HK1178199A

HK1178199A - Method for constructing chimeric rat using rat embryonic stem cells

Info

Publication number: HK1178199A
Application number: HK13104981.8A
Authority: HK
Inventors: 落谷孝广; 川又理树
Original assignee: 日本国立癌症研究中心; Ds制药生物医学株式会社
Priority date: 2009-12-01
Filing date: 2010-11-30
Publication date: 2013-09-06

Description

Method for constructing chimeric rat by using rat embryonic stem cell

Technical Field

The present invention relates to a method for preparing a chimeric embryo having improved germ line transmission efficiency using rat pluripotent stem cells, particularly rat embryonic stem cells (hereinafter, referred to as "ES cells"), a method for preparing a chimeric rat using the chimeric embryo, a chimeric rat prepared by the method, and a medium useful for preparing the chimeric rat.

Background

ES cells are an almost totipotent cell line and are very useful for the preparation of genetically modified animals and the like. For example, a chimeric animal can be produced by injecting ES cells in which a specific gene has been disrupted into a normal host blastocyst to be mixed with the cells of the host embryo, and returning the mixture to the uterus, and a genetically modified animal (knock-out animal) in which a specific gene has been disrupted can be produced by crossing the resulting chimeric animal or progeny and selecting animals born.

Rats are mammals having a size more suitable for manipulation than mice, and are one of the most useful experimental animals widely used in various fields including medicine. Therefore, it has been desired to establish rat ES cells and use the cells to produce chimeric rats. However, a technique for efficiently preparing chimeric rats, in which ES cells are differentiated into germ cell lineages and genetic information from the ES cells is transferred to the next generation without being affected by strains of rat ES cells and strains of host embryos (i.e., chimeric rats in which germ lines are transferred), has not been established so far.

Regarding the preparation of chimeric rats in which germ line transmission was confirmed, the study group of Qi-Long Ying et al and the study group of Austin Smith et al were reported in 2008, respectively (patent document 1, non-patent documents 1 and 2). In these 2 reports, rat ES cells were used to prepare chimeric rats; however, in both cases, chimeric rats of the transmitting germ line were successfully prepared from only one of the various combinations of ES cell lines and host embryo lines. It has been suggested that in the conventional practice of making knockout rats, it is important to use various combinations of ES cell lines and host embryo lines to achieve the preparation of chimeric rats, which poses a problem to be solved in the future.

In addition, despite the fact that some practical cases of preparing chimeric rats delivering germ lines have been reported, there is no report on establishing genetically modified rats (e.g., knockout rats, knock-in rats) targeted by genes, although they are limited as described above. This is probably due to the influence of the characteristics of the starting rat ES cells.

Although the use of MEK inhibitors, GSK3 inhibitors, and TGF β receptor type I Alk5 inhibitors (a-83-01) would establish rat iPS cells capable of producing chimeras, germ line transmission has not been achieved (non-patent document 3). In addition, it has also been reported that the combination of a MEK inhibitor and an Alk5 inhibitor significantly improves the efficiency of iPS cell production from human fibroblasts (non-patent document 4).

In recent years, Watanabe et al have found that Rho-binding kinase (ROCK) inhibitor Y-27632 blocks apoptosis of human ES cells and induces growth after isolation of single cells by enzyme treatment (non-patent document 5), and have not reported the usefulness of ROCK inhibitors for human stem cell culture (patent document 2).

[ File List ]

[ patent documents ]

Patent document 1: WO2008/015418

Patent document 2 WO2008/035110

[ non-patent document ]

Non-patent document 1: Cell, 135, 1299-

Non-patent document 2 Cell, 135, 1287-1298 (2008)

Non-patent document 3 Cell Stem Cell 4, 16-19 (2009)

Non-patent document 4: nat. Methods, 6, 805-

Non-patent document 5: mol. Pharmacol. 57, 976-.

Disclosure of Invention

Problems to be solved by the invention

It is an object of the present invention to provide a method for preparing a novel chimeric embryo, which allows a chimeric rat of a transfer line to be efficiently obtained without limiting the combination of a rat pluripotent stem cell line and a host embryo line, a method for preparing a chimeric rat using the chimeric embryo, and a medium therefor, and thereby provides a chimeric rat of a transfer line comprising a combination of a rat pluripotent stem cell line and a host embryo line, which has not been conventionally possible to prepare.

It is another object of the present invention to provide a rat ES cell which maintains the ability to produce chimeric rats, particularly the ability to transfer genetic modifications to the germline, even after undergoing genetic modification after ES cell preparation, and to provide a genetically modified, particularly gene-targeted, genetically modified rat using the rat ES cell.

Means for solving the problems

The inventors have conducted extensive studies to develop a method for preparing chimeric rats using rat ES cells, and as a result, have found that a chimeric embryo having improved germ line transmission efficiency can be efficiently prepared by injecting rat ES cells into a host embryo in the presence of an ES cell differentiation inhibitor, and that chimeric rats transmitting germ lines can be prepared by using the same. It was found that when a small amount of rat ES cells were injected into host embryos, better results were obtained because the adhesion strength of ES cells to blastocysts was increased when injection treatment was performed in the presence of ROCK inhibitors in addition to ES cell differentiation inhibitors. Furthermore, the inventors have newly found that chimeric embryos with improved germ line transmission efficiency can be prepared by using various combinations of rat ES cell lines and host lines by using an ES cell differentiation inhibitor or by using an ES cell differentiation inhibitor in combination with a ROCK inhibitor, and that chimeric rats transmitting germ lines can be efficiently prepared using the chimeric embryos.

Furthermore, the inventors succeeded in establishing rat ES cells, which maintain the ability to produce chimeric rats even after undergoing genetic modification, by adding an ES cell differentiation inhibitor and a ROCK inhibitor to the medium at the time of establishing rat ES cells, and also successfully preparing chimeric rats, which have the contribution of gene-targeted ES cells, using the ES cells.

The inventors have conducted further studies based on these findings, which led to the completion of the present invention.

Therefore, the present invention is as follows:

(1) a method of making a chimeric embryo, the method comprising the following steps (a) and (b):

(a) a step of contacting a fertilized host embryo collected from a female rat with a rat pluripotent stem cell in the presence of an ES cell differentiation inhibitor,

(b) culturing the host embryo contacted with the rat pluripotent stem cell to form a chimeric embryo.

(2) The method of (1), wherein the contacting is effected by injecting rat pluripotent stem cells into host embryos.

(3) The method of (1) or (2), wherein in step (a), the rat pluripotent stem cells are contacted with the host embryo in the presence of an ES cell differentiation inhibitor and a ROCK inhibitor.

(4) The method according to any one of (1) to (3), wherein the rat pluripotent stem cell is a genetically recombinant cell.

(5) The method according to any one of (1) to (4), wherein the host embryo is pre-cultured in the presence of an ES cell differentiation inhibitor before being contacted with rat pluripotent stem cells.

(6) The method of (5), wherein the preculture is carried out in the presence of an ES cell differentiation inhibitor and a ROCK inhibitor.

(7) The method according to any one of (1) to (6), wherein the culturing in step (b) is performed in the presence of an ES cell differentiation inhibitor.

(8) The method according to (7), wherein the culturing in step (b) is carried out in the presence of an ES cell differentiation inhibitor and a ROCK inhibitor.

(9) The method according to any one of (1) to (8), wherein the ES cell differentiation inhibitor consists of at least 2 drugs selected from the group consisting of: MEK inhibitors, GSK3 inhibitors, TGF β receptor inhibitors and FGF receptor inhibitors.

(10) The method of (9), wherein the ES cell differentiation inhibitor consists of a MEK inhibitor, a GSK3 inhibitor, and a TGF β receptor inhibitor.

(11) The method according to any one of (1) to (10), wherein the pluripotent stem cells are ES cells.

(12) The method according to any one of (1) to (11), wherein the rat pluripotent stem cell is a pluripotent stem cell prepared from a rat strain: in step (a), the rat line does not produce chimeric rats of the transmitting germ line when contacted with a host embryo in the absence of an ES cell differentiation inhibitor.

(13) The method of any one of (1) to (12), wherein the host embryo is derived from a rat strain: in step (a), the rat line does not produce chimeric rats of the transmitting germ line when contacted with rat pluripotent stem cells in the absence of an ES cell differentiation inhibitor.

(14) A method of making a chimeric rat, the method comprising: the chimeric embryo prepared according to the method of any one of (1) to (13) is transplanted into the uterus or oviduct of a pseudopregnant female rat to give birth to an offspring rat.

(15) The method of (14), further comprising: germline transmission in chimeric rats was confirmed.

(16) A germ line-transferred chimeric rat obtained by transplanting the chimeric embryo prepared according to the method of (12) or (13) into the uterus or oviduct of a pseudopregnant female rat to give birth to an offspring rat.

(17) A method of preparing a rat having a contribution of rat pluripotent stem cells to the entire body, the method comprising: chimeric rats having germline transmission confirmed according to the method described in (15) were mated with heterozygote rats.

(18) A culture medium for use in the preparation of chimeric rats comprising a MEK inhibitor, a GSK3 inhibitor and a TGF β inhibitor.

(19) The medium of (18), further comprising a ROCK inhibitor.

(20) Use of an ES cell differentiation inhibitor for the production of a culture medium for the preparation of chimeric rats.

(21) Use of an ES cell differentiation inhibitor and a ROCK inhibitor for the production of a culture medium for the preparation of chimeric rats.

(22) The use according to (20) or (21), wherein the ES cell differentiation inhibitor is a MEK inhibitor, a GSK3 inhibitor and a TGF β inhibitor.

(23) A germline transmission efficiency enhancer for chimeric rats comprising an ES cell differentiation inhibitor.

(24) The agent of (23), further comprising a ROCK inhibitor.

(25) The agent according to (23) or (24), wherein the ES cell differentiation inhibitor consists of at least 2 drugs selected from the group consisting of: MEK inhibitors, GSK3 inhibitors, TGF β receptor inhibitors and FGF receptor inhibitors.

(26) The agent according to (25), wherein the ES cell differentiation inhibitor consists of a MEK inhibitor, a GSK3 inhibitor and a TGF β receptor inhibitor.

(27) A method for producing ES cells using an ES cell differentiation inhibitor and a ROCK inhibitor.

(28) The method of (27), wherein the ES cell differentiation inhibitor comprises: a combination of a MEK inhibitor, a GSK3 inhibitor and a TGF β receptor inhibitor, or a combination of a MEK inhibitor, a GSK3 inhibitor and an FGF receptor inhibitor.

(29) A method for producing ES cells using the medium according to (19).

(30) The method according to any one of (27) to (29), wherein the ES cells are rat ES cells capable of maintaining the ability to produce chimeric rats even after undergoing genetic modification.

(31) The method of (30), wherein the genetic modification is modification by gene targeting.

(32) The method of (30) or (31), wherein the chimeric rat is a transmission germ line chimeric rat.

(33) A rat ES cell capable of maintaining the ability to produce chimeric rats even after undergoing genetic modification.

(34) The method of (33), wherein the genetic modification is modification by gene targeting.

(35) The cell of (33) or (34), wherein the chimeric rat is a transmission germ line chimeric rat.

(36) A genetically modified chimeric rat having a contribution of the genetically modified rat ES cell according to any one of (33) to (35).

(37) The rat of (36), which has a contribution of genetically modified ES cells to a germ line.

(38) A rat obtained by mating the rat according to (37), which has a contribution of the genetically modified ES cell to the whole body.

(39) A culture medium for preparing rat ES cells capable of maintaining the ability to produce chimeric rats even after undergoing genetic modification, comprising an ES cell differentiation inhibitor and a ROCK inhibitor.

(40) A culture medium for preparing rat ES cells capable of maintaining the ability to produce chimeric rats even after undergoing genetic modification, comprising a MEK inhibitor, a GSK3 inhibitor, a TGF β receptor inhibitor and a ROCK inhibitor.

(41) A genetically modified rat having a contribution of rat ES cells undergoing biallelic gene modification.

(42) A method of making a genetically modified rat, the method comprising the step of biallelic modification of rat ES cells.

Effects of the invention

According to the present invention, a chimeric embryo having improved germ line transmission efficiency can be prepared regardless of the strain of rat pluripotent stem cells or the strain of the host embryo; by using the chimeric embryo, a germ line-transferred chimeric rat can be efficiently prepared. Therefore, genetically modified rats (knockout rats, knock-in rats, etc.) which can be widely used for various pharmacological or physiological studies, as well as for regenerative medicine studies, etc. can be easily prepared.

Drawings

FIG. 1 is a photograph showing growth halos of blastocysts in (a) a YPAC-free medium and (b) a YPAC-added medium for ES cells. Blastocysts of 4.5 days were inoculated on division-inactivated MEFs. The zona pellucida was removed using Tyrode buffer. (c) Is a graph showing the results of quantitative PCR analysis of Venus, Oct4, Nanog, Sox2, and Rex1 in the Inner Cell Mass (ICM). 7 days after inoculation, RNA was extracted from dome-like fractions of 7 or 4 blastocyst-induced ICMs without addition of YPAC factor and with addition of YPAC factor. Data are the average of 3 independent experiments, relative gene expression levels in REF, ICM and ICM + YPAC factors, respectively.

[ FIG. 2-1 ]]The figure shows the results of characterization of rat ES cells. (a) The photograph of the colonies shown shows the effect of Y-27632. The isolated single cells (1X 10)⁵TgWW1 cells, passage 6) were seeded into 6-well plates. The left panel shows colonies obtained by adding MEF + YPAC factor to the medium, the middle panel shows colonies obtained by adding PAC factor, and the right panel shows colonies obtained by adding Y factor. (b) Photographs showing alkaline phosphatase (ALP) staining of the same various cells. (c) Is a photograph of a Giemsa stain of 50 TgWL2 cells (passage number: 7). TgWL2 (passage number: 7) shows a karyotype of 42 chromosomes, including the XX sex chromosome. (d) Are graphical representations of gene expression patterns compared by scatter plot analysis. TgWW1 and REF are shown in the left diagram and TgWW1 and LL are shown in the right diagram. Microarray analysis was performed using an Agilent gene chip (whole rat genome microarray kit). The center line represents the equivalence curve; the line above and below it indicates that the gene expression levels of the samples differ by a factor of 2. (e) Are graphical representations comparing the gene expression patterns of REF, TgWL1, TgWW1 and LL. These are the results of Q-PCR of Venus, Oct4, Nanog, Sox2 and Rex1 in rat ES cell lines. Data are the average of 3 independent experiments, relative gene expression levels of REF, TgWL1, TgWW1 and LL, respectively. (f) Is a photograph showing teratoma formation from rat cells. This is achieved by mixing 2.6X 10⁶TgWW1 (passage number: 5) cells were subcutaneously transplanted into teratomas generated in immunodeficient mice.

FIG. 2-2 (g) is a graph showing the results of microarray analysis and hierarchical cluster analysis. A gene expression analysis (Agilent Technology) based on a single color microarray containing 41,000 genes was used. The numerical values on the graph indicate the correlation coefficients. (h) Immunostaining patterns of Oct4, Nanog, and Sox2 in rat ES cells are shown (lower panel). The upper panel shows DAPI stained images. (i) Cross-sectional views of the 3 germ layers of teratomas induced from the TgWW1 ES cell line are shown.

FIG. 3 is a graph showing the results of examination of the effects of embryoid bodies (Eb) and YPAC factors. Photographs of embryoid bodies (TgWL 1) taken 3 days and 7 days after culturing ES cells in basal medium in the absence of Y-27632 are shown: (a) no PAC factor is added, and (b) PAC factor is added. (c) Is a graph showing the results of Q-PCR analysis of Venus, Oct4, Nanog, Sox2, and Rex1 in EB. Data are the average of 3 independent experiments, gene expression levels in ES cells at 0, 3 and 7 days of culture without inhibitor addition (open circles) and PAC addition (filled squares), respectively.

FIG. 4-1 is a schematic representation of germline chimera preparation by YPAC injection method. (a) Is a graphical representation of the expression of AmCyan1 in stably transformed clones generated by introducing the p-CAG-AmCyan1 plasmid into nucleic acids. (b) Is a photograph showing the results of examination of the effect of YPAC factor in the injection method. The image obtained by adding YPAC factor to ES basal medium is shown in the right panel, and the image obtained without addition is shown in the left panel. The upper panel is a photograph taken after 5 hours of incubation; the lower panel is a photograph taken after 30 hours of incubation. (c) Photographs showing fetal germ line chimeras. TgWW1+ C cells were injected into Wistar blastocysts. Venus and AmCyan1 fluorescence was detected throughout the fetus, kidney and testis at day 18. (d) Shows the generation of chimeric colored rats prepared by YPAC injection method. (e) Germ line transmission in adult chimeras is shown. The chimera (TgWL 1) was mated with female Wistar. Germ line individuals are identified by their coat color (4/16). (f) Genotyping analysis of F1 of the female chimera (TgWL 2) is shown. After tail genomic DNA was collected, the Venus region was amplified by PCR. 3 of the 6 individuals were identified as germline individuals. The Venus gene has a size of 199 bp; m indicates a 100 bp DNA marker. Lanes 1, 2 and 3 show germline individuals with spiny hair color, and lanes 4, 5 and 6 show individuals with spiny hair color negative (albino variant). (g) Photographs of single cells injected into blastocysts (TgWW 1) are shown. Photographs were taken of blastocysts at 3 hours of incubation after injection. Each arrow indicates injected cells. (h) Photographs showing chimeric rat fetuses with confirmed germ line transmission. Venus-positive germ cells were detected on the 16 day gonad side. The ratio bar is 100 μm.

[ FIG. 4-2] (i) shows germline transmission in ES cells subcultured for long periods. Injecting long-term cultured ES cells (TgWL 2: generation number: 22) into blastocysts; after 17.5 days, Venus fluorescence on gonads was detected (bar: 300 μm). The top panel shows a bright field image and the bottom panel shows a fluorescent image. (j) An example of a coat color chimera obtained by injection without addition of YPAC factor is shown. (k) An example of a gross chimera obtained by injection with addition of YPAC is shown.

FIG. 5 is a diagram relating to the preparation of Tg rats from ES cells. (A) And (B) shows the cloning and expression of Oct4-Venus transformant cells. The Oct4-Venus transgene was introduced into ES cells at passage 5 (LL 2) and Venus-positive clones were subcultured without drug selection. (A) Arrow in (d) indicates that Venus expression is inhibited. (B) Shows uniform expression of Oct4-Venus in ES cells. (C) Tg rats obtained from ES cells showing uniform expression of Oct4-Venus are shown. Arrows indicate esTG rats obtained from chimeric rats by germ line transmission. (D) Venus fluorescence on gonads of esTg rats at day 16 of the embryo is shown. (E) Shows the outgrowth of esTg blastocysts in YPAC medium. (F) Rat ES cell lines derived from esTg blastocysts are shown. Even after 10 passages, no inhibition of Oct4-Venus expression was observed. (ratio bar: 300. mu.m (A, B, D), 100. mu.m (E, F))

Fig. 6 is a diagram for the preparation of knock-in rats by homologous recombination via ZFNs. (A) Is a schematic illustration of Oct4 targeting. The ZFN pair will recognize exon 1. Each grey box indicates a coding region and each open box indicates a non-coding region. Bald lines indicate homology arms. Each P is a primer. (B) Genotyping PCR analysis of gene-targeted ES cell clones was shown. (C) The gene-targeted ES cell clone number 11 (passage number: 14) is shown (open arrow: undifferentiated cell, closed arrow: differentiated cell).

Fig. 7 is a diagram for the preparation of knockout rats by homologous recombination via ZFNs. This is a schematic for p53 targeting. The ZFN pair will recognize exon 4. Bald lines indicate homology arms.

FIG. 8 (A) shows genotyping PCR analysis of gene-targeted ES cell clones (WT: wild-type, KI: p53 gene-targeted allele). (B) Showing AmCyan1 at homologous recombination p53^+/-Expression in ES cell clones.

FIG. 9 (A) shows p53 gene knock-out rats obtained by mating female chimeras with wild type LEA-line rats (arrows indicate germ line transmitting rats). (B) Genotyping analysis of rats produced as a result of mating female chimeras with wild type LEA-line rats (KI: p53 gene targeted allele) is shown.

Fig. 10 is a diagram for the preparation of knockout rats by homologous recombination via ZFNs. (A) Is a schematic for p53 targeting. The ZFN pair will recognize exon 4. Bald lines indicate homology arms. (B) Genotyping analysis of gene-targeted ES cell clones (WT: wild-type, KI: p53 gene-targeted allele) was shown. Lanes are numbered 1-7 from the left. (C) Display p53^-/-ES cells [ cell line: p53^C/Z]Details of gene targeting of (a). (D) Showing AmCyan1 at homologous recombination p53^+/-Expression in ES cell clones.

In FIG. 11, (A) shows genotyping analysis (WT: wild-type) of the gene-targeted ES cell clone. (B) Shows AmCyan1 and tdTomato at homologous recombination p53^-/-Expression in ES cell clones.

FIG. 12 shows (A) the reaction from p53^-/-ES cells [ cell line: p53^C/R2]The developed chimeric rat fetus. (B) Shows a starting point p53^-/-ES cells [ cell line: p53^C/R2]The head of the developed chimeric rat fetus is malformed (on the left side of the figure). (C) The expression of AmCyan1 in the head of the fetus of (B) is shown.

Detailed Description

The present invention provides a method for efficiently preparing chimeric rats delivering germ lines using rat pluripotent stem cells without being limited to a specific strain of rat pluripotent stem cells or a strain of host embryos. The rat pluripotent stem cell, the method for preparing a chimeric embryo and a chimeric rat, the method for preparing a genetically modified rat, and the kit for preparing a chimeric rat according to the present invention are described below.

1. Pluripotent stem cells of rat

In the present invention, "pluripotent stem cell" means a cell that maintains an undifferentiated state and pluripotency, and is represented by ES cells and induced pluripotent stem cells (iPS cells). The ES cells may be ES cells reprogrammed from the nucleus of somatic cells, but are preferably prepared from rat early embryos by a method described later. Examples include, in addition to ES cells: embryonic germ cells (EG cells) derived from primordial germ cells, pluripotent germline stem cells (mGS cells) isolated from the testis, and the like. Preferably, the rat pluripotent stem cell to be used in the present invention is a rat ES cell or a rat iPS cell, more preferably a rat ES cell.

1-1. rat ES cells

The rat ES cells used for producing chimeric rats in the present invention may be derived from any rat strain as long as it can produce chimeric rats of the transmission line. Although the strain of the rat is not particularly limited, for example, it is selected from rat strains such as the following: wistar Kyoto strain (WKY), Brown Norway strain (BN), Goto-Kakizaki strain (GK), SD strain, F344/Du strain (Fischer), Wistar strain, Wistar Hannover strain, Long-Evans rat lick strain (LEA), ACI strain, etc. In addition, in the present invention, the line may be a pure line, or a line obtained by crossing 2 lines or more. In view of the features of the present invention that enable efficient production of chimeric rats of germline regardless of the rat ES cell line, the present invention is particularly useful when using ES cells prepared from rat lines: in the step of contacting the host embryo with rat ES cells described below, the rat strain does not produce chimeric rats of the transmitting germ line when rat ES cells are contacted with the host embryo in the absence of an ES cell differentiation inhibitor.

Rat ES cells capable of producing chimeric rats delivering germ lines can be obtained in the given case from the cell lines exemplified above, or can also be produced according to methods known per se. Examples of the production method of rat ES cells include the method of Buehr M et al (Cell 2008; 135: 1287-1298), and the like.

Rat ES cells having the ability to prepare chimeric rats delivering germ lines can also be established and prepared by performing a method comprising the following steps (A) to (C):

(A) dissociating the inner cell mass formed by culturing rat blastocysts,

(B) a step of culturing primary embryonic stem cells obtained from the culture of the dissociated inner cell mass until it can be passaged,

(C) dissociating the primary embryonic stem cells that have become passable, preserving the state of cell aggregates, and culturing them.

The culture in the above-mentioned production method is preferably carried out using a medium containing at least 2 ES cell differentiation inhibitors, more preferably using a medium containing at least 2 ES cell differentiation inhibitors and a ROCK inhibitor. Here, the "ES cell differentiation inhibitor" refers to a substance other than Leukemia Inhibitory Factor (LIF): which can inhibit differentiation of ES cells into other cells or tissues, etc., and in the present invention, may be any substance having an effect of inhibiting differentiation of rat ES cells. ES cell differentiation inhibitors include, for example, MEK inhibitors, GSK3 inhibitors, TGF beta receptor inhibitors, FGF receptor inhibitors, and the like. For this culture, specifically, a medium containing a MEK inhibitor and a GSK3 inhibitor is preferably used; more preferably a medium containing a MEK inhibitor, a GSK3 inhibitor, an FGF receptor inhibitor and a ROCK inhibitor; more preferably, a medium containing a MEK inhibitor, a GSK3 inhibitor, a TGF β receptor inhibitor and a ROCK inhibitor is used; particularly preferably, a medium containing a MEK inhibitor, a GSK3 inhibitor, a TGF β receptor inhibitor and a ROCK inhibitor is used.

By using a medium containing an ES cell differentiation inhibitor and a ROCK inhibitor, it is possible to prepare rat ES cells which can maintain the ability to produce chimeric rats, particularly those of the transmission line, even after undergoing genetic modification after ES cell establishment.

The MEK inhibitor is not particularly limited as long as it has an effect of inhibiting the function of MEK (MAP kinase-ERK kinase), and for example, AZD6244, CI-1040 (PD 184352), PD0325901, RDEA119 (BAY 869766), SL327, U0126 (both from Selleck above), PD98059, U0124, U0125 (both from COSMO BIO co., Ltd above), and the like can be mentioned. The concentration of the MEK inhibitor to be added to the medium may be appropriately selected from the following ranges: for example, 0.01 to 100. mu.m, preferably 0.1 to 5. mu.M.

The GSK3 inhibitor is not particularly limited as long as it has an action of inhibiting the function of Glycogen Synthase Kinase (GSK) 3, and for example, SB216763 (seleck), CHIR98014, CHIR99021 (all from Axon Medchem above), SB415286 (Tocris Bioscience), Kenpaullone (COSMO BIO co., Ltd.), and the like can be mentioned. The concentration of the GSK3 inhibitor to be added to the medium may be appropriately selected from the following ranges: for example, 0.01 to 100. mu.m, preferably 1 to 10 μm.

The TGF-beta receptor inhibitor is not particularly limited as long as it has an effect of inhibiting the function of a Transforming Growth Factor (TGF) -beta receptor, and for example, 2- (5-benzo [1,3] dioxol-4-yl-2-tert-butyl-1H-imidazol-4-yl) -6-methylpyridine, 3- (6-methylpyridin-2-yl) -4- (4-quinolyl) -1-phenylthiocarbamoyl-1H-pyrazole (A-83-01), 2- (5-chloro-2-fluorophenyl) pteridin-4-yl) pyridin-4-ylamine (SD-208), 3- (pyridin-2-yl) -4- (4-quinolyl) ] -1H-pyrazole, 2- (3- (6-methylpyridin-2-yl) -1H-pyrazol-4-yl) -1, 5-naphthyridine (all from Merck above), SB431542 (Sigma Aldrich), and the like. TGF β receptor inhibitors also include TGF β receptor antagonists. The concentration of the TGF β receptor inhibitor to be added to the medium may be appropriately selected from the following ranges: for example, 0.01 to 10 μ M, preferably 0.1 to 1 μ M.

The FGF receptor inhibitor is not particularly limited as long as it has an effect of inhibiting the function of a Fibroblast Growth Factor (FGF) receptor, and for example, SU5402 (COSMO BIO co., Ltd.), PD173074 (STEMGENT), and the like can be mentioned. FGF receptor inhibitors also include FGF receptor antagonists. The concentration of the FGF receptor inhibitor to be added to the medium may be appropriately selected from the following ranges: for example, 0.005 to 500. mu.m, preferably 0.07 to 50 μm.

The ROCK inhibitor is not particularly limited as long as it has an effect of inhibiting the function of Rho-binding kinase. Examples of ROCK inhibitors include: GSK269962A (Axon Medchem), fasudil hydrochloride (Tocris Bioscience), Y-27632, H-1152 (all from Wako Pure Chemical Industries, Ltd.), and the like. The concentration of the ROCK inhibitor to be added to the medium may be appropriately selected from the following ranges: for example, 0.0001 to 500. mu.m, preferably 1 to 50 μm.

The basal medium to be used for establishing and culturing rat ES cells is not particularly limited as long as it can be used for culturing animal cells. Examples thereof include: BME Medium, BGJb Medium, CMRL 1066 Medium, Glasgow MEM Medium, modified MEM stretch Medium, IMDM Medium, Medium 199 Medium, Eagle MEM Medium, α MEM Medium, DMEM Medium, hamF12 Medium, RPMI 1640 Medium, Fischer's Medium, and mixed media thereof, and the like.

The medium may be a serum-containing medium or a serum-free medium. When using unconditioned or unpurified serum, the serum must be added to the medium to the following extent: the rat ES cells did not lose their ability to produce chimeric rats of the transmission germ line due to the influence of serum. When 5% or more (e.g., about 10 to about 20%) serum is added, it is desirable to use conditioned or purified serum for ES cell culture. Such serum for ES cell culture (e.g., bovine fetal serum) is commercially available.

The medium may also contain fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, inorganic salts, and the like.

In the case of rat ES cells in culture, Leukemia Inhibitory Factor (LIF) may also be used. The LIF is particularly preferably rat-derived LIF (rLIF). The LIF may be used by adding a medium to the culture in the steps (B) and (C) of the above-mentioned production method as appropriate. Meanwhile, in step (a), in order to increase the inner cell mass formation efficiency, the rat ES cell establishment efficiency, and the rat ES cell mass, the concentration of rLIF added to the medium is very preferably not more than 100 units/mL of the medium, and more preferably, the rat ES cells are cultured using the medium containing no LIF at least until the inner cell mass formation stage, preferably through the whole step (a). For rLIF, commercial products (Chemicon Company, etc.) can be purchased and used.

Preferably, rat ES cells are further produced and cultured using feeder cells. The feeder cells may be cells derived from any species available to those of ordinary skill in the art, and are preferably normal fibroblasts, rather than established feeder cell lines. In particular, normal mouse embryonic fibroblasts may be mentioned. More specifically, primary cultured cells (normal fibroblasts) of mouse embryonic fibroblasts between day 12 and day 16 of pregnancy can be mentioned. Examples of normal fibroblasts include normal fibroblasts from day 12.5 ICR fetal mice. Feeder cells can be prepared by conventional methods. Commercially available products (mouse fibroblasts; Asahi Techno Glass Corporation, etc.) may also be used. Preferably, feeder cells inactivated by mitomycin C treatment or the like are used.

The culture vessel for culturing rat ES cells is not particularly limited as long as it can be used for cell culture; such culture vessels include, for example, flasks, tissue culture flasks, plates, petri dishes, tissue culture dishes, petri dishes, microplates, microwell plates, multi-well plates, chamber slides, petri dishes, test tubes, trays, culture bags, roller bottles, and the like. The culture vessel may be non-cell adherent or cell adherent. A cell-attached culture vessel whose surface is coated with a cell-supporting substrate layer for the purpose of improving cell attachment can be used; such cell supporting basal layers include, for example, collagen, gelatin, matrigel, poly-L-lysine, laminin, fibronectin, and the like.

The culture may be, for example, in CO₂In an incubator, in about 1 to about 10%, preferably about 2 to about 5%, more preferably about 5% CO₂Concentration atmosphere, at about 30 to about 40 deg.C, preferably about 35 to about 37.5 deg.C, more preferably about 37 deg.C.

As other components in the medium, components conventionally used for culturing ES cells are suitably contained, by combining them within the ordinary knowledge of those skilled in the art.

The specific composition of the medium is exemplified below.

1) Culture medium (culture medium) for rat ES cell establishment

The medium used in the step from blastocyst to inner cell mass formation is referred to as "medium for rat ES cell establishment".

(specific examples of compositions)

DMEM including 110 mg/L sodium pyruvate and 200 mM GlutaMax (GIBCO)

20% FBS（ES CELL QUALIFIED FBS）（GIBCO）

0.1 mM 2-mercaptoethanol (Sigma)

1% stock solution of non-essential amino acids (GIBCO)

1% 1 × antibiotic antimycotic (GIBCO)

10 μM Y-27632（WAKO）

1 μM PD0325901（Axon Medchem）

0.5 μM A-83-01（TOCRIS）

3 μM CHIR99021（Axon Medchem）

2) Culture medium of rat ES cells

The medium used in the culture after the formation of the inner cell mass (including the culture of established rat ES cells) is referred to as "the culture medium of rat ES cells".

(specific examples of compositions)

The culture medium for ES cells is similar to that used for the production of rat ES cells, and may additionally contain rat leukemia inhibitory factor (rLIF). rLIF is preferably added and mixed just prior to use.

(1) Rat ES cell establishment method

One specific example of a method for establishing rat ES cells of the present invention is shown below.

1) Oocyte (embryo at blastocyst stage) sampling

As rats for oocyte sampling, rats from the following strains can be used: such as the previously described Wistar Kyoto (WKY) strain, Brown Norway (BN) strain, Goto-Kakizaki (GK) strain, SD strain, F344/Du (Fischer) strain, Wistar Hannover strain, Long-Evans rat lick strain (LEA), and ACI strain. Rats in the age range of 8-40 weeks, preferably 10-20 weeks, more preferably 10-12 weeks, may be used.

Oocyte sampling may be performed by conventional methods known to those of ordinary skill in the art. Specifically, the rats were naturally crossed, and female rats for oocyte sampling were sacrificed about 4 to 5 days after the pessary test to extirpate the uterus. The uterus is perfused with a suitable medium to recover fertilized oocytes (embryos). As used hereinThe medium includes, for example, mw medium (640.0 mg/100 ml NaCl, 35.6 mg/100 ml KCl, 16.2 mg/100 ml KH)₂PO₄、29.4 mg/100 ml MgSO₄-7H₂O、190.0 mg/100 ml NaHCO₃100.0 mg/100 ml glucose, 2.5 mg/100 ml sodium pyruvate, 46.0 mg/100 ml calcium lactate, 5.0 mg/100 ml streptomycin, 7.5 mg/100 ml penicillin, 0.5% phenol red (0.2 ml), 20 mM beta-ME (10. mu.l), 100 mM EDTA-2Na (10. mu.l), 300.0 mg/100 ml BSA), M2 medium (0.251 g/L calcium chloride-2H)₂O, 0.143G/L magnesium sulfate, 0.356G/L potassium chloride, 0.162G/L potassium phosphate, 5.532G/L sodium chloride, 4.0G/L albumin, 1.0G/L D-glucose, 4.969G/L HEPES, 0.01G/L phenol red-Na, 0.036G/L pyruvic acid-Na, 0.35G/L sodium bicarbonate, 0.06G/L penicillin G, 0.05G/L streptomycin sulfate, 4.349G/L D, L-lactic acid), etc.

Recovered embryos can also be cultured in media such as mw media, M2, M16, and the like. By this culture, a blastocyst (an embryo at the blastocyst stage) develops from a fertilized oocyte (embryo) through the morula. To promote development towards this stage, the culture is typically at 5% CO₂The incubation was carried out in an incubator at 37 ℃ overnight. Microscopic observation confirmed that development had progressed to the blastocyst stage. Preferably, the development is carried out to a late blastocyst stage.

2) Formation and isolation of inner cell masses

The blastocyst obtained in the above 1) was confirmed by a microscope, and the zona pellucida was removed. The zona pellucida was removed using acid Tyrode (pH 2.5), hyaluronidase, pronase, etc. Then, the feeder cells treated with mitomycin C were seeded on a culture dish, the rat blastocysts from which the zona pellucida was removed were transferred to the dish, and culture was started using the medium for rat ES cell establishment.

Between day 1 and 4 of culture, the zona pellucida removed rat blastocysts (late stage) were attached to feeder cells. 5 to 10 days after the attachment, the inner cell mass emerging from the blastocyst is mechanically separated using a 200. mu.l pipette or the like. The separated inner Cell mass is mechanically dissociated using a pipette or a protease such as trypsin-EDTA, Accutase (registered trademark), etc. This dissociation step is preferably performed using a pipette or the like until the inner cell mass becomes a cell aggregate consisting of about 5 to 20 cells. The state of the maintained cell aggregates can be confirmed with a microscope.

3) Establishment of ES cells

The inner cell mass dissociated in 2) above was cultured in a culture medium of rat ES cells in a gelatin-coated culture dish in which feeder cells were seeded. Primary ES cell colonies typically appear between days 2 and 4 after the start of culture. The appearance of colonies of primary ES cells was confirmed by microscopic observation (the appearing ES cells are referred to as "primary ES cells"). By continuing the culture for about 5-10 days thereafter, the primary ES cell colonies become in a state capable of being passaged. As used herein, "a state capable of being passaged" refers to a state in which: wherein the number of cells constituting the formed primary ES cell colony has reached about 200-600, and the cell spacing has become compact, and the cell mass has been shown to have a shiny dome-like form. While confirming it with such a morphology with a microscope, ES cell colonies were isolated using a 200. mu.l pipette or the like. The separated ES cell colony is transferred to a sterilized tube or the like containing a culture medium of rat ES cells, and mechanically dissociated until it becomes a cell aggregate consisting of about 5 to 20 cells, or dissociated by using a protease such as Accutase (registered trademark). Mechanical dissociation is preferred. Dissociated ES cell colonies were subjected to primary culture (cells at passage 1) in rat ES cell medium in gelatin-coated culture dishes seeded with feeder cells. The ES cell colonies appeared about 2-4 days after the start of culture and became in a state capable of being passaged about 5-10 days.

After removing the medium, the ES cell colonies which had become passable in the above and the entire surface thereof were coated with Accutase (registered trademark) or 2.5% trypsin which had been incubated at 37 ℃ in advance. Protease treatment was immediately stopped when it was microscopically confirmed that 70% or more of all ES cell colonies were being detached from the feeder cells. The protease treatment can be stopped as follows: for example, a medium containing 10% fetal bovine serum or a large amount of serum-free medium is added. Then, the ES cell colonies were further mechanically disrupted using a 5 ml pipette or the like, the cell suspension was centrifuged (about 3 minutes at room temperature, 1000 rpm) to separate the cells from the medium, and only the cells were recovered. The cells were suspended in the culture medium of rat ES cells, and after confirming with a microscope that the cells formed aggregates of 5-20 cells (instead of becoming completely single cells), the cells were transferred to a culture dish in which feeder cells were seeded, and cultured (passage 2 cells).

Thereafter, since the cells become a state capable of being passaged every about 3 to 5 days, they can be continuously passaged and cultured by dissociating the cells with a protease such as Accutase (registered trademark) or mechanically dissociating the cells.

It was confirmed that rat ES cells retain the ES cell performance, that is, the desired ES cells maintain the undifferentiated state (totipotency), for example, by examining the expression of ES cell-specific genes (e.g., Oct3/4 gene, Nanog gene), alkaline phosphatase activity, embryoid body-forming ability, expression of SSEA-1 and SSEA-4, chromosome number, condition after subculture, in vitro pluripotency, ability to differentiate into cells of 3 germ cells, teratoma-forming ability, and the like. Their verification can be achieved using techniques known per se (see, for example, WO 2005/085427), some specific examples of which are given in the examples below.

1-2. rat iPS cells

Rat iPS cells can be prepared according to the method described, for example, in Cell Stem Cell 4, 16-19 (2009). As the starting material, rat somatic cells, fetal, infant or adult somatic cells collected from the aforementioned rat ES cell line can be used. Specifically, tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, sperm stem cells, and the like; (ii) tissue progenitor cells; cells that have differentiated such as lymphocytes, epithelial cells, muscle cells, fibroblasts, and the like; and the like. As the reprogramming factors for nuclear reprogramming, in addition to those used in the foregoing references, reprogramming factors that can be used for establishing iPS cells in mice, humans, and other mammals can also be appropriately selected and used within a feasible range. As the MEK inhibitor, GSK3 inhibitor and TGF β inhibitor for improving the efficiency of nuclear reprogramming, the same substances as those described above with respect to the establishment of rat ES cells can be appropriately selected and used. By the above method, it was confirmed that the established rat iPS cells maintain an undifferentiated state (totipotency).

2. Method for preparing chimeric embryo and chimeric rat

The present invention provides a method for preparing a chimeric embryo with improved germ line transmission efficiency, and a method for preparing a chimeric rat by obtaining a progeny rat from a rat subjected to a chimeric embryo transfer. By using the method of the present invention, germ line-transferred chimeric rats can be efficiently provided without being limited by strains of rat pluripotent stem cells or host embryos.

Rats are mammals with experimentally suitable dimensions (about 10 times the size of mice) and are advantageous in the following respects: (1) the drug can be easily administered into a blood vessel within a cell, (2) a surgical operation or a transplantation experiment can be performed, (3) a large amount of tissue can be collected, and the like. Although many disease model rats have been developed and found in the past, genetically modified rats, particularly those requiring gene targeting (such as knockout rats and knock-in rats), have not been prepared since there has been no established method for efficiently preparing chimeric rats of the delivery germ line and not limited by the strain of rat ES cells and the strain of host rats.

Using chimeric rats prepared by the method of the present invention, genetically modified rats can be easily produced without limitation by the rat pluripotent stem cell lineage and the host rat lineage. Herein, "genetically modified rat" refers to any genetically modified rat known to one of ordinary skill in the art, such as chimeric rats, knockout rats, knock-in rats, transgenic rats, and silent rats.

More specifically, the method for producing a chimeric embryo of the present invention comprises the following steps (a) and (b):

(b) culturing a host embryo contacted with the rat pluripotent stem cell to form a chimeric embryo.

In addition, the method for preparing the chimeric rat of the present invention comprises the following steps: the chimeric embryo prepared by the above method is transplanted into the uterus or oviduct of a pseudopregnant female rat to give birth to an offspring rat.

The rat pluripotent stem cell used for preparing the chimeric rat of the present invention is a cell mentioned in the above "1. rat pluripotent stem cell", preferably a rat ES cell or a rat iPS cell, more preferably a rat ES cell. The rat pluripotent stem cell may be a gene recombinant cell having a specific gene recombined by a publicly known method. Examples include ES cells from which a specific gene has been artificially removed, ES cells into which a specific gene has been artificially introduced, and the like.

The rats used to obtain the host embryos of the present invention may be derived from any rat strain. For example, the rat strains include the Wistar Kyoto strain (WKY), Brown Norway strain (BN), Goto-Kakizaki strain (GK), SD strain, F344/Du strain (Fischer), Wistar strain, Wistar Hannover strain, Long-Evans rat lick strain (LEA), ACI strain, and the like. In the present invention, the line may be a pure line, or a line obtained by crossing 2 or more. In view of the features of the present invention that allows for the efficient production of chimeric rats of germline transmission regardless of the rat ES cell line, the present invention is particularly useful when using host embryos collected from rat strains: in the step of contacting the host embryo with the rat pluripotent stem cell, the rat strain does not produce chimeric rats of the transmitting germ line when the rat ES cell is contacted with the rat pluripotent stem cell in the absence of an ES cell differentiation inhibitor.

Oocyte sampling may be performed using female rats in the 8-40 week age range, preferably using rats of 10-20 weeks of age, more preferably using rats of 10-12 weeks of age.

The starting host embryo is not particularly limited as long as it is a fertilized early embryo collected from female rats; often, early embryos prior to the blastocyst stage (e.g., 8-cell stage embryos, 16-cell stage embryos, morula stage embryos, blastocyst stage embryos) and the like may be mentioned.

Host embryos can be collected from the mated female rats by methods known per se. The mating may be spontaneous mating, or may be performed after ovulation induction. The method for collecting the host embryo is not particularly limited; to explain specifically, female rats for egg collection are mated with male rats of the same strain spontaneously or after administration of gonadotropins (follicle stimulating hormone, then luteinizing hormone) to induce superovulation, after which female rats for egg collection are killed at appropriate times (e.g., 3.5 days post mating for 8-cell stage embryos, 3.75 days post mating for 16-cell stage embryos, 4 days post mating for morula stage embryos, 4 days post mating for blastocyst stage embryos, 4.5 days post mating), the uterus is extirpated, and the uterus is perfused with an appropriate medium, whereby early embryos can be recovered. Here, examples of the medium for perfusion include the above-mentioned medium for establishing rat ES cells, the above-mentioned mw medium also for recovering early embryos, M2 medium, and the like.

The harvested host embryos can be cultured prior to contacting with the rat pluripotent stem cells. As a basic medium for the preculture, a medium similar to that used for establishing rat ES cells can be used. The medium may be a serum-containing medium or a serum-free medium. The medium may also contain fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, inorganic salts, and the like.

In a preferred embodiment, the ES cell differentiation inhibitor is added to the above-mentioned pre-culture medium. As the ES cell differentiation inhibitor, at least 2 kinds of drugs selected from the following can be mentioned: MEK inhibitors, GSK inhibitors, TGF β receptor inhibitors and FGF receptor inhibitors. As MEK inhibitors, GSK inhibitors, TGF-beta receptor inhibitors and FGF receptor inhibitors, the above-mentioned substances for culturing rat ES cells can be used. The ES cell differentiation inhibitor is preferably a combination comprising a MEK inhibitor and a GSK inhibitor, and a more preferred embodiment is a combination further using a TGF β receptor inhibitor.

The concentration of the MEK inhibitor to be added to the medium may be appropriately selected from the following ranges: for example, 0.01 to 100. mu.m, preferably 0.1 to 5. mu.M. The concentration of the GSK3 inhibitor to be added to the medium may be appropriately selected from the following ranges: for example, 0.01 to 100. mu.m, preferably 1 to 10 μm. The concentration of the TGF β receptor inhibitor to be added to the medium may be appropriately selected from the following ranges: for example, 0.001 to 10 μ M, preferably 0.1 to 1 μ M. The concentration of the FGF receptor inhibitor to be added to the medium may be appropriately selected from the following ranges: for example, 0.005 to 500. mu.m, preferably 0.07 to 50 μm.

In another preferred embodiment, a ROCK inhibitor may be additionally added to the medium for preculture of host embryos in addition to the ES cell differentiation inhibitor. Specific examples of the ROCK inhibitor include the above-mentioned substances for rat ES cell culture. The concentration of the ROCK inhibitor to be added to the medium may be appropriately selected from the following ranges: for example, 0.0001 to 500. mu.m, preferably 1 to 50 μm.

The preculture can be carried out, for example, in CO₂In an incubator, in about 1 to about 10%, preferably about 2 to about 5%, more preferably about 5% CO₂Concentration atmosphere, at about 30 to about 40 deg.C, preferably about 35 to about 37.5 deg.C, more preferably about 37 deg.C.

Rat pluripotent stem cells to be contacted with the host embryo, preferably rat ES cells described in 1-1 above, are selected as appropriate from those described in 1 above. To facilitate the preparation of chimeric rats, it is desirable that the rat pluripotent stem cells are recombinant rat pluripotent stem cells having a reporter gene (e.g., GFP (including modified GFP such as EGFP and Venus), β -gal, luciferase, etc.) previously introduced therein by a conventional method. In selecting chimeric rats using only coat color, germ line transmission will be demonstrated in the next generation; however, if reporter pluripotent stem cells are used, germline transmission can be demonstrated in the current generation of chimeric rats by detecting expression of the reporter gene in germ cells of the chimeric rats. Specifically, transgenic rats into which reporter genes have been introduced may be prepared in advance, and pluripotent stem cells may be obtained from the rats; alternatively, an expression vector containing a transformant cell selection marker gene such as a drug resistance gene and a reporter gene may be introduced into rat pluripotent stem cells prepared by electroporation or the like as described above, and the rat ES cells into which the reporter gene has been introduced may be selected by drug selection or the like.

The rat pluripotent stem cells may be contacted with the host embryo by methods known to the skilled artisan. For example, by micromanipulation, rat pluripotent stem cells are transplanted into the blastocoel of a rat blastocyst or into a morula-stage or 16-cell stage embryo, and either with or as part of an inner cell mass (microinjection method: Gordon J.W. et al,Proc. Natl. Acad. Sci. USA., 777380 and 7384 (1980)). Alternatively, zona pellucida was removed from 2 8-cell embryos, pluripotent stem cells were injected, and the embryos were co-cultured to form aggregates. When in useWhen the resulting aggregate was cultured, one blastocyst was obtained (cell aggregate method: Dvorak P. et al,Int. J. Dev. Biol., 39: 645-652(1995)). When injecting rat pluripotent stem cells, it is preferable to coat rat ES cells with mineral oil, oil droplets, liquid paraffin, or the like, and to perform injection into host embryos.

As a medium for contacting the rat pluripotent stem cells and the host embryos, a medium similar to that used for establishing rat ES cells can be used as a basal medium. The medium may be a serum-containing medium or a serum-free medium. The medium may also contain fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, inorganic salts, and the like.

2 or more ES cell differentiation inhibitors are added to the contact medium. The at least 2 ES cell differentiation inhibitors are selected from: MEK inhibitors, GSK inhibitors, TGF β receptor inhibitors and FGF receptor inhibitors. Examples of MEK inhibitors, GSK inhibitors, TGF β receptor inhibitors, and FGF receptor inhibitors include: the above-mentioned substances for culturing rat ES cells. The at least 2 ES cell differentiation inhibitors preferably include: including combinations of MEK inhibitors and GSK inhibitors, a more preferred embodiment is the further use of a combination of TGF β receptor inhibitors.

In a preferred embodiment, a ROCK inhibitor may be further added to the contact medium in addition to the ES cell differentiation inhibitor. Specific examples of the ROCK inhibitor include the above-mentioned substances for rat ES cell culture. The concentration of the ROCK inhibitor to be added to the medium may be appropriately selected from the following ranges: for example, 0.0001 to 500. mu.m, preferably 1 to 50 μm.

After contacting with rat pluripotent stem cells, the host embryo may form a chimeric embryo when cultured. Such a medium for post-culture may have the same composition as the aforementioned medium for contact; the medium is preferably a medium containing an ES cell differentiation inhibitor, more preferably a medium containing 2 or more ES cell differentiation inhibitors. Specifically, a medium containing a MEK inhibitor and a GSK3 inhibitor is preferable, and a medium containing a MEK inhibitor, a GSK3 inhibitor and a TGF β receptor inhibitor is more preferable. In addition, ROCK inhibitors may be added to the medium.

The post-culture may be performed under conditions similar to those used for the aforementioned preculture.

The chimeric embryo prepared above is transplanted into the uterus or oviduct of a pseudopregnant female rat (prepared by natural xenogenesis with a male rat after the vasectomy treatment) to allow the rat to produce a progeny rat, whereby a chimeric rat can be obtained.

The fact that the chimeric rat prepared by the method of the present invention has a tissue derived from rat pluripotent stem cells can be confirmed by, for example, expression of hair color of a rat strain from which rat pluripotent stem cells are derived in the chimeric rat, expression of a reporter gene in a tissue section of the chimeric rat, and the like. In particular, differentiation into germ line is detected by expression of a reporter gene in germ cells, whereby the presence or absence of germ line transmission can be rapidly and conveniently confirmed. Final confirmation of germline transmission can be achieved as follows: for example, DNA is extracted from cells of a progeny rat obtained by mating siblings of a chimeric rat, and the presence of a reporter gene is detected by southern blotting, genomic PCR, or the like.

By mating chimeric rats with confirmed germ line transmission with heterozygote rats, rats having rat pluripotent stem cells contributing to their entire body, i.e., rats having: all of the cells carry genetic information derived from rat pluripotent stem cells. For example, a progeny rat obtained by mating a chimeric rat of the transmission line with a wild-type rat will have only one homologous chromosome derived from a rat pluripotent stem cell, while a progeny rat obtained by mating a sibling of a chimeric rat will have 2 homologous chromosomes derived from a rat pluripotent stem cell.

3. Method for preparing genetically modified rat

As described above, by mating the chimeric rat of the transmission line with the heterozygote rat, a rat having rat pluripotent stem cells contributing to its entire body can be prepared; thus, by genetically modifying rat pluripotent stem cells, genetically modified rats can be established that: all cells thereof have a genetic modification. Accordingly, the present invention also provides a method of preparing a genetically modified rat using the chimeric rat prepared by the above-mentioned "method for preparing a chimeric embryo and a chimeric rat", and a genetically modified rat prepared by the method. Herein, "genetically modified rat" refers to any genetically modified rat known to those of ordinary skill in the art, such as a knockout rat, a knock-in rat, a transgenic rat, and a silent rat.

Preferably, the chimeric rat used to prepare the genetically modified rat is a chimeric rat: it has rat ES cells described in 1-1 above, which have contributed thereto, established using an ES cell differentiation inhibitor and a ROCK inhibitor.

A knockout rat refers to a mutant rat in which the target gene has been artificially disrupted, and is also referred to as a gene-targeted rat. Knockout rats can be prepared, for example, according to Donehouwer, A.L. et alNature, 356215 and 221 (1992) and the likeA preparation method of a knockout mouse. Specifically, a vector for homologous recombination (targeting vector) is constructed based on the genomic DNA sequence of the target gene. At this time, a drug resistance gene (such as G418 resistance gene, hygromycin resistance gene, etc.) was introduced as a marker gene for selecting recombinant clones. The constructed targeting vector is introduced into rat pluripotent stem cells by an electroporation method or the like. From the resulting cells introduced with the targeting vector, colonies in which homologous gene recombination has occurred are selected based on a marker gene or the like. Using the thus obtained genetically recombinant rat pluripotent stem cells, a chimeric rat is produced according to the chimeric rat production method of the present invention. By crossing the chimeric rat with a wild-type rat, a heterozygous knockout rat can be generated in its offspring rat, and when the heterozygous knockout rat is crossed, a homozygous knockout rat can be generated in its offspring rat.

In recent years, it has become possible to easily perform biallelic modification of a target gene at the cellular level. Specifically, this may be facilitated using techniques such as Zinc Finger Nucleases (ZFNs), for example. Therefore, for rat ES cells, homotypic knock-out rats can be easily produced by causing a biallelic deficiency in the target gene using these publicly known techniques, and using the biallelic deficient ES cells.

The aforementioned class of knockout rats includes conditional knockout rats. "conditional knock-out" refers to a system for site-specific or time-specific knock-out of a gene using the Cre/loxP system or the FLP/FRT system. Specifically, both ends (3 '-end and 5' -end) of the gene to be targeted are replaced with a gene flanked by a loxP sequence or FRT sequence, and Cre or FLP protein is supplied to cleave the gene flanked by the aforementioned loxP sequence or FRT sequence (Sternberg N., et al,J. Mol. Biol., 150: 487-507（1981））。

"knock-in rat" refers to a mutant rat: wherein an artificially prepared foreign gene having homology with the target gene has been introduced into its site. The introduction of the foreign gene may or may not disrupt the function of the target gene. For example, in order to monitor the expression of a target gene, a marker gene such as lacZ gene, GFP gene, or the like may be introduced, or the gene may be replaced with a gene into which a mutation has been introduced.

Knock-in rats can be produced, for example, according to the methods described in Pewzner-Jung, y. et al,J. Immunol., 1614634-4645 (1998) and the like. Basically, the rats can be produced by the same principle as in the previously described knockout rats.

"transgenic rat" refers to a rat into which a foreign gene has been artificially introduced. Transgenic animals are routinely prepared by injecting a gene of interest into the male pronuclei of fertilized oocytes harvested from a donor animal by micromanipulation (microinjection method). The fertilized egg is transplanted into the oviduct of a recipient animal, and the naturally born animal becomes a transgenic animal. The requirements for chimeric rats were not as high as in the case of the previously described knockout and knock-in rats. However, the method for producing a chimeric rat of the present invention can be effectively used to increase the efficiency of introduction of a foreign gene and the efficiency of production of transgenic animal individuals.

Transgenic rats can be produced, for example, according to Yamamoto H.et al,Cancer Res., 621641-1647 (2002), etc.

The aforementioned transgenic rat species include conditionally transgenic rats. "conditionally transgenic" refers to a system that expresses genes site-specifically or time-specifically using the Cre/loxP system or the FLP/FRT system. Specifically, the target gene is expressed by inserting a cassette in which loxP sequence or FRT sequence is flanked at both ends (3 '-end and 5' -end) of a drug resistance gene or the like into the target gene, thereby suppressing the expression of the target gene, and supplying Cre or FLP protein to cleave the gene flanked by the loxP sequence or FRT sequence (Sternberg, N.et al,J. Mol. Biol., 150: 487-507（1981））。

by "silent rat" is meant a rat: wherein a short double-stranded RNA (siRNA, which is an intermediate for RNAi (RNA interference)) or an antisense nucleic acid has been artificially introduced and expressed, and the action of the siRNA or the antisense nucleic acid suppresses the expression of a target gene. Establishment of expression System for siRNA based on vector System: (Science 296: 550-553（2002）, Nature Biotech. 20500, 505 (2002), etc.), the preparation of such silent animals has been achieved.

Silent rats can be produced, for example, according to the methods described in Tiscornia, g.Proc. Natl. Acad. Sci. USA. 1001844, 1848 (2003), etc. Basically, the rats can be produced by the same principle as in the aforementioned transgenic rats.

4. Kit for preparing chimeric rat

The present invention provides a kit comprising a culture medium for the preparation of chimeric rats of the delivery germ line. The culture medium (e.g., broth) contained in the kits of the invention may also be used to establish rat ES cells that have the ability to produce chimeric rats of the transmission germ line.

One feature of the kit of the present invention is that it contains a medium containing an ES cell differentiation inhibitor as a component. The ES cell differentiation inhibitor may be any substance as long as it has the effect of inhibiting the differentiation ability of rat ES cells in the present invention. Inhibitors of ES cell differentiation include, for example, MEK inhibitors, GSK3 inhibitors, TGF β receptor inhibitors, and FGF receptor inhibitors. In the present invention, at least 2 of them are used as ES cell differentiation inhibitors. Preferably, the ES cell differentiation inhibitor comprises: a combination of a MEK inhibitor and a GSK inhibitor, a combination of a MEK inhibitor, a GSK inhibitor and a TGF β receptor inhibitor, and a combination of a MEK inhibitor, a GSK inhibitor and an FGF receptor inhibitor; MEK inhibitors, GSK3 inhibitors and TGF β receptor inhibitors are more preferred because of the high capacity to produce germ line-transmitting chimeric rats. In another preferred mode of embodiment, a ROCK inhibitor may be further added in addition to the ES cell differentiation inhibitor. The details of MEK inhibitors, GSK3 inhibitors, TGF β receptor inhibitors, FGF receptor inhibitors and ROCK inhibitors are as described above.

The kit of the present invention may contain the aforementioned ES cell differentiation inhibitors alone or in combination with at least 2 or more (as appropriate). When 2 or more ES cell differentiation inhibitors are contained in combination in the kit, a plurality of ES cell differentiation inhibitors may be mixed and packaged in a single container, but they are preferably packaged in separate containers.

The kit may comprise as a kit component rat pluripotent stem cells as defined in "1. rat pluripotent stem cells" above. The rat pluripotent stem cells are preferably rat ES cells.

The aforementioned kit may further contain feeder cells as a constituent component. The feeder cells may be cells derived from any species available to those of ordinary skill in the art, and are preferably normal fibroblasts, rather than established feeder cell lines. Specifically, primary cultured cells (normal fibroblasts) of mouse embryonic fibroblasts between day 12 and day 16 of pregnancy can be mentioned. Examples of normal fibroblasts include normal fibroblasts from day 12.5 ICR fetal mice. Feeder cells can be prepared by conventional methods. Commercially available mouse embryonic fibroblasts (Asahi Techno Glass Corporation) may also be used.

Examples

Examples of the present invention are explained below, which should not be construed as limiting.

Example 1 preparation of fluorescent reporter transgenic rat

To establish and validate ES cells, transgenic rats were prepared for monitoring Oct4 gene expression by fluorescence. Oct4 promoter region DNA was amplified by PCR from Wistar rat genomic DNA using KOD Ver.2 DNA polymerase (Toyobo) and inserted into the pCS2-Venus plasmid. Oct4 promoter-Venus DNA was injected into pronuclei of fertilized eggs of Wistar rats to obtain Oct4-VENUS transgenic rats.

Example 2 preparation of rat ES cells

(1) Preparation of reagents and feeder cells

First, 4 inhibitors were prepared, i.e.,Y27632 as ROCK inhibitors (WAKO Company),PD0325901 as MEK inhibitor (Axon Medchem Company),A-83-01 as TGF-beta receptor type I inhibitors (TOCRIS Company) andCHIR99021 acts as an inhibitor of GSK3 (Axon Medchem Company) (hereinafter, these 4 inhibitors are referred to as "YPAC factors").

A basal medium for ES cells was prepared by adding 20% FBS for ES cells (batch No. 1204059: GIBCO Company), 0.1 mM 2-mercaptoethanol (Sigma Company), 1% nonessential amino acid stock solution (GIBCO Company), and 1% 1 × antibiotic antimycotic agent (GIBCO Company) to DMEM (GIBCO Company) containing 110 mg/L sodium pyruvate and 200 mM GlutaMax.

YPAC factor was added to this basal medium for ES cells to give the following concentrations: 10 μ M of ROCK inhibitor Y-27632, 10 μ M of MEK inhibitor PD0325901, 0.5 μ M of TGF- β receptor type I inhibitor A-83-01, and 3 μ M of GSK3 inhibitor CHIR99021, and the medium was used for the experiment (hereinafter, this medium is referred to as "YPAC medium").

The feeder cells used were mitomycin C treated neomycin resistant mouse embryonic fibroblasts (MEF: Millipore) maintained using media prepared as follows: 10% FBS (EQUITECH-BIO Company, batch No. SFB 30-1502) and 1% 1 × antibiotic antimycotic (GIBCO Company) were added to DMEM.

(2) Establishment of ES cells

Rat blastocysts were obtained by perfusing the uterus of pregnant rats, which were pregnant for 4.5 or 5 days, with a basal medium of ES cells. After removing the zona pellucida using Tyrode buffer solution (Ark Resource Company), the blastocysts were transferred to 6-well plates and cultured using a basal medium (YPAC medium) of ES cells obtained by adding YPAC factor in (1) to feeder cells (MEF). After about 7 days, the outgrowth of the blastocysts was re-inoculated into YPAC medium; ES Cell colonies were enzymatically isolated using Accutase (innovative Cell Technologies company) and cultured. The established ES cells were cultured under MEF-YPAC medium conditions and passaged every 3 to 4 days. DMSO (dimethyl sulfoxide) was added to YPAC medium, and ES cells were frozen and thawed according to a conventional method. TgWL1 (ES cells) and TgWL2 (ES cells) were cultured with the addition of 1000U/mL rLIF until passage numbers reached 3 or 4.

(3) Studies Using multiple rat strains

Using the method for establishing rat ES cells described in (2) above, a study was conducted to determine whether rat ES cells of a non-Wistar strain of rat could be established. As a result, it was possible to establish rat ES cells of all lines studied, as shown in Table 1.

TABLE 1

a. Different media were used (FBS: MEF culture, EQUITECH + BIO)

b. Outgrowth indicates elongation of the ICM.

c. Cell lines indicate that the culture lasted at least 7 passages. Single cell subculture was started between passage 1 and passage 3. Dome colonies of undifferentiated cells were formed continuously from single cells.

TgWL transgenic hybrids between Wistar and LEA

TgWW transgenic hybrids between Wistar and wild-type Wistar

WW wild type Wistar

LL: LEA。

Example 3 validation of rat ES cells

The rat ES cells obtained in example 2 and their preparation methods were verified.

(1) Outgrowth of Inner Cell Mass (ICM) in YPAC Medium

Using the Inner Cell Mass (ICM) of blastocysts from transgenic rats prepared in example 1, studies were performed with and without addition of YPAC factor to the basal medium of ES cells of example 2. Oct4-Venus fluorescence was detected 3 days after plating in the absence of YPAC factor; however, after 7 days, dome-like growth similar to the mouse ES colonies appeared, but subsequently disappeared. In the presence of YPAC factors, the ICM cells grew rapidly while maintaining Oct4-Venus fluorescence even after 7 days (FIGS. 1a and b). Checking the gene expression of pluripotency factors Oct4, Nanog, Sox2 and Rex1 by quantitative PCR; higher levels were found with the addition of YPAC factors than in the absence of YPAC factors. In the presence of YPAC factor, Oct4 mRNA was decreased, as was the expression of Venus mRNA and fluorescence intensity (FIG. 1 c). In this experiment, the blastocysts used were those derived from transgenic Wistar (TgWW, Arbino), wild type Wistar (WW, albion), LEA (LL, spiny) or transgenic Wistar/LEA (TgWL, spiny) hybrids. It was confirmed by immunostaining that rat ES cells established in the presence of YPAC factors expressed Oct4, Nanog and even Sox2 (fig. 2 h).

(2) Validation of the Effect of factor Y (ROCK inhibitor) in culture Medium

When established rat ES cells were cultured using ES cell culture medium that did not contain a ROCK inhibitor (hereinafter, referred to as "factor Y"), but supplemented with a MEK inhibitor, a TGF-beta receptor type I inhibitor, and a GSK3 inhibitor (hereinafter, these 3 inhibitors are referred to as "PAC factor"), the resulting colonies maintained an undifferentiated state, but became sparse colonies (FIG. 2 a). Meanwhile, when cultured using ES cell medium supplemented with Y factor alone, rat ES cells attached to MEFs and propagated, but could not maintain an undifferentiated state and exhibited no alkaline phosphatase activity (fig. 2a, b).

(3) Karyotyping in ES cells

The karyotype of the 50 rat ES cells obtained was analyzed by giemsa staining; almost all cells showed normal 42-chromosome karyotype (FIG. 2 c). The results were: TgWL1 (70%, XX, P14), TgWL2 (84%, XX, P7, fig. 2 c), TgWW1 (92%, XX, P5), and LL1 (84%, XX, P6).

(4) DNA microarray analysis

(i) Microarray analysis 1

As a result of microarray analysis, TgWW1 and LL ES cells were found to have similar gene expression, but differences were observed between TgWW1 and Rat Embryonic Fibroblasts (REF). Statistical analysis of ES cells revealed a correlation coefficient of 0.968; although the expression levels of the pluripotency markers Oct4, Sox2, Dppa3, Tbx3, Fbxo15 and Cdh1 (also known as E-cadherin) were similar in TgWW1 and LL (fig. 2 d), but higher than in REF (fig. 2E).

(ii) Microarray analysis 2

As a result of microarray analysis, TgWL1, TgWW1 and LL1 ES cells were found to have similar gene expression, but differences were observed between the ES cells and Rat Embryonic Fibroblasts (REF) (fig. 2 g). Statistical analysis of ES cells revealed a correlation coefficient of 0.971 between TgWL1 and TgWW1 and a correlation coefficient of 0.961 between TgWW1 and LL 1.

(5) Formation of teratomas

TgWW1 cells (ES cells) prepared in example 2 were dispersed into single cells using Accutase, after which the cells were washed 2 times with 10 mL of PBS, and 2.6X 10 cells suspended in 100. mu.L of PBS were added⁶Cells were injected subcutaneously into immunodeficient mice; teratomas were detected in this mouse at day 34 (fig. 2 f). Fixing the teratomas in paraffin and performing histological staining using hematoxylin-eosin; differentiation into endoderm, mesoderm and ectoderm 3 germ layers was confirmed (fig. 2 i).

(6) Formation of ES cell embryoid bodies (embryoid body: EB)

Rat ES cells prepared in example 2 were dispersed into single cells using Accutase, after which the cells were cultured using ES cell culture medium prepared by adding 3 inhibitors (PAC factors) containing no Y factor to the ES cell culture medium of example 2 on a low adhesion dish (NUNC Company) to obtain Embryoid Bodies (EBs); the cells aggregated efficiently, forming distinct three-dimensional EBs (figure 3 b). The Oct4-Venus fluorescence intensity increased at 7 days, and expression of Venus, Oct4, Nanog, Sox2, and Rex1 was also maintained as confirmed by quantitative PCR (FIG. 3 c). Meanwhile, in the absence of PAC factor addition, EB production efficiency was significantly lower than mouse EB (fig. 3 a), but expression of various pluripotency marker genes was reduced (fig. 3 c).

Example 4 preparation of germ line-transmitting chimeric rats by YPAC injection Using multiple strains

Germ line transferred chimeric rats were prepared using multiple ES cell lines as described below.

(1) Preparation of blastocysts

Blastocysts obtained from pregnant rats that were 4.5 days pregnant were incubated in injection medium (YPAC medium without antibiotics) for 2-3 hours and then used for microinjection.

(2) Preparation of ES cells

10-20 dome-like colonies of the ES cells in example 2 were collected using a glass tube and treated with Accutase for 5 minutes, and then they were dispersed into single cells in the injection medium. Next, the cells were transferred to 500. mu.L injection medium and incubated at room temperature for 30-60 minutes, then the ES cells were centrifuged and transferred to injection medium and covered with mineral oil (SIGMA Company).

(3) Injection of ES cells into blastocysts

10-15 ES cells were injected into blastocysts and the original state of the embryos was restored, and the blastocysts were incubated at 37 ℃ for 3-5 hours in the injection medium. 10-20 embryos were transferred to the uterine horn of a pseudopregnant rat pregnant for 3.5 days. Chimeric rats were identified by gross color, and germline transmission was identified by the gross color of born F1 rats (as a result of mating chimeric rats) and by Oct4-VENUS fluorescence of fetal germ cells. Genotyping was achieved by PCR on the tail DNA.

Example 5 verification of germline transmission chimeric rats and methods for their preparation

The utility of YPAC injection methods and germ line transmission was verified as described below.

(1) Preparation of rat ES reporter cells (cyan)

To monitor rat ES cells, 5. mu.g of pCAG-AmCyan1 was introduced into 3.2X 10 using mouse ES cell Nucreofector kit (Amaxa Company)⁶TgWW1 cells (ES cells); colonies of CAG-AmCyan1/Oct4-VENUS positive ES cells emitting cyan/green fluorescence were collected using glass tubes and expanded without drug selection, and these were used as monitor cells (TgWW 1+ C). In addition, 10. mu.g of pOct4-Venus was introduced into 2.4X 10 cells⁶LL cells (ES cells), and reporter ES cells (LL 1+ V) were prepared in the same manner.

(2) Verification of Effect of YPAC factor-added injection Medium

Studies were conducted with and without addition of YPAC factor to the injection medium. After 5 hours of incubation, there was no difference between the results obtained by injection in the absence of YPAC factor and the results obtained by injection in the presence of YPAC factor (hereinafter, referred to as "YPAC injection"); a plurality of cyan positive cells attached to the Inner Cell Mass (ICM) and vegetative ectoderm (TE). However, after 30 hours incubation in the absence of YPAC factors, a small number of apoptotic cyan-positive cells were present in the blastocyst, whereas in the presence of YPAC factors, some cells accumulated on the ICM and the shape of the blastocyst was maintained (FIG. 4 b).

(3) Verification of the ability to generate germ line chimeric rats

The ES cells TgWW1+ C from example 5 (1) were injected into blastocysts and incubated for 3.5 hours before they were transferred to the uterus of mice pseudopregnant for 3.5 days. 2 of 9 individuals in day 18 embryos were found to be cyan positive in the epidermis and kidneys and Oct4-Venus negative. Oct4-Venus positive cells were specifically detected in gonad germ cells (FIG. 4 c). For all other ES cells, germ line chimera formation was also confirmed by detecting Oct4-Venus fluorescence on fetal gonads. Germ line chimeras were identified in 2 of 12 mice, even in long-term cultured TgWL2 cells over 22 passages. Furthermore, germline transmission was also confirmed in LL1+ V, prepared by introducing the Oct4-Venus gene into LL cells induced from the LEA rat strain.

To identify chimeras by gross color, TgWL1 cells were injected into Wistar rats with the addition of YPAC factor. In 23 individuals, 8 chimeric colored rats were formed from TgWL1 cells over 11 or 12 passages (fig. 4 d). Meanwhile, when injection was performed without addition of YPAC factor, it was difficult to prepare chimeric colored rats, only 1 out of 44 individuals being chimeric colored rats, despite using the same cell line with a small passage number of 6 or 8; the chimera rate was low. In all 4 cell lines, the use of YPAC factor to make chimeric colored rats was successful. To confirm germ line transmission in F1, TgWL1 chimeras and Wistar rats were mated; one individual in the female chimera produced 4 out of 16 germline transmitted individuals containing the TgWL1 cell line, rodent colored offspring (fig. 4 e). In addition, F1 germline transmission was also demonstrated in cells derived from the TgWL2 cell line. It has been found that when YPAC factors are used to make chimeric rats, as shown in tables 2 and 3, chimeric rats of the transmission line can be made without limitation by the strain of rat ES cells and the strain of host rats.

The cell lines used were: TgWL1 and TgWL2, which are ES cells established from TgWL rats (hybrid between transgenic Wistar rats and LEA rats), TgWW1 and TgWW2, which are ES cells established from TgWW rats (TgWW: hybrid between transgenic Wistar rats and wild type Wistar rats), WW1, which is ES cells established from WW rats (wild type Wistar rats), and LL1 and LL2, which are ES cells established from LL rats (LEA rats).

To identify the Venus region by gene analysis, tail genomic DNA was collected and amplified by PCR. The Oct4-Venus gene of the F0 chimera was inherited by mendelian law to 2 of 3 murky F1 germline individuals (fig. 4F). As shown in Table 2, when long-term cultured ES cells (TgWL 2: passage number: 22) were injected into blastocysts, Venus fluorescence was detected on the gonads of 17.5-day embryos (FIG. 4 i).

In addition, the effect of YPAC factors on chimera ratio was studied. When chimeras were prepared without addition of YPAC when TgWL1 rat ES cells (passage number: 6) were injected into blastocysts and when the blastocysts were cultured (FIG. 4 j), the proportion of the rodent hair color chimeras was found to be low as shown by the arrows in FIG. 4 j. At the same time, chimeras of TgWW1 and LL1 ES cells were prepared in the presence of YPAC factors (A: TgWW1 cells were injected into LEA blastocysts; B: LL1 cells were injected into Wistar blastocysts); as shown in fig. 4k, the rate of the rodent hair color chimera was found to be higher. The arrows in fig. 4k indicate the degree of contribution of ES cells to the chimera.

TABLE 2

ES cells were established using a separate medium.

M, male, F, female

TgWL transgenic Wistar-LEA hybrids

TgWW transgenic Wistar-wild type Wistar hybrids

WW wild type Wistar

LL: LEA

TgWW1+ C indicates a cell line stably expressing the transgene.

LL1+ V indicates a cell line stably expressing the transgene Oct 4-Venus.

TABLE 3

a the range of the hair color is narrow.

(4) Preparation of germline chimeras from Single rat ES cells

To confirm pluripotency of rat ES cells, single cells were injected into blastocysts (fig. 4 g). The 9 th generation TgWW1 cells were injected into Wistar blastocysts and then they were incubated for 3 hours using YPAC factor injection medium to allow single cells to bind to the inner surface. At day 16, 8 fetuses developed from 35 blastocysts, and germ line transmission was achieved in 1 fetus (fig. 4 h).

(5) Study of production conditions of germ line-transferred chimeric rat

The results of the study for making chimeric rats using "YPAC factor" consisting of ROCK inhibitor, MEK inhibitor, TGF-beta receptor type I inhibitor and GSK3 inhibitor, or "PAC factor" consisting of 3 inhibitors, namely, MEK inhibitor (PD 0325901), TGF-beta receptor type I inhibitor (a-83-01) and GSK3 inhibitor (CHIR 99021) are shown in table 4. All rES cells were established using YPAC medium.

The results of the study confirmed that chimeric rats can be prepared using not only YPAC factor but also PAC factor, and that the resulting chimeric rats have undergone germ line transmission. In addition, when the number of rES cells to be injected into host blastocysts is small, the addition of a ROCK inhibitor to YPAC or PAC factors in the step of injecting rES cells into host embryos increases the attachment strength of ES cells to blastocysts, which is considered desirable.

Although the use of rat ES cells or mouse ES cells for the preparation of chimeric animals has been reported so far, no report has been made on the addition of a compound (MEK inhibitor, GSK3 inhibitor, etc.) that inhibits ES cell differentiation when ES cells are injected into host embryos. Rat ES cells prepared in example 1 are considered to have similar capabilities to those described in the prior art documents, since they are rat ES cells capable of germ line transmission; however, in the absence of added inhibitor, no germ line-transmitting rats could be prepared. Thus, it has been found that a method for preparing chimeric rats using a drug which inhibits differentiation of ES cells makes it possible to prepare rats of the transmission line far more efficiently than the conventional method.

[ Table 4]

Example 6 preparation of chimeric rats derived from the transmitting germline of genetically modified ES cells

Genetically modified ES cells were used to prepare chimeric rats that passed germ lines as described below.

(1) Preparation of genetically modified rat ES cells

Mu.g of pOct4-Venus was introduced into 3X 10 cells using mouse ES cell Nucleofector kit (Amaxa Company)⁶LL2 cells (ES cells established with YPAC factors) were plated onto MEFs in culture vessels coated with 2% matrigel (BD Bioscience Company). Colonies of Oct 4-Venus-positive ES cells emitting green fluorescence were collected using a glass tube and expanded with drug selection and used as Oct 4-Venus-positive ES cells (LL 2). After passage 2, the green fluorescence from 13 of the 15 ES cell colonies was not uniform, while the other 2 emitted uniform fluorescence (fig. 5A, B).

(2) Preparation of chimeric rats derived from the transmitting germline of genetically modified ES cells

ES cell LL2, which stably emits green fluorescence, was injected into Wister-derived blastocysts in the presence of YPAC factor and incubated for 3-5 hours, followed by transfer to the uterus of mice pseudopregnant for 3.5 days to generate transgenic rats (esTG rats). Germ line transmission in the prepared esTG rats was confirmed by the fur color of F1 rats (fig. 5C, table 3) and Venus fluorescence on gonads of 16-day-embryonic esTG rats (fig. 5D). In addition, esTG rats grew while retaining normal reproductive capacity.

(3) Validation of ES cells derived from esTG rats

The Venus fluorescence expression pattern of ES cells from esTG rats was verified. Venus fluorescence expressed by outgrowth of blastocysts from esTG rats (fig. 5E) was identical to outgrowth of blastocysts from conventional transgenic rats (cvTG) prepared in example 1 (fig. 1 b). Even after 10 passages, the cells were successfully cultured while keeping Venus fluorescence stably (fig. 5F). Judging from these results, it was found that good quality esTG rats stably expressing Venus could be prepared.

Example 7 preparation of chimeric rats Using Gene-targeted rat ES cells

A ZFN pair (Sigma Company) was designed which recognized 124 bp downstream of the start codon of the first exon of Oct4 gene and targeted donors constructed with the short homology arm shown in fig. 6A were prepared from rat genomic DNA by PCR using KOD ver.2 DNA polymerase (Toyobo).

Accurate targeting of this donor, containing the AmCyan1-IRES-NEO cassette, induced expression of AmCyan1 and neomycin phosphotransferase under the control of the endogenous Oct4 promoter. Mu.g of targeted donor and 5. mu.g of ZFN-encoding mRNA were added to 6X 10 together with mouse ES cell Nucreofector kit (Amaxa Inc.)⁶In one ES cell (Long-Evans rat (LEA), passage number: 7) to achieve nuclear transfection. ES cells were seeded on MEF in a 2% matrigel (BD Bioscience) coated culture vessel and cultured in YPAC factor-supplemented medium without drug selection, and AmCyan 1-positive cells were collected using a glass tube. There were no AmCyan-1-positive colonies into which only the donor was inserted, and the expression of AmCyan1 was confirmed among 18 colonies into which ZFN-encoding mRNA and donor were introduced. These 18 colonies were subcultured, and 11 colonies survived. As a result of genotyping analysis, gene targeting was found to have been accurately achieved in 8 out of 11 clones (73%) (fig. 6B).

These 8 heterozygous (Oct 4 +/-) clones showed uniform expression of AmCyan1 in undifferentiated cells, but not in differentiated cells (fig. 6C). For the 4 expanded clones, 15-18 ES cells were injected into blastocysts obtained from pregnant rats at 4.5 days of pregnancy with addition of YPAC factor and transplanted into uterine horn of pseudopregnant rats (species: Wister) at 3.5 days of pregnancy. From 1 (No. 11) clone, 3 hair color chimeras were developed.

Thus, it was found that rat ES cells established using YPAC factors have the ability to produce chimeric rats even after undergoing genetic modification such as gene targeting after their establishment.

TABLE 5

Development of chimeric rats from Gene-targeted ES cell clones

Donor ES cells Long-Evans Agouti (LEA)

Receptor blastocyst Wister

Example 8 use of rat ES cells deficient in p53 Gene (p 53) ^+/- ) Preparation of p53 knockout rat

(1) Rat ES cells deficient in p53 gene (p 53)^+/-) Preparation of

ZFN expression plasmid (Sigma Company) and ZFN-encoding mRNA that recognized rat p53 gene (exon 4) were designed and targeted donors constructed with the short homology arms shown in fig. 7 were prepared from rat genomic DNA by PCR using KOD ver.2 DNA polymerase (Toyobo co., Ltd.).

Mu.g of targeted donor and 5. mu.g of ZFN-encoding mRNA were added with the mouse ES cell Nucreofector kit (Amaxa Company) to 6.5X 10 cells established in example 6 (1)⁵An Oct 4-Venus-positive ES cell (LEA-strain rat) [ LL 2]](passage number: 5) to achieve nuclear transfection. ES cells were seeded on MEF in a culture vessel coated with 2% matrigel (BD Bioscience Company) and cultured in a medium supplemented with YPAC factor. After 1 day of culture, geneticin was added to the medium at a concentration of 0.2. mu.g/ml. In thatAt 11 days of culture, geneticin-resistant ES cell colonies were collected into glass tubes and cultured. As a result of genotyping analysis, 6 of the 7 colonies collected were found to have undergone homologous recombination, lacking the p53 gene (p 53), as shown in FIG. 8A^+/-ES cells). No homologous recombination occurred in 1 colony (clone No. 3). Cyan fluorescence also confirmed p53^+/-Homologous recombination with the p53 locus in ES cells (FIG. 8B).

(2) Rat ES cells deficient in p53 gene (p 53)^+/-) Preparation of chimeric rats

With the addition of YPAC factor, 1 p53 found to undergo homologous recombination^+/-ES cell clones (passage number: 9) were injected into 90 blastocysts (12 cells/1 blastocyst) obtained from Wistar-strain rats and transplanted into the uterine horn of pseudopregnant rats (species: Wister) pregnant for 3.5 days. As a result, 9 chimeric colored rats (male chimera: 5 chimeras, female chimera: 4 chimeras) were formed in 13 animals.

Mating the female chimera with a wild type LEA-line rat; in live-born rats, genotyping analysis was performed on chimeric colored rats; 2 of 3 male chimeric colored rats (rats with germ line transmission) were found to have p53 knockdown (p 53)^+/-) (FIG. 9).

It was thus found that rat ES cells established using YPAC factors have the ability to produce chimeric rats, even after undergoing genetic modification such as gene targeting after their establishment, are capable of producing chimeric rats with germ line transmission, and thus can be used to prepare knockout rats.

Example 9 use of rat ES cells deficient in p53 Gene (p 53) ^-/- ) Preparation of chimeric rats

(1) Rat ES cells deficient in p53 gene (p 53)^+/-) Preparation of

A ZFN expression plasmid (Sigma Company) recognizing rat p53 gene (exon 4) and ZFN-encoding mRNA were designed and targeted donors (CAG-AmCyan 1-IRES-Neo-pA) constructed with the short homology arm shown in fig. 10A were prepared from rat genomic DNA by PCR using KOD ver.2 DNA polymerase (Toyobo co., Ltd.).

Add 10. mu.g of targeted donor and 5. mu.g of ZFN-encoding mRNA to the 4.5X 10 together with mouse ES cell Nucreofector kit (Amaxa Company)⁶Oct 4-Venus-positive ES cells (Wistar-strain rats) (passage number: 3) to achieve nuclear transfection. ES cells were seeded on MEF in a culture vessel coated with 2% matrigel (BD Bioscience Company) and cultured in a medium supplemented with YPAC factor. After 1 day of culture, geneticin was added to the medium at a concentration of 0.2. mu.g/ml. At 9 days of culture, 46 geneticin-resistant colonies were collected and cultured. As a result of genotyping analysis, p53 gene-deficient ES cells in which homologous recombination had occurred were identified (p 53)^+/-ES cells [ cell line: p53^+/C]) (FIG. 10B, lane 2, FIG. 10D). It was found that 1 of 46 colonies had undergone biallelic homologous recombination (p 53)^-/-ES cells [ cell line: p53^C/C]) (2.2%) (FIG. 10B, lane 7, FIG. 10D). It was found that homologously recombined and homologously frameshifted alleles occurred in 7 of 46 colonies (p 53)^-/-ES cells [ cell line: p53^C/Z]) (15%) (FIG. 10B lanes 3 and 4, FIG. 10C, FIG. 10D). As a control, 10. mu.g of targeted donor nuclear transfection (nucleofectect) alone was performed at 4.5X 10⁶Oct 4-Venus-positive ES cells (Wistar-strain rats) (passage number: 3). ES cells were seeded on MEF in a culture vessel coated with 2% matrigel (BD Bioscience Company) and cultured in a medium supplemented with YPAC factor. After 1 day of culture, geneticin was added to the medium at a concentration of 0.2. mu.g/ml. At 9 days of culture, 14 geneticin-resistant colonies were collected and cultured. As a result of genotyping analysis, it was found that these colonies did not undergo homologous recombination and had randomly integrated thereinTargeting donors (p 53^+/+ES cells [ cell line: p53^+/+（-ZFN）]) (FIG. 10B, lane 5).

(2) P53 Gene-deficient rat ES cells (p 53)^-/-) Preparation of

ZFN expression plasmid (Sigma Company) recognizing rat p53 gene (exon 4), and ZFN-encoding mRNA were designed, and a targeted donor constructed with the short homology arm shown in FIG. 10A (containing tdTomato as a reporter instead of AmCyan 1; CAG-tdTomato-IRES-Neo-pA) was prepared from rat genomic DNA by PCR using KOD Ver.2 DNA polymerase (Toyobo Co., Ltd.).

Mu.g of targeted donor and 5. mu.g of ZFN-encoding mRNA were added with mouse ES cell Nucreofector kit (Amaxa Company) to 2.5X 10 prepared in example 9 (1)⁶A p53^+/-ES cells [ cell line: p53^+/C](passage number: 9) to achieve nuclear transfection. ES cells were seeded on MEF in a culture vessel coated with 2% matrigel (BD Bioscience Company) and cultured in a medium supplemented with YPAC factor. After 6 days of culture, 8 colonies expressing red fluorescence of tdTomato were collected and cultured (FIG. 11B). As a result of genotyping analysis, 3 colonies (clone numbers 1,3 and 8) were found to have undergone homologous recombination (38%) (p 53)^-/-ES cells [ cell line: p53^C/R]) (FIG. 11A).

(3) Rat ES cells deficient in p53 gene (p 53)^-/-）（p53^+/-) Preparation of chimeric rats

P53 prepared in example 9 (1) with addition of YPAC factor^+/-ES cells [ cell line: p53^+/C]And p53 prepared in examples 9 (1) and (2)^-/-ES cells [ cell line: p53^C/C, p53^C/Z, p53^C/R](12 ES cells each) were injected into blastocysts obtained from rats pregnant for 4.5 days, and transplanted into pseudopregnant rats (species: LEA) pregnant for 3.5 daysThe uterine horn.

As a result, when p53 was used^+/-In ES cells, a gross mosaic appears. In addition, the normal growth of the chimeric rat fetus was confirmed at a high frequency, p53^+/-The percentage contribution of ES cells was also high.

Meanwhile, when p53 is used^-/-ES cells malformations occurred in 184 out of 209 chimeric rat fetuses. At 6 p53^-/-These abnormalities were found in the ES cell clones (FIG. 12). Most of the chimeric rat fetuses with malformations suffered from miscarriage (fig. 12A). From 2 p53^-/-ES cell clone (p 53)^C/C1、p53^C/R2) The developed chimeric rat fetuses have malformations in their heads (left side in fig. 12B). This phenotype was attributable to p53, as evidenced by cyan fluorescence^-/-High contribution of ES cells to the head (left side in fig. 12C). In 25 out of 209 normal growing chimeric rat fetuses (right side in FIG. 12B), p53^-/-The contribution of ES cells to the head was low (right side in fig. 12C). And use of p53^+/-ES cells (25.2. + -. 5.7%) when using p53^-/-Abnormal growth of chimeras occurred at a significantly higher frequency with ES cells (85.5. + -. 2.6%).

TABLE 6

Study of development of chimeric rats from different cell clones

Thus, it was found that rat ES cells established using YPAC factors have the ability to produce chimeric rats even after undergoing genetic modification such as gene targeting in 2 cycles after their establishment.

Conventionally, because it takes much time and cost to select genetically modified ES cells, single-allele modified ES cells have been used to prepare genetically modified animals having a double-allele modified target gene. For this reason, there is absolutely no report on the preparation of genetically modified animals by biallelic deletion of a target gene in ES cells. In 2010, it was also reported that as a result of mating a monoallelic deficient p53 knockout rat (prepared from a p53 gene monoallelic deficient ES cell), a monoallelic deficient p53 knockout rat (Nature vol.467, 211-215 (2010)) was born, but it was not reported that any malformation occurred as described in example 9 above.

In the present invention, it is considered that the time required for preparing a genetically modified animal can be shortened by producing the genetically modified animal at the rat ES cell stage by modifying the target gene (gene disruption, mutation, etc.) bi-allerily, and therefore, the analysis of gene function at the individual level is accelerated. It is also expected that the reliability and accuracy of gene function analysis will be improved compared to conventional analysis, and a disease animal model that more accurately reflects the actual condition will be prepared.

Industrial applicability

By using the method of the present invention, chimeric embryos with improved germ line transmission efficiency can be prepared regardless of the strain of rat ES cells or the strain of host embryos; by using the chimeric embryo, a germ line-transferred chimeric rat can be efficiently prepared. It is thus possible to easily prepare genetically modified rats (knockout rats, knock-in rats, etc.) which can be widely used for various pharmacological or physiological studies, as well as for regenerative medicine studies, etc.

The present application is based on patent application No. 2009-274008 filed on 12/1/2009 in japan and patent application No. 2010-166571 filed on 7/23/2010 in japan, the contents of which are incorporated herein in their entirety.

Claims

1. A method of making a chimeric embryo, the method comprising the following steps (a) and (b):

2. The method of claim 1, wherein said contacting is effected by injecting rat pluripotent stem cells into host embryos.

3. The method of claim 1 or 2, wherein in step (a), the rat pluripotent stem cells are contacted with the host embryo in the presence of an ES cell differentiation inhibitor and a ROCK inhibitor.

4. The method of any one of claims 1-3, wherein the host embryo is pre-cultured in the presence of an ES cell differentiation inhibitor prior to contacting with rat pluripotent stem cells.

5. The method of claim 4, wherein the pre-culturing is performed in the presence of an inhibitor of ES cell differentiation and a ROCK inhibitor.

6. The method of any one of claims 1-5, wherein the culturing in step (b) is performed in the presence of an ES cell differentiation inhibitor.

7. The method of claim 6, wherein the culturing in step (b) is performed in the presence of an inhibitor of ES cell differentiation and a ROCK inhibitor.

8. The method of any one of claims 1-7, wherein the ES cell differentiation inhibitor consists of at least 2 drugs selected from the group consisting of: MEK inhibitors, GSK3 inhibitors, TGF β receptor inhibitors and FGF receptor inhibitors.

9. The method of claim 8, wherein the ES cell differentiation inhibitor consists of a MEK inhibitor, a GSK3 inhibitor, and a TGF β receptor inhibitor.

10. The method of any one of claims 1-9, wherein the pluripotent stem cells are ES cells.

11. The method of any one of claims 1-10, wherein the rat pluripotent stem cell is a pluripotent stem cell prepared from a rat strain that: in step (a), the rat line does not produce chimeric rats of the transmitting germ line when contacted with a host embryo in the absence of an ES cell differentiation inhibitor.

12. The method of any one of claims 1-11, wherein the host embryo is derived from a rat strain: in step (a), the rat line does not produce chimeric rats of the transmitting germ line when contacted with rat pluripotent stem cells in the absence of an ES cell differentiation inhibitor.

13. A method of making a chimeric rat, the method comprising: a chimeric embryo prepared according to the method of any one of claims 1-12 is transplanted into the uterus or oviduct of a pseudopregnant female rat to give birth to an offspring rat.

14. A method of preparing a rat having a contribution of rat pluripotent stem cells to the entire body, the method comprising: chimeric rats were mated with heterozygotic rats.

15. A culture medium for use in the preparation of chimeric rats comprising a MEK inhibitor, a GSK3 inhibitor and a TGF β inhibitor.

16. The culture medium of claim 15, further comprising a ROCK inhibitor.

17. A genetically modified rat having a contribution of rat ES cells undergoing biallelic gene modification.

18. A method of making a genetically modified rat, the method comprising the step of biallelic modification of rat ES cells.