EP0716690A1 - Cellules souches pluripotentes d'embryons de rats et leur procede d'obtention et d'utilisation - Google Patents

Cellules souches pluripotentes d'embryons de rats et leur procede d'obtention et d'utilisation

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Publication number
EP0716690A1
EP0716690A1 EP94927285A EP94927285A EP0716690A1 EP 0716690 A1 EP0716690 A1 EP 0716690A1 EP 94927285 A EP94927285 A EP 94927285A EP 94927285 A EP94927285 A EP 94927285A EP 0716690 A1 EP0716690 A1 EP 0716690A1
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European Patent Office
Prior art keywords
rat
stem cells
cells
embryonic stem
cell
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EP94927285A
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German (de)
English (en)
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Philip M. Iannoccone
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Northwestern University
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Northwestern University
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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/235—Leukemia inhibitory factor [LIF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"

Definitions

  • the present invention relates to pluripotent embryonic stem cells derived from rat, to a method of obtaining and culturing the rat embryonic stem cells, and to cell culture media and conditions appropriate therefor, as well as to the use of such cells in the production of chimeric and transgenic rats.
  • gain-of-function mutations can be obtained by simple transfection of embryonic stem cells, or microinjection into oocytes of an extra DNA copy of the gene.
  • the phenotype produced by the extra copy will not be stable.
  • the allele will not exhibit Mendelian inheritance.
  • gene therapy for defective genes in some cases it may be necessary to replace the defective gene with the normal gene at the appropriate locus, since for proper expression, the gene may need to be integrated in a region of "active chromatin".
  • such gene replacement may be necessary to maintain the appropriate number of regulatory sequences in the cell by preventing an increased number of gene promoters from diluting out regulatory molecules.
  • the technology for targeted gene modification was available for only purportedly 'simple' species such as bacteria or yeast. More recently, the technology has become available for higher-level eukaryotes, e.g., the fruit fly, and has been extended to the mouse.
  • the advent of targeted in vivo gene mutagenesis in mouse was made possible in part by the isolation and establishment in culture of pluripotent cells from in vitro cultures of mouse blastocysts (Evans et al., Nature. 292. 476-480 (1981)).
  • pluripotent cells have a normal karyotype and are able to differentiate in vitro, or after inoculation into a mouse. More importantly, these pluripotent cells can be employed as a vehicle for the transfer into the mouse genome of mutant alleles, which are either selected in cell culture, inserted into the cells via transformation with specific DNA fragments, or integrated into the genome of the pluripotent stem cells. Moreover, the ability of these cells to colonize the germ line can be further exploited by coupling this capability with methods for insertional mutagenesis and targeted disruption of specific genes. The resultant phenotype can be examined in the living organism.
  • colonization of the embryo including the germ line with pluripotent stem cells can be used to generate a chimeric animal
  • introduction of exogenous DNA into the pluripotent cells prior to colonization, or insertional mutagenesis of these cells can be used to generate a transgenic animal.
  • a transgenic animal is one which possesses an alteration in its DNA as a result of intentional experimental intervention. The production of a transgenic animal may be greatly facilitated if a library of chromosomal genes from the species is available. In certain species, transgenic animals can be produced by simply microinjecting DNA into the zygote, or by transfecting the embryo with recombinant retroviral vectors incorporating the transgene.
  • embryonic stem cells as a vehicle for gene transfer has many advantages over these approaches.
  • employment of embryonic stem cells allows extensive in vitro genetic manipulation, selection, and screening prior to actual generation of the transgenic animal.
  • this approach circumvents the tandem, head-to-tail integration of exogenous DNA at a single chromosomal site which can be observed using other approaches.
  • pluripotent cell route to chimera formation is available for mouse, the approach has been hampered in other species due to an inability to obtain pluripotent cells, or due to an inability to obtain pluripotent cells capable of contributing to chimera formation.
  • pluripotent cells have been isolated from mink (Sukoyan et al., Mol. Reprod. Dev.. 33. 418-431 (1992)) and hamster (Doetschman et al., Dev. Biol.. 127. 224-227 (1988)).
  • the mink cells are apparently limited in their pluripotential capability, as such cells are unable to contribute to chimera formation.
  • the present invention provides pluripotent embryonic stem cells derived from rat. These cells are capable of prolonged growth in culture in the absence of overt differentiation. Methods and cell culture media and conditions appropriate for the isolation of the cells, as well as morphological details enabling recognition of the cells, are provided herein.
  • the present invention also provides methods and cell culture media and. conditions for the maintenance of the pluripotent embryonic stem cells in vitro. Under appropriate culture conditions, however, the cells are capable of differentiation into an array of cell types which predominate in the developing embryo. This propensity of the embryonic stem cells attests to their pluripotent nature. Accordingly, the present invention further provides methods for inducing differentiation of the pluripotent embryonic stem cells.
  • the pluripotent nature of the embryonic stem cells is further corroborated by their ability to contribute to chimera formation.
  • the present invention therefore, further provides methods for chimera production, as well as methods for the generation of transgenic rats.
  • Figures 1A-H are a series of photomicrographs demonstrating the various stages of rat embryonic stem cell (RESC-01) isolation:
  • D £ F) higher magnification showing endoderm differentiation (Nomarski optics, bar 20 ⁇ m) ;
  • Figure 2 is a graph of time (day) versus cell number (x 10 5 ) for RESC-01 cells grown on HREF embryonic fibroblasts (solid line) , STO mouse embryonic fibroblasts (long dashes) , gelatin-coated plastic (stippled line) , or plastic (short dashes) .
  • the growth curves were obtained by plating RESC-01 cells in the presence of 500 units/ml of LIF.
  • Figure 3 is a graph of time (day) versus differentiated colonies (%) for RESC-01 cells grown on HREF embryonic fibroblasts (solid line) , STO mouse embryonic fibroblasts (long dashes) , gelatin-coated plastic (stippled line) , or plastic (short dashes) .
  • Figures 4A-B are graphs of time (day) versus cell number (x 10 5 ) for RESC-01 cells grown on HREF embryonic fibroblasts (A) and gelatin-coated plastic (B) .
  • the growth curves were obtained by plating RESC-01 cells in the presence of a LIF concentration of 0, 100, 500, 1000 or 2000 units/ml.
  • Figures 5A-B are graphs of time (day) versus differentiated colonies (%) for RESC-01 cells grown on HREF embryonic fibroblasts (A) and gelatin-coated plastic (B) .
  • RESC-01 cells were plated in the presence of a LIF concentration of 0, 100, 500, 1000 or 2000 units/ml.
  • An injection pipette shown on the right contains a RESC-01 cell.
  • mammalian embryogenesis proceeds in a remarkably similar fashion across species.
  • numerous species-specific differences in development can be tabulated. While a single variation between species may appear subtle and of little consequence, when considered in the aggregate, such variations evince dramatic species- specific differences in embryogenesis.
  • procedures for the isolation of embryonic stem cells and use of such cells for the production of chimeric or transgenic animals may have been developed for other species, important rat-specific differences in embryologic development preclude the verbatim application of such procedures for the isolation of rat pluripotent embryonic stem cells and for the production of chimeric and transgenic rats.
  • timing is a critical concern in the present invention, in terms of the appropriate time for isolation of embryos from which pluripotent embryonic stem cells can be obtained, the length of time such embryos should be maintained in culture prior to isolation of cells, and even down to the smallest detail of recognizing when to passage or refeed cells.
  • tissue culture conditions are of importance in this invention, not only for enhancing the efficiency of pluripotent rat embryonic stem cell isolation, and incorporation of such cells into an early stage embryo, but also for maintaining embryonic stem cells in an undifferentiated state. Moreoever, recognition of the different development stages and appropriate manipulation at each stage is a relevant factor in the present invention.
  • one of the key elements in embryonic stem cell isolation is to disrupt development of the isolated embryo prior to extensive differentiation, but at a point when the stem cell component is sufficiently large to survive.
  • the present invention provides, among other things, substantially pure pluripotent embryonic stem cells from rat. More specifically, the present inventive embryonic stem cells are obtained from a preimplantation embryo.
  • a preimplantation embryo is an organism in an early stage of development occurring in the period immediately following fertilization of the egg, up until implantation into the wall of the uterus, such as, for example, the eight-cell, morula or blastocyst stage.
  • an "embryonic cell” is any cell that can be obtained from such a preimplantation embryo.
  • the present invention also provides a method for obtaining the rat pluripotent embryonic stem cells from a preimplantation embryo. Since the appropriate timing for isolation of the preimplantation embryo, as well as. isolation of putative stem cells from this embryo, is important to this invention, the present invention accordingly provides information concerning the stage of development at which the preimplantation embryo can be isolated and placed in culture for the purpose of isolating pluripotent embryonic stem cells, as well as the length of time of maintaining the embryo culture which is sufficient to allow the cultured preimplantation embryo to obtain an appropriate size and stage of development from which potential stem cells can be separated by disruption, and the manner in which this disruption and subsequent culture of disrupted fragments is to be conducted.
  • the preimplantation embryo is a blastocyst.
  • the substantially pure pluripotent embryonic stem cells are obtained by removing a preimplantation embryo, preferably a blastocyst, from a rat uterus.
  • a rat blastocyst obtained between days 4 and 5 of pregnancy, particularly day 4.5, is at an appropriate developmental stage to allow isolation of pluripotent embryonic stem cells. This is in distinct contrast to other species, in which embryonic development proceeds differently than in rat, resulting in a difference in time when preimplantation embryos are isolated.
  • embryos appropriate for isolation of stem cells are obtained on day 3.5 of pregnancy (Doetschman et al., Dev. Biol.. 127.
  • the present invention provides preferred culture conditions, for example, a preferred feeder cell layer, which are appropriate to employ for obtaining rat pluripotent embryonic stem cells from isolated preimplantation embryos.
  • the invention also provides conditions for maintaining such cells in culture in the absence of cell differentiation.
  • a preferred method of culturing embryonic stem cells comprises maintaining the cells in the presence of leukemia inhibitory factor (LIF) , or culturing the cells on a feeder cell layer. LIF has been shown to inhibit the differentiation of mouse embryonic stem cells in culture, even in the absence of embryonic fibroblast feeder layers (Pease et al., Exp. Cell Res.. 190.
  • feeder cell layer of the present invention is comprised of rat embryonic fibroblasts, one skilled in the art will recognize that additional means and agents can similarly be utilized to impede differentiation of rat pluripotent embryonic stem cells.
  • rat stem cells The isolation of rat stem cells is facilitated through use of a feeder cell layer for culturing rat blastocysts which differs from the feeder cell layers employed for murine, hamster, and ungulate species.
  • the feeder layers on which the blastocysts are placed is preferably comprised of primary embryonic fibroblasts isolated from midgestation Holtzman strain fetuses (i.e., obtained on the 14th day of pregnancy) by maceration and trypsin treatment of the embryo carcass.
  • the HREF (Holtzman strain Rat Embryonic Fibroblast) cells are maintained under suitable conditions, e.g., in a 5% C0 2 atmosphere at 37°C in DMEM containing 10% FBS (Intergen Co.), 2 mM L-glutamine and penicillin/streptomycin. Growth arrest of the fibroblasts may be achieved by any suitable means, e.g., incubating the cells with fresh medium containing mitomycin-C (10 ⁇ g/ml; Sigma, St. Louis, MO, M- 0503) for 4 hours. The cells may then be plated after suitable exposure to the growth arrest medium, e.g., 24 hours after exposure to mitomycin-C, at a suitable density.
  • the preimplantation embryos particularly blastocysts, which are removed from the rats are placed on rat embryonic fibroblast feeder layers in appropriate culture dishes, e.g., organ culture dishes.
  • a medium needs to be employed for in vitro culture which will facilitate the growth of the preimplantation embryos, e.g., blastocysts (Van Winkle et al., Dev. Biol.. 142. 184-193 (1990); Ng et al., In: Current Topics in Developmental Biology. R.A. Pederson, ed. (San Diego: Academic Press, 1992) 235-274) .
  • a cell culture medium such as Markert's modification of Whittingham's medium (Yamamura et al., Dev. Genet.. 2, 131-146 (1981)) is preferred, particularly when further supplemented with 20% FBS (lot-screened for mouse ES cell growth, Intergen Co.
  • a cell culture medium such as DMEM supplemented as indicated for Markert's modification of Whittingham's medium may be employed to replace the supplemented version of Markert's modification of Whittingham's medium, either entirely, or only in the later stages of isolation of rat stem cells.
  • DMEM cell culture medium
  • These media which were found to be appropriate for growth of rat blastocysts and isolation of rat stem cells, are different from the supplemented DMEM medium that is employed for isolation of murine (Robertson, In: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. E.J.
  • the preimplantation embryos attach within a reasonable period of culturing, e.g., within about 48 hours of culturing the blastocysts, and then hatch from the zona pellucida.
  • the medium is preferably changed every day after the first 48 hours of culturing.
  • DMEM supplemented as indicated previously for Markert's modification of Whittingham's medium has been found to be best suited for sustaining rat embryo growth at this stage, as evidenced by the increased survival and decreased differentiation of rat blastocysts cultured using DMEM.
  • the rat blastocysts After a suitable period of time, generally about 72 hours, some of the attached blastocysts will have expanded inner cell mass (ICM) populations. Between that time and another about 12-24 hours, i.e., between about 72 and 94 hours of incubation, the rat blastocysts will have typically achieved an appropriate size and level of development to allow disaggregation of an ICM-derived component. It is important to isolate ICM cells only after the rat blastocysts have reached a stage where substantial ICM proliferation has occurred but cells have not yet differentiated into endoderm cells. Thus, the ICM outgrowth will preferably be disrupted by suitable means, e.g., by pipetting, at about 96 hours following incubation.
  • suitable means e.g., by pipetting
  • the cells are preferably fed a few hours, e.g., about two hours, prior to this disruption.
  • the ICM outgrowth can be dissociated with use of trypsin, as is done for murine and ungulate species.
  • trypsin as is done for murine and ungulate species.
  • this is not a preferred means of dissociation, as trypsinization of rat blastocysts, for a reason which is unclear, deleteriously impacts upon the ability to isolate pluripotent stem cells from the rat blastocyst fragments. Accordingly, in the case of the rat, trypsinization needs to be done with extreme caution.
  • the individual disrupted ICM outgrowths are then transferred to separate dishes, e.g., organ culture dishes, precoated with HREF feeder layers and are maintained in supplemented DMEM. Additional fresh medium is added as needed, e.g., about every 24 hours, and the medium is preferably changed every 48 hours.
  • the growing colonies derived from the initial ICM are disrupted, preferably mechanically, over a suitable period of time, e.g., every day within the same dish for 5 days. This repetitive disruption, preferably mechanical disruption, of colonies derived from the ICM is yet another step which appears important in the isolation of rat embryonic stem cells, but not stem cells from other species.
  • the HREF feeder layers When a significant portion, e.g., about 50%, of the HREF feeder layers are covered with colonies, single cell suspensions of the embryonic cells from the rat blastocysts are made with trypsin and placed on new HREF feeder layers.
  • the rat embryonic cells are expanded to a suitable level. e.g., about 75% confluence, trypsinized, and passaged on HREF feeder layers in suitable tissue culture plates.
  • the cells can be frozen in freezing vials in a suitable medium, e.g., Gibco freezing medium, after the addition of fresh DMEM medium, preferably about 2 hours after the addition of fresh DMEM medium.
  • the cultures are preferably refed a suitable period of time, e.g., 2 hours, prior to exposure to trypsin.
  • the cells may also be frozen on plates by feeding cells with supplemented DMEM and then, after a suitable period of time, e.g., 2 hours later, replacing the DMEM with a suitable freezing medium, e.g., with about 400 ⁇ l of Gibco freezing medium.
  • the plates should be tightly wrapped in thin plastic film and stored at a suitably low temperature, e.g., in a -70°C freezer.
  • the cells may be thawed by any suitable means, e.g., by adding 600 ⁇ l of prewarmed DMEM medium, immediately aspirating off the medium, and adding 1 ml of prewarmed DMEM
  • the resultant rat embryonic stem cells derived from rat blastocysts are typically rather small, e.g., about 10- 20 microns across, and are flat-appearing when observed with Nomarski optics. Under optimal culture conditions differentiation of the embryonic stem cells does not occur.
  • the rat embryonic stem cells have a prominent nucleus containing one or more nucleoli and typically contain a minimal amount of cytoplasm. The cells can be demonstrated to be diploid by karyotype analysis, and there are no obvious borders between the cells in culture.
  • the identification of the rat embryonic stem cells may be validated by carefully observing the growth of the putative colony.
  • the preferred rat embryonic stem cell colony of the present invention exhibits growth and an absence of overt differentiation, as well as an ability to contribute to chimera formation.
  • These characteristics of the rat embryonic stem cells are indicative of pluripotent stem cells.
  • the rat pluripotent embryonic stem cells of the present invention may be maintained in culture and grown on various substrata.
  • the stem cells can be plated onto feeder layers in DMEM supplemented as previously described for the isolation of rat embryonic stem cells.
  • the stem cells are preferably grown on HREF fibroblast feeder layers, since, while they grow on STO mouse fibroblast feeder layers, as well as on gelatin-coated plastic and even plastic, they do not grow as well on these other materials.
  • the present inventive rat embryonic stem cells can be maintained in culture in the undifferentiated state using highly purified LIF.
  • the proportion of differentiated rat embryonic stem cells decreases as the concentration of LIF is increased, particularly when the stem cells are plated on HREF feeder layers, as LIF more effectively retards differentiation when the rat embryonic stem cells are plated on HREF feeder layers, without, however, influencing the proliferation of the HREF embryonic fibroblasts.
  • the supplemented DMEM used in the present invention preferably contains a suitable amount of LIF, e.g., at least about 500 units/ml LIF, preferably at least about 1000 units/ml LIF, most preferably at least about 2000 units/ml LIF, and as high as 10000 units/ml LIF or more.
  • LIF low-density lipoprotein
  • the apparent necessity of such high concentrations of LIF to retard differentiation has also not been reported for other species.
  • the present inventive rat pluripotent embryonic cells are useful in that they can be employed to generate chimeric, as well as transgenic, rats.
  • One skilled in the art will recognize that selection of the rat strain to use for chimera formation is important, but that a variety of strains may be suitably employed, both in terms of the strain appropriate for embryonic stem cell isolation and the strain appropriate for isolation of the preimplantation embryo into which the embryonic stem cells will be incorporated.
  • the present invention provides a preimplantation embryo or embryonic cell into which one or more of the rat embryonic stem cells, or nucleuses of the cells, have been introduced, as well as a method of incorporating one or more rat pluripotent embryonic stem cells into a rat preimplantation embryo and, in particular, a rat blastocyst.
  • the present invention also provides a chimeric or transgenic rat which is the progeny of such a preimplantation embryo or embryonic cell.
  • the introduction of the present inventive rat embryonic stem cells, or nucleuses of the cells, into a preimplantation embryo or embryonic cell may be accomplished by any suitable means.
  • the stem cells may be injected into rat blastocoel cavities as described for injection into mouse blastocoel cavities (Bradley, In: Teratocarcinomas and embryonic stem cells: a practical approach. E.J. Robertson, ed. (Oxford: IRL Press, 1987) 113-151) , with necessary modifications being made to accommodate species-specific differences in embryological processes.
  • rat blastocysts appropriate for injection are obtained on day 4.5 of pregnancy as compared with day 3.5 for mice.
  • the rat morula stage embryo differs distinctly from that of the mouse in being planar in shape (Yamamura et al., Dev. Genet.. 2., 131-146 (1981); Weinberg et al., J. Cell Sci.. 89., 423-431 (1988)).
  • This geometry is maintained in the rat blastocyst which is characteristically ovoid in shape.
  • the mouse blastocyst undergoes a period of prolonged expansion prior to implantation in the uterus
  • the rat blastocyst implants quite rapidly following a short period of expansion. Accordingly, based on the characteristic ovoid shape of blastocysts isolated from the rat, it may be necessary to expand the blastocyst prior to microinjection of the stem cells, e.g., for rat chimera formation.
  • the expansion of rat preimplantation embryos, especially blastocysts, prior to introduction of the embryonic stem cells may be accomplished by any suitable means, preferably by incubation in a cell culture medium comprised of a suitable carbon source, minerals, buffers, proteins, carboxylic acids, and carboxylic acid derivatives.
  • the expansion is most preferably effected by incubating the blastocyst at 37°C in 5% C0 2 atmosphere for about two hours in Brinster's medium (Brinster, In: Pathways to Conception (Springfield: Charles G.
  • tissue culture grade reagents 554.5 mg NaCl, 35.6 mg KC1, 16.2 mg KH 2 P0 , 14.3 mg anhydrous MgS0 4 , 210.5 mg NaHC0 3 , 100 mg dextrose, 5 mg streptomycin sulfate, 6 mg penicillin G, 100 mg albumin (BSA Pentax fraction V), 5.6 mg sodium pyruvate, and 0.38 ml of D,L-sodium lactate.
  • the medium is brought to a final volume of 100 ml.
  • the expanded blastocysts are placed in the microdrop of the medium along with the stem cells.
  • a suitable number of stem cells e.g., about 10 to 30 stem cells, can be injected into the blastocoel using conventional means, preferably a DeFunbrune pump.
  • one or more pluripotent embryonic stem cells may be incorporated into a rat embryo by coculturing a rat preimplantation embryo with the pluripotent embryonic stem cells.
  • This preferred method is similar in certain respects to an approach used in mice (Wood et al., Proc. Natl. Acad. Sci.. 90. 4582-85 (1993)).
  • the cells are preferably prepared in supplemented DMEM medium containing LIF, for example, in a concentration of at least 500 units/ml, most preferably in a concentration of about 2000 units/ml, to which is then added the rat embryos, preferably morula stage rat embryos isolated from pregnant rats at day 3.5 of pregnancy.
  • the embryos are preferably cultured in a suitable medium, e.g., in particular in an organ culture dish coated with heat-inactivated rat serum and containing Markert's modification of Whittingham's medium that had been adjusted with water to an osmolality of 295 mOsm/kg H 2 0.
  • a suitable period of culturing time e.g., about 20-30 hours, rat embryos are obtained to which have been introduced the embryonic stem cells of the present invention.
  • a nucleus of a pluripotent embryonic cell may be introduced into a rat embryonic cell by replacing the pronuclei of a fertilized rat egg with the nucleus from the pluripotent embryonic stem cell.
  • the preimplantation embryo could be transferred to a pseudopregnant surrogate mother. This approach has been suggested for mouse (Palmiter et al., Ann. Rev. Genet. f 20. 465-99 (1986)).
  • rat embryonic stem cells may be employed to introduce rat embryonic stem cells into the rat preimplantation embryo in the event that strain-specific differences, or other factors, render the method of expansion of the preimplantion embryo and, in particular, the method of expansion of the blastocyst, ineffective for a particular strain.
  • the preimplantation embryos are transferred to the uteri of surrogate mothers on day 3 or 4, preferably day 3.5, of pseudopregnancy of the rats. This is another example of a difference between the rat and other species, inasmuch as such transfer is routinely performed on day 2.5 for the mouse.
  • Pseudopregnancy in the surrogate rats may be established by a variety of means, preferably by the mechanical stimulation of the cervix during estrus (Ng et al., In: Current Topics in Developmental Biology. R.A. Pederson, ed. (San Diego: Academic Press, 1992) 235-274) .
  • the timing of pseudopregnancy can be established by noting the day of onset of leukocytes in the vaginal smear (Ng et al., supra) .
  • These novel methodologies developed expressly for the rat facilitated rat chimera production by providing greater control over the manipulation of embryologic stages. It has been established that methods to reliably establish and date pseudopregnancy in the rat will impact upon the ability to produce chimeras with high efficiency (Weinberg et al., Science. 227. 524-527 (1985); Iannaccone et al.. Development. 99. 187-196 (1987); Iannaccone et al., J. EXP. Zool.. 243. 217-223 (1987); Iannaccone et al.
  • the surrogate mothers may be allowed to deliver naturally, thereby resulting in the preparation of rat chimeras.
  • germ line chimeras are obtained as a result of the present invention; otherwise, somatic cell chimeras are obtained.
  • the present invention also provides for the modification of the rat pluripotent embryonic stem cells prior to introduction into the preimplantation embryo, especially for the purpose of preparing transgenic rats which are the progeny of such embryos.
  • the rat embryonic stem cells can be modified prior to incorporation, and appropriate screens can be conducted to select for rat embryonic stem cells exhibiting the desired properties.
  • a genetic marker can be introduced into the rat embryonic stem cells, or the cells can be infected with viruses, or treated with viral, chemical, or physical agents which alter certain properties of the cells.
  • the rat embryonic stem cells can be fused with cells from another species.
  • the present invention provides a preimplantation embryo into which has been introduced rat pluripotent embryonic stem cells which comprise genetic material with at least one change therein, as well as an embryonic cell into which has been introduced a nucleus of such a rat pluripotent embryonic stem cell which comprises genetic material with at least one change therein.
  • rat pluripotent embryonic stem cells which comprise genetic material with at least one change therein
  • an embryonic cell into which has been introduced a nucleus of such a rat pluripotent embryonic stem cell which comprises genetic material with at least one change therein.
  • DNA deoxyribonucleic acid
  • RNA Ribonucleic acid
  • oligonucleotides to the extent that they impact on gene expression, can also be considered genetic material.
  • the present invention also contemplates so-called antisense and triple helix DNA approaches, as well as other approaches, including use of ribozymes, particularly those which act in a sequence-specific fashion, which exert an effect, albeit a transient one, on gene expression.
  • gene expression is defined as including any stage or activity from transcription of nascent mRNA to appropriate modification and transport of translated protein, such as, for example, elongation of initiated message or translocation of nascent message from the nucleus to the cytoplasm.
  • the change in genetic material is selected from the group consisting of addition of a DNA segment, rearrangement of a DNA segment, deletion of a DNA segment, replacement of a DNA segment, methylation of unmethylated DNA, demethylation of methylated DNA, and introduction of a DNA lesion.
  • the DNA segment can be as small as one nucleotide, can be single-stranded or double-stranded, and can be a mixture of single-stranded and double-stranded regions.
  • the addition of a DNA segment to the rat pluripotential embryonic stem cell can be done by the actual physical integration of the segment into the genome as well as by introduction of the segment in an autonomously replicating vector, as is known in the art. Such addition can be accomplished using molecular or genetic techniques, or a combination of techniques.
  • a DNA lesion can include but is not limited to a missing base or altered base (e.g., an alkylated base), a cyclobutyl dimer, DNA strand breaks, and cross-linking of DNA strands.
  • rat embryonic stem cells can be transfected with mammalian expression vectors, enhancer trap vectors, promoter-probe vectors, vectors in which the subcloned DNA is under the control of its own cis-acting regulatory elements, and vectors which are designed to facilitate gene integration or gene replacement in host cells.
  • homologous recombination requires correspondence of portions of the exogenous DNA with segments of the endogenous DNA (i.e., correspondence of segments flanking both 5' and 3' ends of the gene for double-strand crossover events resulting in gene replacement, and correspondence with segments flanking either the 5' or 3' end of the gene for single-strand crossover events resulting in gene integration)
  • homologous recombination is facilitated using a gene or chromosomal library of genes subcloned into a vector containing portions of the long and short arm of the chromosome which flank the relevant gene, as well as containing an additional selectable gene which confers upon host cells some particular characteristic, such as, for example, antibiotic resistance.
  • the present invention also contemplates untargeted mutagenesis, as, for example, by appropriate treatment with mutagens.
  • any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site-directed mutagenesis (Hutchinson et al., J. Biol. Chem. , 253. 6551 (1978)), as well as any commercial kit or product for mutagenesis.
  • a DNA sequence may or may not be subcloned into a vector used for transfection. Potential DNA/ sequences which may be present include but are not limited to: coding sequences for structural or regulatory genes and non-coding sequences important in the regulation of gene expression, or important in the processing or transport of nascent DNA or protein.
  • the DNA sequences may be those found in nature or may be entirely or partly engineered.
  • the introduced nucleic acid may be RNA.
  • any means including any type of plasmid or non- plasmid vector, such as a cosmid or modified virus, may be employed to introduce the DNA sequence into the rat pluripotent embryonic stem cells.
  • the DNA may be introduced as a liposome-DNA complex or attached to an adenoviral capsid.
  • the vector system must be compatible with the rat pluripotent embryonic stem cells.
  • Vectors can be introduced into the stem cells via transformation, transfection, infection, electroporation, etc. Accordingly, the present invention provides a method of making a preimplantation embryo into which has been introduced rat pluripotent embryonic stem cells which comprise genetic material with at least one change therein.
  • the present invention provides a method of making a rat embryo into which has been introduced rat pluripotent embryonic stem cells which comprise genetic material with at least one change therein. Also, the present invention provides a method of introducing into a rat embryonic cell a nucleus of a rat pluripotent embryonic stem cell which comprises genetic material with at least one change therein.
  • the present invention includes the method of producing a chimeric or, more specifically, transgenic rat by transferring the preimplantation embryo, particularly the blastocyst, or transferring the embryonic cell, to which the rat embryonic stem cells, or nuclei of these cells containing altered genetic material have been incorporated, to the uteri of pseudopregnant rats.
  • the techniques appropriate for both inducing and dating pseudopregnancy in the rat, as well as the timing of embryo transfer, for such altered embryonic stem cells are the same as for the unaltered embryonic stem cells.
  • the present invention provides a method of producing a rat containing a particular allele in the homozygous state, e.g., rats which are homozygous for DNA sequences introduced into or altered in the rat pluripotent embryonic stem cells.
  • a particular allele in the homozygous state e.g., rats which are homozygous for DNA sequences introduced into or altered in the rat pluripotent embryonic stem cells.
  • Such rats can be produced by mating with each other chimeric rats which have been produced using the aforesaid methods of the present invention and which are germ line chimeras.
  • the skilled artisan will know the appropriate breeding experiments to perform to verify germ line chimerism.
  • the allele is lethal when homozygous, as for example are certain mutations of essential genes, the allele can be maintained in the heterozygous state.
  • Example 1 This example confirms that the blastocyst of the rat can be manipulated to give rise to a culture of pluripotent stem cells which can be maintained in vitro.
  • Pluripotent stem cells were derived from blastocysts obtained from the inbred PVG strain of black-hooded rats (Festing et al.. Transplantation. 16. 221-245 (1973)) carrying the RT1 C haplotype.
  • PVG rats were used because of the availability of congenic strains which can be distinguished at a major histocompatibility class I locus by using particular monoclonal antibodies (Howard et al.. Immunology. 41. 131-141 (1980) ) . Moreover, this strain can easily be distinguished visually from the strain selected as host for chimera formation.
  • the blastocysts (PVG-J? ⁇ c x PVG-i?T2 ⁇ ) were removed from the rat uterus on day 4.5 of pregnancy by sacrificing the animal and flushing out the uterine horns with a balanced salt solution (T6', which may alternatively be designated T6 or T6'M310; Van Winkle et al., Dev. Biol.. 142. 184-193 (1990); Ng et al., In: Current Topics in Developmental Biology. R.A. Pederson, ed. (San Diego: Academic Press, 1992) 235-274).
  • T6' which may alternatively be designated T6 or T6'M310; Van Winkle et al., Dev. Biol.. 142. 184-193 (1990); Ng et al., In: Current Topics in Developmental Biology. R.A. Pederson, ed. (San Diego: Academic Press, 1992) 235-274).
  • blastocysts were placed on rat embryonic fibroblast feeder layers in organ culture dishes using rat embryo medium, particularly Markert's modification of Whittingham's medium (Yama ura et al., Dev. Genet.. 2., 131- 146 (1981)) supplemented with 20% FBS (lot-screened for mouse ES cell growth, Intergen Co.
  • the feeder layers on which the blastocysts were placed were comprised of primary embryonic fibroblasts isolated from midgestation Holtzman strain fetuses (i.e., obtained on the 14th day of pregnancy) by maceration and trypsin treatment of the embryo carcass.
  • the HREF cells were maintained in a 5% C0 2 atmosphere at 37°C in DMEM containing 10% FBS (Intergen Co.), 2 mM L-glutamine and penicillin/streptomycin. Growth arrest of the fibroblasts was achieved by incubating the cells with fresh medium containing mitomycin-C (10 ⁇ g/ml; Sigma, St. Louis, MO, M- 0503) for 4 hours.
  • the cells were then plated 24 hours after exposure to mitomycin-C at a density of about 5 x 10 5 cells per 60 mm culture dish.
  • the feeder layers were at a density of about 10 6 cells per culture dish at the time of coculturing with blastocysts.
  • the blastocysts attached within 48 hours of culturing, and subsequently hatched from the zona pellucida.
  • the medium was changed every day after the first 48 hours of culturing, with the medium being DMEM supplemented as indicated previously for Markert's modification of Whittingham's medium. This medium was employed in all subsequent examples, with only the concentration of LIF being varied as indicated. Moreover, it is conceivable that this medium may be employed even in the initial stages of rat embryonic stem cell isolation, in replacement of Markert's modification of Whittingham's medium.
  • the rat blastocysts had expanded inner cell mass (ICM) populations as exhibited in Figure IA.
  • ICM inner cell mass
  • the rat blastocysts had achieved an appropriate size and level of development to allow disaggregation of an ICM-derived component, e.g., the rat blastocysts had reached a stage where substantial ICM proliferation had occurred but the cells had not yet differentiated into endoderm cells.
  • the ICM outgrowth was disrupted by pipetting at about 96 hours following incubation. The cells were fed two hours prior to this disruption.
  • the individual disrupted ICM outgrowths were transferred to separate organ culture dishes precoated with HREF feeder layers, and were maintained in supplemented DMEM. Additional fresh medium was added every 24 hours, and the medium was changed every 48 hours. The growing colonies derived from the initial ICM were mechanically disrupted every day within the same dish for 5 days. The appearance of the colonies following this mechanical disruption is shown in Figure IC.
  • Cells were frozen in freezing vials in Gibco freezing medium 2 hours after the addition of fresh DMEM medium. The cultures were routinely refed 2 hours prior to exposure to trypsin. Cells were also frozen on plates by feeding cells with supplemented DMEM and 2 hours later, replacing the DMEM with 400 ⁇ l of Gibco freezing medium. The plates were tightly wrapped in thin plastic film and stored in a -70 ⁇ C freezer. Cells were thawed by adding 600 ⁇ l of prewarmed DMEM medium, immediately aspirating off the medium, and adding 1 ml of prewarmed DMEM medium.
  • the resultant rat embryonic stem cell line derived from the blastocyst was designated RESC-01, for Rat Embryonic 85tem cells, C haplotype.
  • RESC-01 Rat Embryonic 85tem cells
  • C haplotype Rat Embryonic 85tem cells
  • a flat-appearing RESC- 01 colony is exhibited in Figure IB.
  • Example 2 This example sets forth some of the identifying characteristics observed for rat embryonic stem cells in culture.
  • Rat embryonic stem cells obtained in Example 1 were typically rather small. The cells ranged in size from about 10-20 microns, with most cells being about 13 microns. As exhibited in Figure IB, colonies of the cells appeared flat when observed with Nomarski optics. Under optimal culture conditions differentiation of the embryonic stem cells did not occur. Differentiation could be observed initially, as shown in Figure IB, by a surrounding at the periphery of the colony of endoderm cells.
  • the rat embryonic stem cells had a prominent nucleus containing one or more nucleoli.
  • the cells typically contained a minimal amount of cytoplasm.
  • the cells were demonstrated to be diploid by karyotype analysis. There were no obvious borders between the cells in culture.
  • the identification of the rat embryonic stem cells was validated by carefully observing the growth of the putative colony.
  • a rat embryonic stem cell colony exhibited growth and an absence of overt differentiation, as well as an ability to contribute to chimera formation, as described in subsequent examples. These characteristics of the rat embryonic stem cells are indicative of pluripotent stem cells.
  • Example 3 This example confirms the method of maintaining rat embryonic stem cells in culture and validates the growth of these cells on various substrata.
  • Example 1 The RESC-01 cells of Example 1 at passage 6-7 were plated onto 60 mm plastic, gelatin-coated plastic, or plastic dishes precoated with either STO mouse fibroblast feeder layers or HREF fibroblast feeder layers. In this example as well as subsequent examples, feeder cell layers were growth arrested prior to use as described in Example 1.
  • RESC-01 cells were harvested with trypsin and maintained at 4°C during cell manipulation. About 5 x 10 4 RESC-01 cells were then plated onto feeder layers in DMEM supplemented as previously described for isolation of rat embryonic stem cells. The supplemented DMEM also contained 500 units/ml LIF, to allow growth curves to be obtained. Under these plating conditions, the RESC-01 cells attach within 24 hours as single cells. Duplicate plates were set up for each time point, and the medium was changed daily. The cells were harvested with trypsin and counted with a hemocytometer on days 1, 2, 3, 4, and 5.
  • the RESC-01 cells grow best on the HREF feeder layers.
  • the RESC-01 cells grew less well on the STO feeder layers, even less well on gelatin-coated plastic, and the worst on plastic.
  • the differences between the growth curves on the different substrata were statistically significant.
  • rat embryonic stem cells can be maintained in culture. Furthermore, the experiments validate that rat embryonic stem cells of the present invention differ from previously described pluripotent embryonic stem cells in exhibiting a preference for growth on the HREF feeder layers on which these cells were derived.
  • This example corroborates the growth and differentiation of rat embryonic stem cells in culture on various substrata.
  • Duplicate plastic 60 mm plates were prepared for use by coating plates with gelatin, or by plating on growth arrested fibroblast feeder layers.
  • the RESC-01 cells of Example 1 were harvested with trypsin and were maintained at room temperature during cell manipulation. About 5 X 10 4 RESC-01 cells were plated on HREF feeder layers, STO feeder layers, gelatin-coated plastic, or plastic in medium containing either 500 units LIF/ml or no LIF at all. Under these plating conditions, the RESC-01 cells attach within 24 hours in clumps of 2 to 3 cells. Cell aggregates were examined with a 2OX phase contrast objective. Each day fifty randomly chosen colonies were examined, and the percentage which demonstrated any evidence of differentiation was recorded. Fifty colonies were scored in six determinations made on duplicate plates. A colony in which any differentiated cells (epithelial, mesenchymal, or endodermal morphologies) were observed was scored as differentiated.
  • the RESC-01 cells demonstrate slow differentiation toward endoderm-like cells. In distinct contrast to the slow differentiation of RESC-01 cells observed on feeder layers, the cells exhibit rapid differentiation when cultured on plastic or gelatin-coated plastic.
  • RESC-01 cells grown on plastic or gelatin- coated plastic differentiate into cells which are morphologically distinct from undifferentiated RESC-Ol cells. As exhibited in Figures 1D-F, under these conditions, the obtained differentiated culture was comprised of round refractile endoderm-like cells on the surface of RESC-01 colonies, flat polygonal epithelial cells, and fusiform-shaped mesodermal cells.
  • the proportion of differentiated colonies of RESC-01 cells grown on STO feeder layers was significantly less than the proportion grown on gelatin-coated plastic on days 2, 3, and 4 (p ⁇ 0.001) , and than the proportion grown on plastic on days 2, 3, and 4 (p ⁇ 0.001).
  • the proportion of differentiated colonies of RESC-01 cells grown on gelatin-coated plastic was significantly less than the proportion grown on plastic on day 2 (p ⁇ 0.003).
  • Example 5 This example corroborates the lack of a negative effect of different concentrations of LIF on growth of rat embryonic stem cells plated on various substrata.
  • the RESC-01 cells of Example 1 were plated on either HREF feeder layers or gelatin-coated plastic in the presence of a LIF concentration of 0, 100, 500, 1000 or 2000 units/ml. As shown by the growth curves presented in Figures 4A-B, the presence of LIF in the medium at concentrations ranging from 0 to 2000 units/ml did not diminish proliferation of RESC-01 cells on either HREF embryonic fibroblasts or gelatin-coated plastic. Unlike mouse pluripotent embryonic stem cells, there was no growth plateau evidenced for RESC-01 cells at even the highest LIF dose of 2000 units/ml.
  • LIF can be employed in tissue culture medium to maintain rat embryonic stem cells in an undifferentiated, pluripotent state at a concentration up to 2000 units/ml without negatively impacting on proliferation. Since no decrease in proliferation was observed at the even the highest LIF dose, a LIF dose of much greater than 2000 units/ml may be necessary to completely abrogate rat embryonic stem cell proliferation.
  • Example 6 This example validates the concentration-dependence of LIF-mediated inhibition of differentiation of rat embryonic stem cells plated on various substrata.
  • the ability to maintain the RESC-01 cells of Example 1 in an undifferentiated state by addition of different concentrations of LIF was investigated using various substrata.
  • the experiments were conducted as in Example 5, except that, instead of counting cells, the percentage of differentiated cells in a random sample was determined.
  • the proportion of differentiated colonies of RESC-01 cells grown on HREF in the absence of LIF was significantly greater than the proportion obtained in the presence of 500 units/ml LIF on days 2 and 3 (p ⁇ 0.001), than the proportion obtained in the presence of 1000 units/ml LIF on day 2 (p ⁇ 0.001), and than the proportion obtained in the presence of 2000 units/ml LIF on days 2 and 3 (p ⁇ 0.006).
  • This example confirms the ability of rat embryonic stem cells to spontaneously differentiate in suspension culture, resulting in the formation of embryoid bodies, or cystic structures comprised of several cell layers, which are reminiscent of the early embryo.
  • the RESC-01 cells of Example 1 were lightly trypsinized, and clumps of cells were transferred with a wide-bore pipette into 100 mm bacteriological Petri dishes (Baxter, Deerfield, IL) for 30 minutes to allow attachment of fibroblasts.
  • the unattached RESC-Ol cells were then placed in sterile 100 mm plastic bacteriological Petri dishes containing 10 ml of DMEM, which was supplemented as described in Example 1.
  • the cellular aggregates were cultured in suspension for 4 to 5 days without further addition of any medium. The cultures were passaged after this amount of time by first settling the simple embryoid bodies in a conical tube.
  • the exhausted medium was then aspirated off, the embryoid bodies were split into three plates, and 10 to 12 ml of fresh medium were added to each plate.
  • the embryoid bodies were incubated for another 4 to 5 days, with the cultures being refed every other day. On the days when the medium was not replaced, 5 ml of fresh medium was added.
  • the RESC-01 cells spontaneously formed cystic bodies in culture, which were capable of further growth in suspension culture.
  • the cystic bodies differentiated within 4 to 6 days into structures identical to simple embryoid bodies.
  • some of the rat cystic embryoid bodies acquired complex shapes with cystic fluid-filled cavities comprised of two cell layers ( Figures 1G-H) : one endoderm-like and the other ectoderm-like.
  • Figures 1G-H two cell layers
  • Several of these rat embryoid bodies were observed by phase contrast microscopy to begin rhythmic contractions similar to those produced with mouse embryoid bodies (Sanchez et al., J. Biol. Chem.. 266. 22419-22426 (1991)).
  • These structures can be cultured for many weeks.
  • Example 8 This example demonstrates that rat embryonic stem cells can participate in chimera formation following microinjection into blastocyst stage embryos. The selection of the strain used for chimera formation is an important consideration because certain inbred strains may yield fewer embryos, and the methodology requires an appropriately marked strain such that chimeric and non-chimeric rats can be distinguished. Moreover, certain pairings of strains could conceivably result in the generation of sterile offspring. Accordingly, for these experiments, the Holtzman strain was selected as host for chimera formation.
  • Example 1 The RESC-Ol cells of Example 1 were microinjected into Holtzman strain rat blastocysts isolated as in Example 1.
  • the RESC-01 cells were trypsinized for 3 minutes at 37°C, and were subsequently pipetted gently for 2 minutes through a narrow bore pipette to insure a single cell suspension.
  • 6 ml of flushing medium Spindle et al., J. Exp. Zoology. 186. 305- 318 (1973)
  • M2 Specification Media, Lavallett, NJ
  • the RESC-01 cells were injected into rat blastocoel cavities as described for injection into mouse blastocoel cavities (Bradley, In: Teratocarcinomas and embryonic stem cells: a practical approach. E.J. Robertson, ed. (Oxford: IRL Press, 1987) 113-151) , with necessary modifications being made to accommodate species-specific differences in embryological processes. Namely, rat blastocysts appropriate for injection were obtained on day 4.5 of pregnancy as compared with day 3.5 for mice. The rat morula stage embryo differs distinctly from that of the mouse in being planar in shape (Yamamura et al., Dev. Genet.. 2., 131-146 (1981); Weinberg et al., J.
  • rat blastocysts The expansion of rat blastocysts was accomplished by incubating the blastocyst at 37°C in 5% C0 2 atmosphere for about two hours in Brinster's medium (Brinster, In: Pathways to Conception (Springfield: Charles G. Thomas Publishing Company, 1971)) that had been modified to facilitate expansion of the rat blastocyst, and in particular, the Holtzman strain blastocyst.
  • the modified medium was prepared by adding with stirring to 75 ml of nanopure water in a 100 ml volumetric flask the following tissue culture grade reagents: 554.5 mg NaCl, 35.6 mg KC1, 16.2 mg KH 2 P0 4 , 14.3 mg anhydrous MgS0 4 , 210.5 mg NaHC0 3 , 100 mg dextrose, 5 mg streptomycin sulfate, 6 mg penicillin G, 100 mg albumin (BSA Pentax fraction V), 5.6 mg sodium pyruvate, and 0.38 ml of D,L-sodium lactate. Following the addition of 18.8 mg of CaCl 2 , the medium was brought to a final volume of 100 ml.
  • the expanded blastocysts were placed in the microdrop of medium along with the RESC-01 cells. From 10 to 30 RESC-01 cells were injected into the blastocoel using a DeFunbrune pump. After the blastocysts were injected, they were transferred to the uteri of Holtzman strain surrogate mothers on day 3.5 of pseudopregnancy. Pseudopregnancy in the surrogate rats was established by mechanical stimulation of the cervix during estrus (Ng et al.. In: Current Topics in Developmental Biology. R.A. Pederson, ed. (San Diego: Academic Press, 1992) 235-274) .
  • the timing of pseudopregnancy was established by noting the day of onset of leukocytes in the vaginal smear (Ng et al., supra) .
  • the surrogate mothers were allowed to deliver naturally.
  • the procedures developed for rat and outlined herein resulted in a pregnancy rate of 79% and a live birth rate of 39%. Over the course of several experiments, eighty- nine pups were obtained. Eighty-three of the pups were albino, or of the non-chimeric, Holtzman strain.
  • the Holtzman strain is characterized by an albino coat pattern.
  • the PVG strain of black hooded rats is characterized by a complete black coat color in the head and dorsal black pigmentation in the trunk. Accordingly, the coat color patterns observed for the six pups could only have occurred as a result of chimera formation between the injected RESC-01 cells derived from the PVG strain, and the Holtzman strain blastocyst. Thus the patterns represent the effects of mixing in the epidermis and der is of both Holtzman and PVG cells.
  • the patterns observed in the rat RESC-01 cell chimeras are consistent with those previously reported in aggregation chimeras formed by amalgamation of eight- cell embryos from strains genetically similar to the strains employed (Yamamura et al., Dev. Genet.. 2., 131-146 (1981)). Namely, aggregation chimeras produced between completely black and black hooded strains, or between completely black and albino hooded strains, showed large spots and did not demonstrate stripes or stippled patterns. Further, aggregation chimeras produced between completely black and albino hooded strains showed patchy mosaicism in the head similar to the RESC-01 cell chimeras. These similarities provide convincing evidence that the RESC-Ol cells contributed to chimera formation.
  • the population of 89 pups obtained included 46 pups (8 litters) obtained from injections of blastocysts which were not fully expanded. There were no chimeras among these 46 pups. Thirty pups (4 litters) were derived from injections of blastocysts which were fully expanded. Five of these thirty pups were chimeric, and at least one chimera was obtained in each of the four litters. The remaining chimera was from a group of 13 pups (3 litters) , which were derived from injections of blastocysts, only some of which were fully expanded. Thus, in the best series of experiments, 17% (or 5/30) of live births were chimeric.
  • obtained chimera can be mated with the blastocyst donor strain, in this case a Holtzman strain rat, and if the embryonic stem cells have colonized the germ line, then at least some of the offspring should evidence the hooded PVG coat pattern of the pluripotent embryonic stem cells.
  • the obtained chimeras are sterile, which may be the result of the present pairing between the PVG strain and Holtzman strain
  • germ line chimeras can be obtained using a different strain as blastocyst donor, or by isolating pluripotent embryonic stem cells from a different strain.
  • Example 9 This example describes an alternative method of introducing rat pluripotent embryonic stem cells into multicellular rat embryos by coculturing rat embryos with the stem cells, as well as the use of this method in the generation of chimeric rats.
  • the RESC-01 cells of Example 1 were prepared as for microinjection of blastocysts, in supplemented DMEM medium containing LIF at a concentration of 2000 units/ml, as described in Example 8.
  • the cell suspension was placed in a plastic culture dish for 10 to 20 minutes to allow fibroblasts to attach. Unattached cells were collected by centrifugation and resuspended in DMEM containing 5% FBS and 23 mM sodium lactate (Sigma L-4263) . Aliquots of 15 ⁇ l were placed in drops onto a 60 mm tissue culture plate, and 10 ⁇ l of the cell suspension was added to each drop to obtain about 8.5 x 10 3 RESC-01 cells per drop. The drops were overlaid with light mineral oil and equilibrated at 37°C and 6% C0 2 .
  • the zona pellucida was removed from the embryos by brief (30 to 60 seconds) incubation in modified T6' solution containing 1 g/1 sodium bicarbonate and 10 g/1 polyvinyl pyrrolidine, and from which penicillin/streptomycin, phenol red, BSA and HEPES had been omitted.
  • the final pH of the modified T6' solution was adjusted to 2.5, and the osmolality was adjusted with water to 310 mOsm/kg H 2 0.
  • the embryos were placed in the drops of the RESC-01 cells for about 20 minutes to 2 hours until 3-10 RESC-01 cells attached to the embryos.
  • the embryos were subsequently transferred to an organ culture dish which had been coated with heat-inactivated rat serum, and were rinsed with Markert's modification of Whittingham's medium that had been adjusted with water to an osmolality of 295 mOsm/kg H 2 0.
  • the embryos were cultured in the medium for 20 to 26 hours, and then transferred to pseudopregnant surrogate mothers, as in Example 8.
  • rat chimeras will similarly be obtained by the outlined method of coculturing multicellular rat embryos with rat pluripotent embryonic stem cells prior to transfer to pseudopregnant rats. It is further anticipated that this latter approach may prove more effective than microinjection for generation of chimeric rats when certain strains of rat are utilized.
  • This example describes a method of producing transgenic chimeras using the rat pluripotent embryonic stem cells.
  • Examples 8 and 9 methods of producing a rat chimera using rat pluripotent embryonic stem cells were described.
  • the stem cells were not modified in any fashion prior to incorporation in the rat blastocyst or earlier stage embryo.
  • the rat pluripotent embryonic stem cells can be modified prior to incorporation into the embryo or blastocyst, for example, by incorporation into these cells of exogenous DNA.
  • a commercially available expression vector containing 3-galactosidase coding sequences under the control of a RSV promoter (Stratagene, La Jolla, CA) can be employed.
  • the lacZ sequences in this vector are separated from the promoter by a linker sequence containing appropriate restriction sites for subcloning of genes or gene fragments.
  • the expression vector is transfected into the RESC-Ol cell line. Transfectants are confirmed, and expression of jS-galactosidase verified, by replica plating colonies and staining for J-galactosidase activity (S. Gal, Methods Enzvmol.. 151. 104 (1987); Sanes et al., Embo J.. f5, 3133 (1986)). Colonies which express 0-galactosidase are then selected and expanded. The resultant cell line is introduced into a rat blastocyst or early stage embryo, and subsequently transferred to a pseudopregnant rat, as set forth in Examples 8 and 9.
  • transgenic chimeras obtained using this approach can be verified by simple observation of phenotypic properties, as described in Example 8, as well as by fixing tissue sections in glutaraldehyde solution, and analyzing sections for /3-galactosidase activity (Sanes et al., Embo J.. j5, 3133 (1986)). Additional experiments can also be performed, such as Southern and Northern hybridization, or Western blotting, to verify the presence or expression of the exogenous DNA sequences in the chimeric host.
  • rat pluripotent embryonic stem cells which have been altered in some fashion will be obtained. These stem cells can further be employed in the method of Examples 8 or 9 to generate transgenic rats.
  • Example 11 This example describes a method of obtaining rats which are homozygous for DNA sequences introduced into or altered in the rat pluripotent embryonic stem cells.
  • the chimeras obtained by the methods outlined in Examples 8-10 are mosaics, comprised of cells inherited from the RESC-Ol cells of Example 1, as well as cells inherited from the host blastocyst or embryo.
  • the rats produced in Example 10 contain the introduced or altered DNA in the heterozygous state. Rats containing the introduced or altered DNA of the rat pluripotent embryonic stem cells in the homozygous state can be produced by mating with each other germ line chimeras obtained by the methods of Examples 8, 9 or 10, as described in Example 8. Under these conditions, one fourth of the offspring will typically be homozygous for the introduced or altered DNA.
  • the new strain of rat can be bred to carry the alteration in the homozygous state. If the loss or gain of function of the gene causes perinatal mortality, the altered gene can be maintained in the heterozygous state.
  • transgenic rats which are homozygous for a particular allele can be obtained using the outlined method.

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Abstract

L'invention concerne des cellules souches pluripotentes d'embryons de rats qui servent à obtenir des rats chimériques et transgéniques. L'invention concerne aussi des procédés ainsi que des milieux et conditions de culture de cellules appropriés à l'obtention de cellules souches pluripotentes d'embryons de rats. En outre, l'invention concerne des détails de morphologie permettant la reconnaissance des cellules ainsi que des procédés permettant d'obtenir des rats chimériques et transgéniques.
EP94927285A 1993-08-30 1994-08-29 Cellules souches pluripotentes d'embryons de rats et leur procede d'obtention et d'utilisation Withdrawn EP0716690A1 (fr)

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