Vol. 14, p. 3795 (1992)). A control construct (designated "MTneo") in which the expression of neo was driven by the promoter of metallothionein was transfected in parallel. The number of colonies observed after selection with G418 is shown in Table 1 below: This indicates that the Oct3 / 4 promoter is active in ES cells but not fibroblasts. 2.2 Effect of Octneo expression on the in vitro differentiation of ES cells under G418 selection The Octneo and MTneo constructs were electroporated into HM-1 ES cells and G418 resistant clones were isolated. Next, a suspension culture method for inducing the differentiation of ES cells was used. This method is described in "Embry Derived Stem Cells (Teratocarcinomas and Embryonic Stem Cells, A Practical Approach)" by Robertson, EJ, "Teratocarcinomas and Embryonic Stem Cells". Embryo-Derived Stem Cells ", EJ. Robertson, published by IRL Press Ltd, Oxford, 1987. Differences from the Robertson method will be described in detail below. Differentiation of transfected clones under selective conditions was examined as follows. Day 0: Two clones of Octneo (Octneo2, 4) and one clone of MTneo (MTneo6) were cloned into Selfridge et al., (Som. Cell and Mol. Gen.), vol. 325-336 (1992)) and grown as a monolayer of undifferentiated ES cells on a gelatinized tissue culture plastic in ES culture. G418 was added at 0.2 mg / ml. The G418 concentration is twice the concentration required to kill untransfected HM-1 cells. Day 1: A monolayer of ES cells was transferred onto a non-gelatinized plastic in ES medium to induce cell aggregation. Day 2: ES cells were transferred as multicellular aggregates in suspension cultures of petri dishes for bacteria. The culture medium used was ES medium with reduced serum content (2.5% fetal serum, 2.5% neonatal serum) and no LIF. After several days under the above-mentioned conditions, ES cell aggregates are likely to form an embryoid body composed of an undifferentiated internal mass of cells and an outer layer of endoderm. The cells of each clone were divided into 4 groups with different concentrations of G418 in the culture medium as follows: 0 mg / ml, 0.5 mg / ml, 1.0 mg / ml, 1.5 mg / ml. Day 8: Embryoid bodies were induced to adhere to the matrix by transferring them to a gelatinized tissue culture dish. This allowed evaluation of the extent of differentiation that had already occurred in suspension culture. However, because reattachment itself produces stimulators that differentiate, the selection with G418 was continued for an additional week to kill the newly differentiated cells. During this period, growth was performed in ES medium containing LIF. The presence of LIF does not affect the viability of differentiated cells. Day 15: The attached embryoid bodies were fixed with methanol / acetic acid and stained with crystal violet. Differentiation was assessed by the presence of differentiated cells, predominantly endoderm halo, spreading on the substrate. The results are summarized in Table 2 and representative examples of embryoid bodies are shown in Figures 1A to 1F. These results indicate that the differentiated ES derivative of Octneo-transfected clones is killed at G418 concentrations of 0.5 mg / ml and 1 mg / ml, whereas the MTneo control differentiated derivative is viable. Be done. At higher concentrations of G418, the more common toxin was observed in both differentiated and undifferentiated cell types. Example 3 Induction of ES cells from embryos of Octneo-transformed mice The effect of drug selection on the efficiency of ES cell induction was examined. Octneo DNA was microinjected into mouse zygotes and 10 transgenic mouse lines (C57B1 / CBA hybrid) were obtained by standard techniques (Hogan et al., Supra). Transformed male progeny of founder animals from each transformed line were spontaneously crossed with females of strain 129 SV. Blastocysts were harvested and explanted intact onto feeder layers using the method described by Robertson (cited above). The feeder layer is Sto-neo cells inactivated with mitomycin C, which is an established fibroblast cell line transfected with a metallothionein-neo construct that confers resistance to G418. Each flush (ie, the contents of one uterus) was divided into a non-selected treatment group and a selected treatment group at a ratio of 1: 2. Selection was performed by including 100 μg / ml G418 in the culture medium. This concentration was previously established as the minimum amount needed to kill untransformed embryos within 10 days. Explants of early embryos were cultivated with 2-5 embryos in one well of a 48-well plate. This culture medium consisted of BHK21 Glasgow MEM supplemented with 10% neonatal serum, 10% fetal serum and mouse leukocyte blocking factor. In all other respects, isolation of ES cells was performed as described by Robertson (cited above). Embryos from 5 of the transformed mouse strains all died under the selective conditions shown below: However, embryos from the other five lines gave rise to the G418 resistant ES cell line. These data are shown below: Males used for mating were hemizygous for the Octneo transgene and half of the explanted embryos did not contain the Octneo transgene and therefore could not survive the selection with G418. Therefore, when comparing the induction rate of ES cells under selective conditions and non-selective conditions, the induction efficiency of ES cells under selective conditions must be doubled in order to explain such lethality. Thus: ES induction efficiency in the absence of G418: 9% Unregulated efficiency in the presence of G418: 13.5% True efficiency explaining death of non-transformed embryos: 27% Statistical significance (unrelated unrelated t test), p <0.05 These results show that selection of Octneo-transformed embryos with G418 significantly improves the induction efficiency of ES cell lines. Example 4 Induction of ES cells not containing Octneo DNA In many cases, it is desirable that ES cells do not have experimentally introduced DNA sequences. By using the selected construct with the modified agent, an ES cell line can be established and then removed from the host's genome. Such an example uses the bacteriophage P1 site-specific recombinase Cre and its target site loxP. The construct used to embody the above is referred to as OctneoTL, which, as shown in Figure 2, is composed of three functional elements: 1. Drug selection cassette (Octneo); Negative selectable marker expressed under the control of a ubiquitously expressed promoter (eg, thymidine kinase (TK) gene of Herpes sinplex virus) (eg, phosphoglycerate kinase (Soriano et al., Cell, 64, pp. 693-702 (1991)); and 3. Bacteriophage P1 site-specific recombination site loxP (Baubonis et al., Nukuru Acidsless) located at either end of the DNA fragment (Nucl. Acids. Res.), Volume 21, p. 2025-2029 (1993)) OctneoTL transformed animal strains were obtained as in Example 3 and ES cells were selected with G418. Induced from transformed embryos by culturing while establishing established ES cell lines by lipofection Site-specific between two loxP sites mediated by a Cre recombinase protein (Baubonis et al., Nucl. A cids. Res., 21: 2025-2029 (1993)) It can be made non-transformant by recombinant means.Cells deficient in the DNA between the two loxP sites are selected by deletion of the TK gene by growing in medium containing gan ciclovir as a drug. . Example 5 Induction of ES cells from large animal species Production of a transformant strain for the purpose of inducing ES cells is not feasible in livestock such as cattle because the period of one generation is long. In this case, the selectable construct is microinjected or transfected into the embryo and then placed directly in culture to induce ES cells. The selection cassette can be introduced as a DNA fragment into the embryo of a domestic animal in the same manner as described in Example 3 or 4, or into a vector designed to be able to replicate regardless of the host genome. Vectors of these types of episomes include bovine papillomavirus (Mathias et al., EMBO J., Vol. 2, pp. 1487-1492 (1983)) and adenovirus (Quantin et al., Proc. National Academia (Proc. Natl'l. Acad. Sci.), 89, pp. 2581-2584 (1992)). The advantage of using such episomal vectors is that most embryos and their cultured derivatives contain long term selective cassettes after microinjection. In contrast, embryos microinjected with known DNA fragments have only 10 to 15% of stable DNA incorporation. Non-integrated DNA is present in other embryos for about 1 week after injection. Removal of episomes from established strains is achieved by selection with the ganciclovir against the TK marker gene.

────────────────────────────────────────────────── ─── Continuation of front page    (72) Inventor Stacey, Alexander, Jarvie             S             Lotian E-Hit, UK 10 5             Peedy, Edinburgh, Dalhousiete             Russ 31 (72) Inventor Schnike, Angelica, Elizabeth             Lotian E-Hit, UK 10 5             Peedy, Edinburgh, Dalhousiete             Russ 31 (72) Inventor Dalrymple, Michael, Alexander             United Kingdom, Lothian E-Hatch 6 4             Etch, Edinburgh, North Four             To Street 21

Claims (1)

[Claims] 1. From embryo to embryo in culture, which consists in selectively killing the differentiated cells of the embryo. A method of selecting stem (ES) cells. 2. The method of claim 1, wherein the differentiated cells are killed by a drug selection method. The method described in. 3. The cells of the embryo are operably linked to a DNA sequence encoding a drug resistance marker. Of undifferentiated cell-specific or substantially specific promoters The method according to claim 2, which comprises an article. 4. The method according to claim 3, wherein the promoter is the Oct-3 / 4 promoter. How to list. 5. The promoter according to claim 3, wherein the promoter is the FGF-4 promoter. Method. 6. The drug resistance marker is neo, hygro or his, dhfr, gp Claims 3, 4 or 3 which can be encoded by the t, gs or ada genes. Alternatively, the method according to item 5. 7. Selective in differentiated cells with DNA sequences whose expression results in cell death The method according to claim 1, which is expressed in. 8. Embryonic cells are engineered to connect to a DNA sequence whose expression leads to cell death. Consists of a promoter that is specific or substantially specific to the committed differentiated cell 8. The method of claim 7, which comprises a construct. 9. The DNA sequence, whose expression causes cell death, encodes a toxin. The method according to claim 8 10. 10. The method according to claim 9, wherein the toxin is diphtheria toxin or ricin toxin. How to list. 11. The embryo according to claim 3, wherein the construct is transformed. The method according to any one of items 6 and 8 to 10. 12. 7. The construct according to claims 3-6, wherein the construct is capable of episomal replication in the embryo. And the method according to any one of items 8 to 10. 13. The method of claim 1, wherein the construct is present in the embryo in a non-integrated and non-replicating form. The method according to any one of 3 to 6 and 8 to 10. 14． (A) Under the control of a promoter that is specifically expressed in undifferentiated ES cells DNA sequences conferring drug resistance in and / or specifically in differentiated cells Is it a DNA sequence whose expression results in cell death under the control of an expressed promoter? An animal embryo comprising an expressible DNA comprising: 15. 15. The embryo according to claim 14, wherein the expressible DNA is transformed. The embryo according to paragraph. 16. Claims wherein the expressible DNA is capable of episomal replication in the embryo The embryo according to paragraph 14. 17． The expressible DNA is present on a bovine papillomavirus expression vector The embryo according to claim 16, which is: 18. The expressible DNA is present on an adenovirus expression vector, The embryo according to claim 16, which is: 19. The expressible DNA is present in the embryo in a non-integrated and non-replicating form. The embryo according to Item 14 of the scope of the invention. 20. Drug under the control of a promoter that is specifically expressed in undifferentiated ES cells A transgene construct consisting of a DNA sequence conferring resistance is integrated into the genome Transformed animal. 21. (A) Under the control of a promoter that is specifically expressed in undifferentiated ES cells DNA sequences conferring drug resistance in and / or specifically in differentiated cells Is it a DNA sequence whose expression results in cell death under the control of an expressed promoter? A DNA construct consisting of 22. Claims 3-6, wherein the construct is further removed from embryonic cells. And the method according to any one of items 8 to 10. 23. Claims wherein the construct is removed using a site-specific recombination system The method according to paragraph 22. 24. The site-specific recombination system is a Crelox system or a FLP system. 24. The method of claim 23, which is a system. 25. The removal of the construct is negative selectable marker hands under the control of constitutive promoters. 25. A method according to claim 22, 23 or 24, which is selected according to a step. the method of. 26. The negative selectable marker is herpes simplex virus 26. The method according to claim 25, which is a protein kinase (TK) gene. 27. The constitutive promoter is a phosphoglycerate kinase promoter 27. The method according to claim 25 or 26.