JP2002527036A - Pluripotent embryonic stem cells and method for obtaining pluripotent embryonic stem cells - Google Patents

Abstract

  (57) [Summary] The present invention provides an isolated population of non-mouse embryonic stem (ES) cells and a method for obtaining these ES cells. In one aspect, the target ES cells are obtained by co-culturing non-target ES cells (eg, mouse ES cells) and embryo cells from a target animal. In one embodiment, rat ES cells are isolated from a co-culture using a positive or negative selectable marker. The invention also includes genetically modified non-mouse ES cells. Chimeric embryos and animals comprising the ES cells or the isolated population of genetically modified ES cells are also provided. In one embodiment, the genetic modification comprises introducing a transgene. In another embodiment, the genetic modification comprises disrupting the function of one or more genes.

Description

DETAILED DESCRIPTION OF THE INVENTION

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application is related to US Provisional Patent Application Ser.
Claim priority of 0 / 066,890. The aforementioned provisional application is hereby incorporated by reference herein in its entirety.

TECHNICAL FIELD [0002] The present invention is in the fields of molecular biology and medicine. More particularly, it relates to novel non-mouse embryonic stem (ES) cells and methods for obtaining these non-mouse ES cells.

BACKGROUND Genetically modified experimental animals are widely used in drug development and as model systems for human disease. Many of these transgenic animals, particularly loss-of-function mutants, are mouse models that have been created by using mouse embryonic stem (ES) cells. Briefly, gene targeting in ES cells uses a homologous recombination event to disrupt or knock out specific gene functions (for techniques involving mouse embryonic stem cells, see US Pat. No. 5,464,764). , 5,48
7,992, 5,631,153, and 5,627,059). The number of mouse mutants created by gene inactivation in ES cells is rapidly increasing. This mutant mouse is created in vivo to understand the function of known genes, discover new genes, and create animal models of human disease (eg, Chisaka et al., (1992) 355: 516-520).
Joyner et al. (1992) POSTIMPLANTATION DEVEL;
OPMENT IN THE MOUSE (Cadwick and Marsh
Ed., John Wiley & Sons, United Kingdom) 277
-297; Dorin et al. (1992) Nature 359: 211-215.
checking).

[0004] Although the development of mouse ES cells has begun a new era in mouse genetics, non-mouse ES cells have proven difficult to isolate. Putative cell lines are derived from hamster, pig and sheep blastocysts (Doetschman et al. (1).
988) Development. Biol. 127: 224-227; Notari
(1990) J. Am. Reprod. Fertil. Addendum 41:51
-56; Notarianni et al. (1991) J. Am. Reprod. Ferti
l. Addendum 43: 255-60; see WO 94/26884). However, it is not clear whether these cells are truly pluripotent ES cells (eg, Ga
rdner and Brook (1997) Int. J. Dev. Biol. 4
1: 235-243).

Report the isolation of human gonad progenitor cells from aborted fetuses (On-line Beardsley (1998) Scientific Am).
erican). When these cells are transplanted into mice without a functional immune system, they clearly give rise to tumors containing various cell types. However, it is not clear that these isolated human cells are pluripotent ES cells.

[0006] Rat pluripotent cells are of particular interest in the transgenic field.
Transgenic rats offer several advantages over the mouse model.
First, rats are larger, which allows for surgical procedures, and they provide a great deal of material for biochemical analysis. A second advantage of the rat model is that a large database of information exists for the rat model, especially for neurodegenerative diseases,
It has been created in the area of cardiovascular research and diabetes. No comparable database exists for mice.

[0007] Rats are also a preferred animal model for drug development assays. Transgenic rats allow sophisticated physiological measurements not possible with transgenic mice. For example, due to their size, rat-containing human transgenes can provide primate-specific analysis in non-primate experimental systems. In some cases, the physiology of the rat allows for the generation of transgenic animals whose phenotype is useful for human disease models, while mice carrying the same transgene do not have the disease phenotype . For example, transgenic models of HLA-B27-related human autoimmune disorders were first attempted in mice, but the pathology did not evolve (Taurog et al. (1988) J. Immunol. 141: 4020-30).
). However, transgenic HLA-B27 rats have a pathology that closely resembles human disease (Hammer et al. (1990) Cell 63: 109.
9-1112). Thus, rat ES cells can be used to develop all of the genetic manipulations currently only possible in mice. When rat ES cell technology is available, silent mutations can be introduced into rat homologues of the human gene and combined with human transgenes to replace "primate" human primates for many studies
Provide animals.

[0008] Despite continued attempts, no one has yet succeeded in inducing rat ES cells. Iannocone et al. (1994) Development Biol.
. 163: 288-292 (see also WO 95/06716) first reported the isolation of rat embryonic stem cells, but recently reviewed this publication following the discovery that this cell line was contaminated with mouse ES cells. (Brenin et al. (1997) D
evelop. Biol. During printing; Brenin et al. (1996) PHARM.
CEREBRAL ISCHEMIA (edited by Krieglstein, Medph
arm Scientific Publishers, Stuttgart)
555-572). Thus, the present invention provides a population of initially isolated rat ES cells.

[0009] The initial success in culturing mouse cells may have been due in part to the accidental selection of mouse strains, and 129 strains of mice were identified in early teratocarcinoma studies due to their tendency to testicular germ cell tumors. Used (Evans & Kaufman (1981) N
atature 292: 154-156; Martin (1981) Proc.
Nat'l. Acad. Sci. ScL USA 78: 7634-7638). The inventor believes that the pluripotent cells in this specific lineage blastocyst intracellular cell mass have the inherent ability to adapt to tissue culture. Other lines, such as the C57BL / 6 mouse, have proven more difficult to derive ES cell lines, and germline competent C57BL / 6 ES cell lines have only recently been reported ( Ledermann et al. (1991) Exp.
7: 254-258). No rat line has a tendency to develop testicular teratocarcinoma, and the lack of such lineage seems to be one of the reasons why it is more difficult to derive the first rat ES cell lineage. Teratocarcinoma is also experimentally induced in rats, usually derived from the yolk sac rather than the germ cells, as compared to the mouse, and these cells are not pluripotent but rather retain the properties of the yolk sac endoderm. This feature may result in rapid dilution of ES-like cells with cells morphologically similar to endoderm cells in cultures derived from blastocysts of several rat strains.

[0010] The present invention provides the first pluripotent rat ES cells. Furthermore, the present invention provides a method that is universally applicable to the generation of ES cells from all species.

SUMMARY OF THE INVENTION The present invention includes an isolated population of non-mouse embryonic stem cells. In one embodiment, the cells are rat ES cells.

In another aspect, the invention provides a method of obtaining embryonic stem cells from a target species, the method comprising the steps of: (a) reducing the growth of embryonic stem (ES) cells from a target species. Co-culturing the non-target embryonic stem cells and cells obtained from the embryo of the target species under supporting conditions; and (b) isolating ES cells from the target species. The non-target embryonic stem cells used in this co-culture are from a species other than the target species (eg, mouse). The target ES cells can be derived from the inner cell mass (ICM) or primordial germ cells (PGC)
Can be

[0013] In another embodiment, the non-target embryonic stem cells used in the co-culture lack a positive selectable marker. The positive selectable marker can be a gene encoding antibiotic resistance or a gene encoding HPRT resistance (HPRT). In yet another embodiment, the embryonic stem cells used in the co-culture have a negative selection marker (
For example, HPRT or herpes simplex virus thymidine kinase (HSV-tk)
Hold. If desired, the embryonic cells can be cultured on a feeder layer of the cells. In one embodiment, the feeder layer is SNL 76/7 cells. In another embodiment, the non-target ES cells used in the co-culture method are mitotically inactivated.

[0014] In yet another aspect, the invention includes a genetically modified non-mouse ES cell. In one embodiment, the genetic modification comprises a transgene insertion.
In another aspect, the genetic modification comprises disrupting the function of one or more genes.

In a further aspect, the invention includes a chimeric embryo comprising an ES cell or an isolated population of genetically modified ES cells. In one embodiment, the chimeric embryo comprises an ES cell that has been genetically modified to include the transgene. In another embodiment, the chimeric embryo comprises ES cells that have been genetically modified to disrupt the function of one or more genes.

[0016] In another aspect, the invention includes an animal containing cells arising from an isolated population of target ES cells. In one embodiment, the animal contains cells derived from rat ES cells that have been genetically modified to contain the transgene. In another embodiment, the animal contains cells derived from rat ES cells that have been genetically modified to disrupt the function of one or more genes.

As will be apparent, preferred features and characteristics of one aspect of the invention are applicable to any other aspect of the invention.

Throughout this application, various publications, patents and published patent applications are referred to by an identifying citation. The disclosures of these publications, patents and published patent specifications referred to in this application are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains. .

The present invention provides pluripotent embryonic stem (ES) cells. This ES cell is typically not a mouse, and isolated rat ES cells are exemplified herein, but the claimed method can be applied to any species. obtain. In general, the invention provides a substantially purified population of non-mouse (eg, rat) ES cells. Methods for producing ES cells include co-culturing embryo cells from a target species (eg, blastocysts or inner cell mass (ICM) from primordial germ cells (PGC)) with non-target ES cells. The non-target ES cells can be from any species (eg, mouse). Preferably, the non-target ES cells contain a negative selectable marker.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, eg, Sambrook, Fritsch and Maniatis, MOLE.
CULAR CLONING: A LABORATORY MANUAL, 2nd
Edition (1989); CURRENT PROTOCOLS IN MOLECULL
AR BIOLOGY, (FM Ausubel et al., Eds., 1987); the
series METHODS IN ENZYMOLOGY (Academi
c Press, Inc. ); PCR2: A PRACTICAL APPRO
ACH (MJ McPherson, BD Hames and GR Ta)
ylor, 1995); ANIMAL CELL CULTURE (RI.
Freshney, ed., 1987); and ANTIBOIDES: A LAB
See ORARARY MANUAL (Harlow et al., Eds., 1987).

Definitions As used herein, certain terms have certain meanings.

The term “embryonic stem cells” or “ES cells” is used to refer to cells of the early embryo that can give rise to all differentiated cells, including germline cells. Although not yet isolated from all species, all animals are considered to have ES cells. Mouse embryonic stem cells (the best characterized ES cells) are derived from the pluripotent inner cell mass of blastocysts, and these pluripotency can be maintained by a suitable culture environment. Mouse ES
The cell line has been established in culture using feeder cells (eg, irradiated fibroblasts) or has been cultured in a medium conditioned by an established teratocarcinoma stem cell line. Some mouse ES cells also have differentiation inhibitory activity (DIA) or leukemia inhibitory factor (L) that prevent spontaneous differentiation of cells in culture.
IF) can be grown without a feeder cell layer.

The terms “non-mouse” and “target” ES cells refer to cells from any animal other than mice. A preferred non-mouse or target cell is a rat. The term "non-target" ES cells refers to cells from any animal other than this target species. A preferred non-target ES cell is a mouse.

When the ES cells are implanted into a host blastocyst, the ES cells contribute to the formation of a chimeric animal, and when the germ cells of the chimera are derived from the ES cells, the progeny of the chimera are derived from the ES cells. Retain the genome ("germline transmission"). “Known” ES cells are:
Cells that are known in the art and have been shown to be pluripotent as determined by the assays and methods described herein. Pluripotent cell lines are not limited to blastocyst-derived lines. Recently, cell lines with at least the in vitro pluripotency of ES cells have been derived from mouse primordial germ cells (Matsu
i et al., (1992) Cell 70: 841-847; Matsui and Hu.
m (1997) Cell 10 (1): 63-8; (1997
) Exp. Cell Res. 232: 194-207).

ES cells can be easily manipulated. Perhaps the factors that ES cells produce and secrete are important in controlling early embryonic development in vivo, so they are useful as models for studying cell differentiation. ES with genetically engineered foreign DNA
The cells can be used to create a true breeding transgenic animal line. Methods of genetic manipulation are known to those skilled in the art.

A “isolated” or “purified” cell population is substantially free of naturally associated cells and materials. Substantially free or substantially purified means that at least 50% of the population is ES cells, preferably at least 70%,
More preferably at least 80%, and even more preferably at least 90%
% Means free of non-pluripotent ES cells that associate with nature.

“Cell line” or “cell culture” refers to a higher eukaryotic cell grown or maintained in vitro. It is understood that the progeny of a cell cannot be completely identical in morphology, genotype, or phenotype to the parent cell.

The term “embryo” refers to tissue obtained from any stage of development of an animal before birth.
During mammalian development, for example, fertilized eggs break apart to form a morula-shaped cell mass called a "morula". During the 8 to 16 cell stage, the morula is called a "blastocyst"-
Transforms into a nearly spherical, liquid-filled structure. The outer cells of the blastocyst are "trophoblast" cells and give rise to placenta and other extraembryonic structures. The embryo itself
It is derived from an "inner cell mass" or "ICM", an accumulation of cells at one pole of the blastocyst. Other sources of embryonic tissue include delayed blastocysts and primordial germ cells.

As described herein, the method developed by the inventor is equally applicable to all species. The term “target animal” or “target species”
Refers to the species from which the isolated ES cells of the present invention are derived. Suitable species are rat, human,
Including, but not limited to, cows and sheep.

The term “selectable marker” refers to a gene whose expression can identify cells that have been transformed or transfected with a vector containing the marker gene. Selectable markers can be "positive" or "negative" and dominant or recessive. "
"Positive selectable marker" refers to a gene that encodes a product that allows only cells carrying this gene to survive and / or grow under certain conditions. For example, plant and animal cells that express the introduced antibiotic resistance gene. Non-limiting example of a suitable antibiotic resistance gene is neomycin resistance (Neo ¹ ) which provides resistance to compound G418, hygromycin resistance and puromycin resistance. Another positive selectable marker is hypoxanthine phosphoribosyltransferase (HPRT)
). Cells carrying this HPRT gene are HAT (aminopterin (a
Cells that grow in (mniopterin), hypoxanthine, thymidine) medium, while being HPRT negative (HPRT), die in HAT medium.

Similarly, the term “negative selectable marker” refers to a gene that encodes a product that can be induced to selectively kill cells bearing this gene. Non-limiting examples of negative selectable markers include herpes simplex virus thymidine kinase (HSV-tk) and HPRT. Ganciclovir or FIAU (1 (1,2-deoxy-2-fluoro-β-D-rabinofuranosyl) -5-iodouracil) was added, and cells retaining mammalian tk were treated with 5-bromo When killed using deoxyuridine (5BdU), the cells carrying the HSV-tk gene are killed. Cells holding this HPRT marker are 6-cells.
It can be selectively killed with thioguanine (6TG). Other examples of selectable markers (both positive and negative) are known to those skilled in the art.

“Positive-negative selection” refers to the process of selecting cells that carry the integrated DNA insert at a particular target location (positive selection), and also the DNA insertion integrated at a non-targeted chromosomal site. Refers to the process of selecting for cells that carry the material (negative selection).

“Transgenic animal” refers to a genetically engineered animal or the progeny of a genetically engineered animal. A "chimeric embryo" is an embryo that has a population of cells having different genotypes. Thus, a transgenic animal or chimeric embryo usually contains material from at least one unrelated organism (eg, from a virus, plant or other animal). The term "transgene" refers to a polynucleotide from one source that has been integrated into the genome of another organism. The transgene may be obtained from any source, for example, isolated from different organisms, species, or made synthetically. The transgene can be a gene, gene fragment or multiple genes. The appropriate size of the transgene can be determined by methods known in the art.

The present invention provides a population of initially isolated rat embryonic stem cells. The present invention also provides a novel method for deriving ES cells from non-mouse species. By using novel culture conditions, we have derived an isolated population of rat ES cells from embryonic tissue. These new methods are equally applicable to target species other than rats. Previous groups have attempted to culture rat ES cells with the amount and / or type of growth factors and feeder cells added to the culture medium with no success. Whatever the medium, previous rat cells cannot be maintained in culture without differentiation. These cultured rat cells are not transmitted to the germline of the chimeric rat. Rat embryo cells appear to have reached a critical stage in culture. At this stage, growth factors and feeder cells are insufficient to promote the induction of undifferentiated pluripotent ES lineages. The present invention overcomes this problem by co-culturing the rat embryo cells with cells of a non-target ES cell line. Contact with the ES cell line appears to support embryonic cells isolated through this crisis in culture. Once through this critical point, rat ES cells
Proliferate on their own and maintain their undifferentiated pluripotent phenotype.

I. Source of Pluripotent Embryonic Stem Cells ES cells can be isolated from any species using the methods described herein.
Examples described herein include ES cells. Long Evans (LE
) Includes ES cells from rat inbred lines (available from Simonsen (16 generations inbred) or from the Sprague-Dawley rat line.
Rats have the appropriate coat color (black head) to identify chimeras when LE ES cell candidates are injected into the white line. Numerous embryos are obtained from this lineage and these cells are well adapted to tissue culture conditions. LE animals can also be time-mated by the supplier, reducing the size of animal communities that must be maintained and the time involved in breeding animals to mating age. Unlike mice, rat strains vary greatly at the time of embryo transfer and each responds differently to superovulation and delayed implantation procedures. To the extent that these variations affect the present invention, they can be readily determined by methods known in the art.

[0036] The non-mouse target ES cells can be derived from any suitable cells. Non-limiting examples are blastocysts (non-delayed), blastocysts with experimentally delayed implantation (delayed blastocysts), and primordial germ cells. PGCs can be isolated from various stages of embryonic development, such as stage 13 embryos. For humans, cells from spontaneous abortions and abortions can be used. Cells can also be obtained from embryos produced by in vitro fertilization techniques.

A. Blastocysts and Delayed Blastocysts Blastocysts can be isolated by any method known in the art. For example, timed-mated females are approximately 4.5 days after mating (0
. 5 is the morning after mating) and the blastocysts are, for example, Br
adley "TERATO CARCINOMAS AND EMBRYONIC
STEM CELLS: A PRACTICAL APPROACH "(E.
J. Robertson, eds., 1987), collected from the uterus by the method described for mice.

[0038] Preferably, the mated female is ovariectomized when the embryo is in the fallopian tube (about 3.5 days after mating). After ovariectomy, Buchanan (1969) J.
Reprod. Fert. Animals receive progesterone injections daily (5 mg / animal / day, subcutaneous injection) as described in 19: 279-283. Between about 6 days and about 11 days, the non-implanted blastocysts are collected from the uterus using any method described in the art (eg, A. Bradley, supra). Delayed blastocysts are usually larger than normal blastocysts and lack the zona pellucida. The blastocysts and delayed blastocysts can be cultured, as described herein, preferably in feeder cells in individual wells of a 24-well plate.

[0039] Embryos produced by in vitro fertilization can be cultured to the blastocyst stage for isolation of ICM.

B. Primordial germ cells Primordial germ cells can also be isolated from embryos. Although the stage of embryonic tissue is not critical, in embodiments involving the production of rat ES cells, timely mated females are sacrificed at about 9.5 days of gestation and embryos are dissected from extraembryonic tissue. You.
At this age, the rat embryo is at stage 13, which is equivalent to a mouse at 8.5 days (stage is determined by the number of somites). The tail region of the embryo, preferably from the last segment to the allantois, is dissociated into a single cell suspension with trypsin (0.5%),
Then gently crush with a micropipette. Cells are plated in 24-well dishes, for example, with or without feeder cells, as described below. At this stage, there are about hundreds of PGCs in each rat embryo.

(II. Culture Conditions) The present invention uses culture conditions that promote ES cell induction.

A. Culture of Embryonic Tissue The primary blastocyst from which the ES cells are derived is grown in any suitable medium under any conditions that allow the growth and proliferation of the ES cells. For example, one suitable medium is mouse ES medium (15% fetal bovine serum (FBS: HyClone), 1 ×
DMEM (Gibco) with glutamine and high glucose supplemented with non-essential amino acids, 0.1 mM 2-mercaptoethanol, and antibiotics.

Primary primordial germ cells (PGC) are preferably exogenous growth factors (eg, LI
F, SCF, and bFGF).
Other growth factors that can be used are known to those skilled in the art. An appropriate concentration of growth factor is
It can be easily determined by one skilled in the art. As described herein, 2000 U /
mL LIF (Gibco); mouse SCF, 60 ng / mL; human bFGF,
20 ng / mL (Genzyme) has been shown to be effective. In addition, cells can be cultured in ES cells prepared from other species, such as ES cells previously prepared as described herein.

[0044] Secondary and subsequent PGC cultures can be cultured in the same medium or in a medium lacking exogenous growth factors. In normal and delayed blastocyst cultures, the inner cell mass (ICM) is visible about 3 days after culture. The ICM is removed under conditions that minimize contamination of other cell types, for example, after about 3-5 days in culture. In one embodiment, the ICM is removed using a micropipette and then 0.25%
Dissociated using trypsin. The ICM is either dispersed in a single cell suspension or, more preferably, gently dispersed to produce a small population of cells. In one embodiment, the ICM culture is cultured in a 6-well dish,
Colonies resulting from the dispersed ICM are then selected by morphological criteria. Exogenous growth factors, such as bFGF, LIF, and SCF, alone or in combination, can be added to the culture.

[0045] Optionally, the ICM culture may be supplemented with a feeder layer (eg, mitotically inactivated SN).
L76 / 7 cells, which are feeder cell lines that produce leukemia inhibitory factor (LIF) and stem cell factor (SCF). Feeder cells that induce differentiation (change in morphology, loss of alkaline phosphatase (AP) staining) should not be used.

B. Co-Culture with ES Cells An important step in deriving embryonic stem cells is to culture embryonic tissue in the presence of pluripotent embryonic stem cells. Without being bound by one theory, the co-culture method
It appears to be effective for cell contact between ES cells and self-adaptation of the culture medium by the ES cells. The inventors noted that purified growth factors were not sufficient to provide pluripotency and optimal maintenance of proliferation of undifferentiated ES cells.
For mouse ES cells to have a high potential for germline transmission, mouse ES cells
It must be cultured on a feeder layer with serum. Most importantly, we also noted that mouse ES cells differentiate much more easily and frequently when they are cultured at low density rather than high density. These observations led the inventors to the claimed co-culture method.

As mentioned above, embryonic tissue can be from any source, preferably a non-mouse donor. In one embodiment, the embryonic cell is an undelayed or delayed blastocyst-derived IC
M. In another embodiment, they are primordial germ cells. In yet another embodiment, they are delayed blastocysts. At least one embryonic tissue cell is used, but between 1 and 50 cells, preferably between about 5 and 10 single cells are also used.
It can be combined into a single culture.

The selected embryonic tissue cells can be isolated and immediately co-cultured with non-target embryonic stem cells. Preferably, the embryonic tissue cells are cultured in vitro for a short time (eg, about 3 days) before adding the non-targeted embryonic stem cells to the culture. In a preferred embodiment, the co-culture is established before the embryonic cells begin to differentiate. If necessary,
Primary embryo tissue cultures contain a feeder layer of cells (eg, STO or SNL 76 /
7 cells).

[0049] Any known pluripotent embryonic stem cells can be used in co-culture conditions. In co-culture, potentially as little as one ES cell may be required, but more (eg, between 10 and 100) may be required. To derive target ES cells, it is preferable to use as few cells as needed. If too much non-target ES cells are added, the target seed embryo cells, which generally divide more slowly, can compete out of view. Preferably, the non-target ES cells are mouse ES cells. Procedures for the isolation of mouse ES cells have been described (eg, Martin, 1981; Lederma).
nn et al., 1991 and Matsui et al., 1992).

In a preferred embodiment, the non-target embryonic stem cells are mouse ES cells having a negative selectable marker gene. Thus, mouse ES cells can be selectively removed from the co-culture. There are a number of suitable selectable markers (including, for example, HPRT and thymidine kinase). Other negative selectable marker genes are known to those skilled in the art.

In a preferred embodiment, the non-target mouse ES cells are cell lines deficient in HPRT, either by knockout or by natural mutation. Suitable ES cell lines are
It cannot revert to the HPRT positive (HAT resistant) phenotype. Co-cultured cells from the target species are HPRT positive and survive HAT treatment. As described in the Examples below, controls (containing only HPRT negative mouse cells) did not survive HAT treatment. In yet another embodiment, the non-target ES cells have been mitotically inactivated by gamma irradiation.

Another means for identifying ES cells from the target species is to use blastocysts from a cross between wild-type female rats and male transgenics as a source of co-culture. Yes, where the transgene is a reporter gene. For example, female Sprague-Dawley rats can be bred to homozygous males with the randomly inserted and stably integrated LacZ reporter gene under the control of the metallothionein promoter. Thus, LacZ protein expression can be induced by metal ions such as zinc or cadmium. Blastocysts isolated from these crosses are then co-cultured with selectable non-target ES cells. After selective killing of the added non-target ES cells, the remaining cells are left in culture in “
It can be induced to express LacZ. As described in the Examples below, the claimed method results in a population of rats that turns blue (ie, expresses LacZ).

III. Characterization of ES Cells ES cells obtained using the methods claimed herein can also be assayed for ES cell phenotype. Representative cell surface markers expressed by ES cells include alkaline phosphatase and anti-SSEA-1. Using embryoid body formation, subsequent culture to produce differentiated cell types, and retinoic acid induction of differentiation,
In vitro assay for differentiation. Putative ES cell pluripotency is also demonstrated by the ability of isolated single cell-derived subclones to differentiate into a wide variety of cell types and by the formation of teratocarcinomas when injected into all animals. Can be done. Cells can be assayed at any stage of the process.

A. Histochemistry Pluripotent ES cells express specific cell surface markers that can be detected histochemically using antibody or colorimetric (or enzyme) assays. As used herein, “antibody” refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulins. Recognized immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as various immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn represent the immunoglobulin classes IgG, I, respectively.
Define gM, IgA, IgD, and IgE.

[0055] Monoclonal or polyclonal antibodies can be used to detect ES cell markers. Anti-SSEA-1 recognizes glycolipids on the surface of undifferentiated mouse stem cells. Alkaline phosphatase (AP) activity is characteristic of primordial germ cells, blastocysts, inner cell mass and ES cells. Figures 6 and 7 show that rat ICM and PCG co-cultured with mouse ES cells are AP positive (see Figures 6D and 7C). Both the AP assay and the SSEA-1 antibody are commercially available.

B. Embryoid Bodies Pluripotent ES cells can differentiate in culture into embryoid bodies containing multiple cell types. ES
The cells are multi-layered and the embryoid bodies resulting from these multi-layered cells contain endodermal, mesodermal, and ectodermal tissues and structures. In contrast, undifferentiated cells (non-ES) have an epithelial layer and develop an embryoid body composed of a single cell type, epithelial cells. For example, in mouse ES cells, removal of certain growth factors or treatment with an inducer (eg, retinoic acid or 3-methoxybenzamide) can result in complex cysts in which the formation of endoderm, mesoderm, and ectoderm can be detected Often achieved by the generation of embryoid-like bodies. One readily observed indicator of pluripotency is the formation of spontaneously contracting heart tissue from embryoid bodies in culture dishes. Generally, the ES cells described herein form embryoid bodies having various cell types, including spontaneously shrinking heart tissue. For example, in Example 4, B. See verse two.

VI. Targeted Mutation of Genes Using ES Cells In theory, any lineage of a particular species can be used as a host blastocyst for injection of that type of ES cells. However, in practice, experiments with mouse ES cells have shown that some combinations do not work as well as others and that host selection may be important in obtaining germline transmission. Was. Rat ES cells are Lon
g When derived from the Evans strain, the preferred host is Fisher 344 strain. This is because this white strain shows LE E by observing pigmentation.
This is because the ES cell contribution from S cells can be evaluated. Alternatively, ES cells from a white rat line (eg, Fisher 344) can be injected into a pigmented host line. Fisher 344 white animals, when injected into white host blastocysts, lack coat color markers to detect the contribution of ES cells in the chimera, where the promising cell line is a colored lineage (eg, Long Evans or Dark Agouti). A) injected into the blastocyst. Hair color markers are more convenient than necessary. This is because ES cell-derived cells can be detected by other means. There are two possible strategies for detecting cells: 1. an endogenous isoenzyme or molecular marker and The introduced protein or genetic marker. Isoenzymes of GPI and other ubiquitous enzymes have been identified for many strains of rat and established techniques (eg, Robert
son, see supra) in blood samples or biopsies.

Another technique that may be more generally useful is the introduction of molecular markers into ES cells in culture. For example, a marked cell line can be created by introducing a β-galactosidase or tyrosinase plasmid. β-galactosidase is detectable by staining and has the advantage of allowing the testing of the chimera as an embryo. The tyrosinase gene converts white cells into cells capable of producing pigment, allowing direct visualization of coat color chimera formation. Methods for transfection of ES cells are described herein and are known in the art, and β-galactosidase and tyrosinase plasmids are commercially available. The advantage of this approach is that any animal strain can be used for the blastocyst host, including the strain from which the ES cells are derived.

The strength of ES cell technology is the ability to genetically modify cells in culture, analyze the cells for precise modifications, and then generate new animal strains from the cells.
In order to explore the powerful tools provided by ES cells, methods for introducing genetic modifications and for analyzing ES cells have been rapidly developed in recent years. Gene targeting strategies allow for inactivation of specific genes by homologous recombination. Selection techniques have been developed to improve the identification of rare targeting events and to introduce minor mutations. (For example, Hasty et al. (
1991) Nature 350: 243-246; U.S. Patent No. 5,629,1.
No. 59). Efficient methods for ES cell analysis using microtiter plates and rapid DNA preparation techniques make it possible to screen thousands of clones for extremely rare events. Known methods for generating transgenic animals in mice, given the non-mouse ES cells described herein, can be readily applied to other species.

[0060] ES cell technology has recently created an important breakthrough in transgenic animal research. In three new reports, including our report, ES cells were used to generate transgenic mice expressing large genomic DNA fragments cloned on the yeast artificial chromosome (YAC). (See, for example, Strauss et al. (1993) Science 259: 1904-1907.
Brinster (1988) Proc. Nat'l Acad. Sci. U
SA 85: 835-40; Choi et al. (1993) 4 (2): 117-23 and 4 (3): 320). The rationale for this technique is the expectation that genomic transgenes that retain the content and organization of the locus appear to be more accurately expressed. However, many genes have exceeded the cloning capacity of traditional plasmids (less than 80 kb) and have traditional transduction pathways (
(By microinjection of the zygote) may have a higher DNA size limit due to the shear forces caused by pushing the DNA with a microinjection pipette. YAC has a cloning capacity of greater than 2000 kb. Transgenic rats by YAC transfection of rat ES cells can be produced using known techniques. In fact, any gene can be disrupted using ES cells or transferred intact using YACs. In rats, genes involved in neurodegenerative diseases (eg, Alzheimer's disease, Parkinson's disease, Huntington's disease, etc.) and cardiovascular diseases are particularly preferred.

VII. Use of ES Cells A. Identification of Genes and Gene Pathways Involved in AD Human and rat ES cells can be prepared by manipulating culture conditions (eg, adding retinoic acid, Serum removal, culture substrate changes, the addition of specific growth factors or growth inhibitors or ligands to cell surface receptors)
It can be made into neuroblasts that can differentiate into neurons in culture. The cells can be made into embryoid bodies, which enhance the differentiation of certain species, and then treated with other factors that enhance neural differentiation. To improve neuronal yield, neuroblasts or neurons first express a neuronal or neuroblast-specific promoter by expressing a selectable marker, a novel cell surface macromolecule, or a reporter gene (green fluorescent protein or LacZ). One can select from other cell types by first transfecting the ES cells with the driving transgene. For cells transfected with a selectable marker, not cells without the transgene,
Addition of a compound to the culture medium that allows cells expressing the transgene to survive and / or grow allows the neurons to be harvested. In the case of cells transfected with the novel cell surface macromolecule gene, expression of this novel cell surface macromolecule can be achieved by a number of means, including but not limited to antibody binding, adhesion to substrates, and the like. And by allowing the selection of neurons by providing a fluorescent tag. In the case of cells transfected with a reporter gene, cells expressing the reporter gene can be collected by FACS. Neurons or neuroblasts can also be separated from other cells by density gradient centrifugation or by using another endogenous property that distinguishes them from other cells.

The gene expression profile of ES-induced cells can be made by mRNA detection. This method includes Northern blot analysis, RNAse protection, reverse transcription PCR
And expressed sequence tag (EST), oligonucleotide or cDNA cDN
A expression arrays or microarrays, but are not limited to these. Prior to such analysis, the mRNA may first be amplified (preferably by linear amplification). Genes that are expressed in neurons but not in their ES progenitor cells are candidates for neuron-specific drug targets. Further analysis of the candidate genes is performed by performing genetic engineering of the precursor ES cells. For example, genes known to be involved in AD (eg, preserinin, apolipoprotein E, amyloid precursor protein (APP)) can be “knocked out” by gene targeting by homologous recombination. Since ES cells are diploid, both copies of the gene can be modified by selecting for a gene conversion event or by targeting both alleles. In another example, the same gene is modified in situ by a “knock-in” method (eg, a cre-lox procedure and a hit and run procedure). Modification of protein domains and "motifs" and the introduction of specific mutations can be made using these methods. In a further example of genetic manipulation of ES cells, the gene is added as a cDNA construct or as a large genomic transgene (eg, BAC and YAC from a genomic library).
Human genes are added to rat cells as well as human cells. The gene to be modified is not limited to an AD-related gene, and may include any desired gene.

The protein expression profile of the cells can be determined, for example, by immunoblot analysis, ELIS
A, can be done using two-dimensional polyacrylamide gel electrophoresis analysis and histochemical analysis. Specific proteins of direct interest for AD are fragments of APP (eg, soluble APP) and A-β amyloid. For example, cells expressing these genes and other genes associated with AD can be used to identify drug candidates and to test hypotheses regarding specific interactions of genes, signaling factors, and proteins in neurons and AD. Can be used for Such cells may provide a scene for gene expression and its regulation in neurons.

B. ES Cells in Testing Drug Candidates Drugs designed to directly affect neurons can be tested in human and rat ES cell-derived neurons. The results have estimates for both preclinical animal studies and clinical trials. Drugs that work through other cell types
Rat or human ES cell-derived neurons can be tested by co-culturing with glial cells, such as macroglia, microglia, and oligodendrocytes. Cell types for co-culture are obtained, for example, as primary cultures, cell lines, and by deriving them from human ES cells. The toxicity of the drug also
It can be determined by using a similar culture. The effect of a drug is assessed by various assays, including, but not limited to, biochemical, immunochemical, or gene expression assays. ES cells can be genetically modified to test the effects of single nucleotide polymorphisms (SNPs) or greater genetic differences on drug efficacy and direct toxicity. Differentiated cells are included to test the efficacy of drugs that must cross the BBB in a model of the blood-brain barrier (BBB).

C. ES-Derived Cells as Transplants into the Brain Among the potential uses of neurons and neuron-associated cells derived from human ES cells is the repair of tissue damaged by neurodegenerative diseases and injuries. Current uses include striatal transplantation of dopaminergic neurons for Parkinson's disease (PD), repair of brain regions damaged by ischemic stroke, and filling spinal cord lesions. Further uses include transplantation of neurons into brain or body regions affected by: AD, peripheral neuropathy, ALS (amyotrophic lateral sclerosis).
, And other neurodegenerative diseases (eg, ataxia (trinucleotide repeat disease))
. The cells to be transplanted include neurons and neuron supporting cells (eg, glial cells or fibroblasts) or a combination of cells that support each other in the transplant.

The cells for such transplantation are not genetically modified or produce specific neurotransmitters (eg, dopamine for PD) or specific growth factors that support themselves or other cells. To be modified.

D. Further Uses of ES Cells In order to reduce the chance of graft rejection, ES cells express molecules that shield cells from the host immune system, such as expressing different HLA types. And / or by knocking out an antigenic macromolecule.

The transgenic ES cells described herein can be used for in vitro screening or testing for compounds. In one aspect, ES cells expressing a gene involved in drug metabolism (eg, the p450 gene) can be used in determining the effect of a compound on development and / or differentiation. Preferably, the ES cells express the human p450 gene.

The genetically modified ES cells described herein can also be used to create animal disease models useful for in vivo screening for potential therapeutics. Such animal models may include "humanized" rats (or other commonly used experimental species). These "humanized" animals are made from ES cells that have been genetically modified to have human genes associated with the disease state. Such generated animal models are useful for the post-discovery preclinical phase of drug development.

The following examples are intended only to illustrate the invention and should in no way be construed as limiting the invention.

EXAMPLES Example 1: Isolation and Culture of Rat Embryonic Tissue A. Rat Strains A small population of bred Norway (BN; Charles River) rats was established. Approximately 40 BN rats were microisolator (mi
croisolators were housed in cages and replaced when animals were used in the experiments. For the primordial germ cell development procedure (see below)
Sprague Dawley rats (SD; Simose)
n) was used. Regularly mated animals (F344 and Long-Evans) from a local supplier (Simonsen) were also used. Simonsen
Long-Evans strain from H.R. is a hooded rat, of which about 50%
Are black and 50% are white. It is a line that was traditionally crossed,
Simonsen animals are effective inbreds that have arisen through 16 generations of sibling-sister mating. For blastocyst injection, the majority of blastocysts were transferred to Fischer34.
The technology to generate from four systems was developed.

The F344 strain and Simonsen LE rats gave rise to the majority of blastocysts and served well for the experimental procedure. The LE animals were not uniformly pigmented, although coat color markers were required for chimeric evaluation. AGUS strains and AB2 rats were selected as host strains for blastocyst injection.
Because these are albino strains without food. Adult mice of these strains were not commercially available in sufficient numbers for use on short-term projects. Instead, a method of blastocyst injection was developed using F344 animals. A rich source of adult, pathogen-free animals (Simonsen) for F344 was available. In combination with LE ES cells, F
Host blastocysts from line 344 readily visualize the contribution of ES cells in chimeras.

B. Generation of Blastocysts by Natural Mating Females mated for blastocyst generation were selected by estrus. For the most effective use of the animals, vaginal plugs found on day 0.5 were checked for the presence of sperm, allowing monitoring of female fertility. The time of occurrence of the BN strain was determined to be 3
. Determined by lavage of the uterus and fallopian tubes from groups of 14 pregnant animals on days 5, 4.5 and 5.5. It was determined that unlike mice, the development of BN rat embryos in individual animals changed from day to day. In general, early cleavage stage embryos were found in the fallopian tubes at 3.5 and 4.5 days, and morula and blastocysts were present in the 4.5 and 5.5 day uterus. Show that in individual animals, embryos range from the zygote to the blastocyst stage, especially in the F344 rat strain and L.
Found in the E rat strain. Animals of these strains are available from the supplier (Simons
en) can be mated regularly and can be removed 3 days after mating. Blastocysts can be harvested on day 4.5 from about 50% of F344 rats and from about 80% of LE rats.

C. Rat Superovulation A method developed by Armstrong and Opavsky to superovulate rats ((1988) Biol. Reprod. 39: 511-).
518) was applied. Young female animals were inoculated with an osmotic minipump (Alza Corporation) containing porcine follicle stimulating hormone (FSH); the pump was removed after 3 days and the animals were mated. This method provides more predictable production of blastocysts in our population, and if the natural mating of the animal does not provide sufficient blastocysts, the host embryo may be injected for injection of ES cell candidates. It can be used for blastocyst production.

D. Delayed Blastocysts Two lines of delayed blastocyst-derived embryonic stem cell lines of mice have been obtained previously. In mice, simple removal of the ovaries at day 2.5, when the embryos descend the fallopian tubes, prevented transplantation after blastocyst development. This procedure had to be considerably modified for use in rats. The original procedure was unsuccessful in rats and delayed blastocysts were ovariectomized on day 2.5 or 3.5 and BN animals tested embryos 3-6 days later Did not get from. The embryo is Depo-
1 was transferred after ovariotomy using a minipump containing Provera (an analog of progesterone used to delay the transfer of mouse blastocysts).
Not obtained from one Sprague Dawley animal. A successful method combined oophorectomy on day 3.5 with daily injection of progesterone (5 mg / animal / day, subcutaneous injection). This method appears to be particularly effective in Long-Evans rats.

E. Primitive germ cells It has recently been reported that cells with all in vitro characteristics of embryonic stem cells can be derived from mouse primordial germ cells at day 8.5 (stage 13). Was. Rat embryos of the same stage (day 9.5) were obtained from BN, SD, F344 and LE rats, and the tail (after the last segment) was dissected and dissociated and placed on the SNL76 / 7 cell support layer. In a medium containing basic FGF, stem cell factor and LIF (Matsui et al.,
1992) (see below for culture conditions).

Example 2 Culture Medium for Rat Cells The culture medium used in most experiments was based on mouse ES cell medium and was prepared using 15% fetal calf serum (Hyclone), 1 × non-essential amino acids (Gibc).
o), and high glucose DMEM (Gibco) supplemented with 2-mercaptoethanol (0.1 mM). Conditioned medium was added to Buffalo rat hepatocytes (
BRL: ATCC), mouse AB-1 ES cells (provided by A. Bradley), and a rat blastocyst-derived cell line (BNR
B-1). The medium was conditioned by culturing the cell line for 2 days, then filtered and frozen for future use. For the experiments, conditioned media was used in a 50% mix with fresh media. The exogenous growth factor used was a leukemia inhibitory factor (
LIF: 1000-2000 units / ml; Gibco / BRL), basic fibroblast growth factor (BFGF: 20 ng / ml; Genzyme), and stem cell factor (SCF: 60 ng / ml; Genzyme).

The normal and delayed blastocysts (shown in FIG. 1) were transformed with LIF-producing mouse fibroblasts (
(SNL76 / 7) or a fibroblast support layer made from embryonic rat fibroblasts and cultured for 3-7 days. All attached blastocysts and almost all inner cell mass (ICM) were visible (FIG. 1d). Culture medium (LIF; L
IF and SCF, or LIF, SCF, and bFGF) did not make a clear difference in early blastocyst culture. The central mass of cells is usually the blastocyst culture 3
After 5 days, they were removed from each culture, dissociated, and subcultured on the same feeder cell type in the same medium. Secondary cultures always contain various forms of colonies,
Some mimicked the compact colony morphology of the cells (FIG. 2). Approximately one week after the secondary culture, colonies mimicking the ES cells were dissociated and subcultured. The subcultured cells were subcultured once and frozen. The cell line is a non-delayed Brow
n Norway blastocysts (BNRB-1), Fischer 344 delayed blastocysts (FRDB-1) and Long-Evans strains.

[0079] Two types of support layers were used in these experiments. SNL76 / 7 is mouse A
B-1 Mouse fibroblast cell line used as a support layer for ES cells (Soriano et al., 1991). SNL76 / 7 cells are neomycin resistant and produce LIF and stem cell factor. Rat embryo fibroblasts (RE
F) has been used for mouse embryos and is described, for example, in Doetschman et al. Emryol. Exp. Morph. 87: 27-45. Feeder cells were mitotically inactivated by treatment with mitomycin C. Mitomycin C is toxic to REF cells at the concentration of 10 μg / ml used for SNL cells, so the concentration was reduced to 4 μg / ml and the cells were treated for a shorter time. Cells were cultured on a support layer in 6- or 24-well tissue culture plates, cover slips, or glass culture slides (LabTek). Alternatively, the support layer can be mitotically inactivated using gamma irradiation (Robertso).
n, see above).

Primordial germ cells were analyzed for Sprague using the method reported for mice.
-Cultured from tissue obtained from incisions of 9.5 day embryonic rats from Dawley and Long-Evans (LE) strains, and the culture was stained for AP as described below. Some of the cultures contained colonies of cells stained with AP (FIG. 3e) and thus appeared like PGCs.

A. Effects of Culture Conditions on Rat Blastocyst-Induced Cells In preliminary experiments, various conditioned media and exogenous growth media were used to enhance the growth or maintenance of cells having the characteristics of embryonic stem cells. Tested in the search. The results are outlined in Table 1. The degree of AP staining was estimated for the cultures shown in FIG. Medium conditioned by BRL cells had no discernable effect on AP staining in rat culture. BRL cells produce LIF, SCF, and other factors that maintain mouse ES cells in an undifferentiated state. Similarly, mouse ES
Cell conditioned media also had no effect on rat cells. High density BN
Medium conditioned by RB-1 cells appears to reduce AP staining in cultures of the same cell line, which may cause the cells to undergo differentiation or inhibit proliferation of undifferentiated cells. It suggests that a factor that produces Of the conditions tested so far, only exogenous bFGF appeared to have a qualitative effect in increasing the number of AP-positive cells.

[0082]

[Table 1] Example 3 Induction of Rat ES Cells During Co-Culture Pluripotent ES cells were induced by co-culturing the above rat cell line with mouse ES cells. Rat ICM for non-delayed blastocysts was
The cells were cultured on the TO support layer for 3 days. After 3 days, about 20-50 cells were present in the ICM. Before differentiation began (usually about 4 days), rat ICMs were transferred to Del 19.2.
Was mixed with a mouse ES cell line called One copy of the HPRT gene (X
This line with a 19.2 kb "knockout" in the
Obtained from Dr. Bradley. Del19.2 cells are HPRT-
And does not survive in HAT medium and cannot return to the HPRT + phenotype. This co-culture of rat ICM: mouse ES cells was performed when confluent,
Passaged at least twice with trypsin. After about 5 days and 2 weeks,
HAT medium was added to kill mouse Del 19.2 cells. The rat cells survive on HAT treatment. Because they are HPRT positive. Hundreds of surviving colonies were obtained. As described in detail below, these colonies maintained markers of pluripotent ES cells.

Example 4 Characterization of Rat Cells Before and After Co-Culture Rat cells were characterized by various methods, both before and after co-culture with ES cells. One cell type was dominant in the first induced cell line (BNRB-1) when not co-cultured. These cells were round, stretchable, and had loose attachment to the underlying layer (FIG. 2C). Unlike mouse ES cells, these cells had little tendency to aggregate into tight clusters (compare FIGS. 3B and 3D). This cell line was tested for the presence of two markers characterizing mouse ES cells (alkaline phosphatase activity and a stage-specific embryonic antigen, SSEA-1). The second cell line (FRDB-
1) formed colonies with a more adherent morphology (FIGS. 2B, 2C) and were analyzed for specific markers.

A. Histochemistry Cells were fixed with 4% paraformaldehyde in PBS for AP or antibody staining. To detect alkaline phosphatase activity, cells were harvested using the Vectastain AP reagent kit (Vector Labs) (AP
(Produces a black reaction product in the presence of). The specificity of the staining was confirmed by levamisole inhibition. Anti-SSEA hybridoma supernatant (D
developmental Studies Hybridoma Bank)
Was used at a 1:50 to 1: 100 dilution, and antibody binding was detected with fluorescein or conjugated anti-mouse IgM (Vector labs). The cells growing on the cover glass were subjected to Nomarski optics (Nikon Diaphot).
Or visual using fluorescence (Leitz Laborlux); cells in tissue culture dishes were photographed using phase optics or brightfield optics. AP
For a quantitative assay of positive cells, cells were trypsinized, fixed with 2% paraformaldehyde, and stained in suspension using the AP reagent kit and counted in a hemocytometer.

1. Alkaline Phosphatase Alkaline phosphatase (AP) activity is characteristic of mouse primordial germ cells, blastocyst cell mass, and ES cells. Rat blastocysts of the inner cell mass have AP activity (FIG. 3A), like mouse ICMs (FIG. 3C) cultured under the same conditions.
). AP activity was consistently observed in cultures of BNRB-1 cells (
FIG. 3B), observed in only a small percentage of cells. Staining is levamisole (
Vector Labs), levamisole also inhibited AP activity in control mouse ES cells.

A quantitative assay was developed that provided a clear measure of the percentage of AP-stained cells. Table 2
As shown in the figure, after multiple passages, the percentage of AP-positive cells in the
It was only 5%. The FRDB-1 line was approximately 50% positive. Mouse ES cell culture (AB-1) was more than 90% positive. Rat cells were analyzed by staining colonies in culture dishes after co-culture. This A
The percentage of P-positive cells appeared to be almost 100% (see FIGS. 6 and 7).

[0087]

[Table 2] B. Rat Cell Pluripotency An important in vitro indicator of cell pluripotency is to indicate that these cells form an embryoid body of multiple cell types. .

(1. Embryoid bodies from rat cells before co-culture) BNRB-1 cells before co-culture showed the ability to differentiate in vitro, but the cells were Did not show the degree. When cultured under conditions that promoted differentiation of mouse ES cells into embryoid bodies, rat cells formed aggregates that developed into sac-like structures (FIG. 5A). These structures mimic a simple embryoid body bound by the endoderm initially formed by mouse ES cells, but unlike mouse cells, the rat embryoid sac is a more complex structure. Did not continue to differentiate.
In most cases, the sac appeared to be joined by a single epithelial layer, but some differentiated into multiple layers. The mouse embryoid bodies differentiated extensively into a variety of cell types when replated on the support layer; the rat embryoid sac did not differentiate when replated. This limited differentiation is consistent with the idea that the predominant cell type of the population will further differentiate into endoderm cells.

Under certain culture conditions, some colonies of BNRB-1 cells underwent morphological differentiation. On the rat embryo fibroblast feeder layer, mouse ES cells lost their AP staining and differentiated into morphologically distinct cell types similar to epithelial cells (FIG. 5C). Similarly, some of the rat cells on the fibroblast feeder layer of rat embryos showed less AP staining and formed colonies similar to epithelial cells (
(FIG. 5B). Similar results were observed when rat cells were cultured in retinoic acid (inducing mouse ES lineage differentiation) (not shown).

2. Embryoid Bodies Formed After Co-Culture Following co-culture, isolated and resuspended rat ES cells have a number of morphologically distinct cell types ("heart tissue" (Including a pulsating mass). The ability to form a beating heart and other embryoid bodies is an indicator of pluripotent ES cells.

Next, these cells are transplanted into an immunodeficient host animal (eg, a nude rat or mouse) to determine if these cells form a teratoma. The cells were subcloned, karyotyped and injected into host blastocysts.

Example 5 Induction of Rat ES Cells During Co-Culture 16 days after mating, 16 blastocysts were obtained from PVG rats, and 1 ml of ES cell culture medium containing 2000 units of mouse LIF ( (Same as above, except containing 20% fetal calf serum). This dish contained a mitotically inactivated STO cell support layer. Blastocysts were cultured for 3 days, revealing clusters of ICM cells. ICM cells were removed from the dish with a glass pipette and incubated for 30 minutes in a PBS solution containing 1 mM EGTA and no calcium and magnesium. The ICM dissociated into small clumps of cells was placed in a 6-well culture dish on a support layer in ES medium (without LIF). 1.5 × 10 ⁵ mouse ES cells (HPRT-Del 19.2 cells) were added to the same well. Control wells contained the same number of mouse ES cells but no rat cells.

After 3 days, cells in both wells were trypsinized (0.25 in 1 mM EDTA).
%; 15 minutes, 3 ° C). Nine tenths of the wells plated with rat ICMs were frozen by standard cell preservation methods, and the remaining tenths were plated in fresh 6-well chambers with the same medium and a support layer. .
Similarly, control wells were passaged at 1/10 dilution. 3 more similar
After three passages and three days of cell freezing (separating the culture containing rat cells in a second passage to make two wells), the two types of cultures are trypsinized, It was then replated (without dilution) on the HAT-containing ES cell medium on the support layer. Thereafter, the medium (containing HAT) was changed every day for 3 days. Medium changes were often made. Because the products of dead cells can often damage live cells in the same dish. On the third day, the culture was carefully observed under a microscope. Control cultures contained STO feeder cells but no visible embryonic stem cells. This means that all of the mouse HPRT-cells
It was shown to be killed by the medium.

The experimental culture (which initially contained the rat ICM) contained a large number of cell colonies.
One of the six wells ("A") contained 38 colonies containing an estimated 100-500 cells; of these, three were completely differentiated cells previously described as "endoderm" Apparently composed of types, six colonies consisted of a morphological mixture of cells, and the remaining 29 consisted only of cells that appeared indistinguishable from mouse ES cells at the microscopic level. Well "B" contained 30 colonies; 7 were "endoderm", 3 were mixtures, and 20 consisted of ES-like cells. The colonies were stained with AP, and colonies containing all ES-like cells were positive for AP, indicating that rat cells did not look like mouse ES cells, and that mouse cells were used as ES cells. It was shown that the marker to be identified was also present (FIG. 7).

To ensure that all mouse cells were killed in the control culture, the medium in the dish was replaced with HAT-free ES medium, and the culture was changed daily by changing the medium. For an additional 10 days. No ES cells appeared even after 10 days. This indicates that all mice E
It was demonstrated that S cells had been removed.

Example 6 Generation of Transgenic Rats Using Rat ES Cells Rat ES cells isolated as described above are tested for pluripotency in vivo by blastocyst injection. Long Evans rat ES cells were transferred to albino Lewi.
Inject into host blastocysts from strain s. The blastocysts are developed in a surrogate mother to term and the offspring are tested. The child has a brown coat spot and eye color,
The contribution from Long Evans ES cells is shown.

As will be apparent to those skilled in the art, various modifications and variations of the above-described embodiments can be made without departing from the spirit and scope of the invention. These modifications and variations are within the scope of the present invention.

[Brief description of the drawings]

FIG. 1, Panels AD are halftone copies of photographs showing rat blastocysts. FIG. 1A
Shows the Nomarski optics 400 times that of normal rat blastocysts from the Brown Norway strain; FIG. 1B shows that Fischer 3 with transplantation delayed for 10 days.
Fig. 4 shows a Normalski optical system 400 times that of rat blastocysts derived from 44 strains. FIG. 1C shows a 200-fold phase difference of a group of delayed rat blastocysts from the Long Evans strain, with transplantation delayed for 8 days. FIG. 1D is a blastocyst 3 days after attachment in culture on a SNL 76/7 fibroblast feeder layer. Inner cell mass (
ICM) is visible in the center of the culture.

FIG. 2, Panels AC are halftone copies of photographs showing ICM-derived cultures of rat blastocysts. 2A and 2B show colonies in a secondary culture of the FRDB-1 cell line. The morphology of this colony is similar to mouse ES cells at this stage of induction. FIG. 2C shows the third passage of cell line BNRB-1. This colony has a more epithelial morphology, representative of endoderm. 2A-C are 200 times phase contrast microscopy.

FIG. 3, Panels A-E, are 200 × halftone reproductions of cultured blastocysts. FIGS. 3A and 3C show alkaline phosphatase (AP) staining of inner cell mass cultured rat and mouse blastocysts, respectively. FIG. 3B shows rat blastocyst-derived cells stained with AP (line BNRB-1), and FIG. 3D shows mouse E stained with AP.
1 shows S cells. FIG. 3E shows AP-positive cells in colonies from Sprague-Dawley embryos on day 9. These cells can be derived from primordial germ cells.

FIG. 4A shows a phase contrast field of Long Evans rat blastocyst intracellular cell mass (ICM) cells cultured for 3 days before staining; FIG. 4B shows 3D stained with anti-SSEA-1 antibody. 1 shows fluorescence microscopy of ICM cells cultured for one day. FIG. 4C shows mouse ES cells stained with anti-SSEA-1. SSEA-1 is heterogeneously expressed. FIG. 4D
Indicates colonies from the BNRB-1 cell line stained with SSEA-1. This rat strain is also heterogeneous for SSEA-1 labeling.

FIG. 5A shows a cystic structure formed by the BRNB-1 cell line after culturing in suspension, similar to a single embryoid body derived from mouse ES cells. FIG. 5B shows morphological changes in the BNRB-1 cell line grown on the rat embryo fibroblast feeder layer; FIG. 5C shows the morphology in mouse ES cells grown on the rat embryo fibroblast feeder layer. Shows a biological change.

FIG. 6, Panels AD are halftone copies of 10 × phase contrast photographs showing the morphology of co-culture of rat blastocysts and mouse ES cells. FIG. 6A shows mouse AB-1.
Delayed Long Evans after 3 days of culture on ES cell feeder layer
3 shows ICM from rat blastocysts. The ICM cells look like mouse ES cells and there is no apparent differentiation. FIG. 6B shows the second passage of LE rat ICM by mechanical separation. Loosely attached spherical cells are present at the edge of the explant. FIG. 6C shows the LEM ICM after 7 passages. This ICM explant looks like mouse ES cells, but globular cells are much more abundant. These fast-growing spherical cells are considered to be endoderm. After several passages of cells with ES morphology, there are very few alkaline phosphatase (AP) -positive cells remaining. FIG. 6D shows AP staining of primary passage rat ICM. Spherical endoderm-like cells are negative.

FIG. 7, Panels AC are halftone copies of photographs of rat primordial germ cells (PGC) in culture. FIG. 7A shows 10 g of PGC cultures obtained by separating hindgut tissue and allantois from LE rat embryos at day 10.5 and co-culturing with mouse ES cells for two passages. A double phase contrast photograph is shown. After two passages (
Mouse ES cell confluence), which were removed by negative selection. The remaining rat cells were passaged one more time and the culture
Inspected on the day. Surviving rat cells are evident. FIG. 7B shows 20 times the cells in FIG. 7A. FIG. 7C shows the same colonies as shown in FIGS. 7A and 7B stained with alkaline phosphatase (AP) after 21 days in culture.
This morphology and staining indicates that these cells have the properties of ES cells.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG , KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW

Claims (32)

[Claims] 1. An isolated population of non-mouse embryonic stem cells. 2. The population of cells according to claim 1, wherein said cells are rats. 3. A method for obtaining embryonic stem cells derived from a target species, comprising: (a) obtaining non-human embryonic stem (ES) cells under conditions that support the growth of embryonic stem (ES) cells derived from the target species; Co-culturing the target embryonic stem cells and cells obtained from the embryo of the target species; and (b) isolating the ES cells from the target species. 4. The method of claim 3, wherein the non-target embryonic stem cells in step (a) are mice. 5. The method according to claim 1, wherein the cell obtained from the embryo of the target species in the step (a) is an inner cell mass (
4. The method of claim 3, which is derived from (ICM). 6. The method according to claim 3, wherein the cells obtained from the embryo of the target species in step (a) are primordial germ cells (PGC). 7. The method of claim 3, wherein said target species is non-mouse. 8. The method of claim 7, wherein said target species is selected from the group consisting of rat, sheep, cow and human. 9. The method of claim 8, wherein said target species is a rat. 10. The method of claim 3, wherein the non-target embryonic stem cells of step (a) lack a positive selectable marker. 11. The method according to claim 11, wherein the positive selection marker is an antibiotic resistance gene or HPR.
11. The method according to claim 10, wherein the method is selected from the group consisting of T resistance (HPRT) genes. 12. The method according to claim 1, wherein the positive selection marker is an HPRT gene.
2. The method according to 1. 13. The method of claim 3, wherein the non-target embryonic stem cells of step (a) carry a negative selection marker. 14. The method of claim 13, wherein said negative selectable marker is HPRT or herpes simplex virus thymidine kinase (HSV-tk). 15. The embryonic cells derived from the target species are cultured on a cell support layer,
The method of claim 3. 16. The method of claim 15, wherein said cell support layer is SNL76 / 7. 17. The method of claim 3, wherein said non-target embryonic stem cells are mitotically inactivated. 18. A non-mouse ES cell that has been genetically modified. 19. The genetically modified ES cell according to claim 18, wherein said cell is a rat. 20. The genetically modified ES cell according to claim 18, comprising one or more transgenes. 21. A chimeric embryo comprising the isolated population of non-mouse ES cells of claim 1. 22. The chimeric embryo according to claim 21, wherein the ES cell is a rat. 23. A chimeric embryo comprising a genetically modified non-mouse ES cell prepared according to the method of claim 3. 24. The chimeric embryo according to claim 23, wherein the function of one or more genes is disrupted. 25. The chimeric embryo according to claim 23, wherein said non-mouse ES cells are rats. 26. The chimeric embryo of claim 23, wherein said genetically modified non-mouse ES cells comprise one or more transgenes. 27. An animal comprising cells derived from the isolated population of non-mouse ES cells of claim 1. 28. The animal of claim 27, wherein said non-mouse ES cells are rats. 29. An animal comprising cells derived from a genetically modified non-mouse ES cell. 30. The animal of claim 29, wherein said non-mouse cells are rats. 31. The animal of claim 29, wherein said genetically modified non-mouse ES cells comprise one or more transgenes. 32. The animal of claim 29, wherein the function of one or more genes is disrupted.