[0001] DESCRIPTION NEW MARKER FOR DETECTION OF INDUCED PLURIPOTENT STEM CELL
[0003] The present invention relates to a new marker for detection of an induced pluripotent stem cell (hereinafter, an iPS cell), and to a method for detection of an iPS cell using the marker.
[0004] Hereinafter, the "induced pluripotent stem cell" may also be simply referred to as "iPS cell."
[0005]BACKGROUND OF INVENTION
[0006] In recent years, mouse and human iPS cells have been established in succession. Takahashi and Yamanaka (I) have established iPS cells by introducing Oct3/4, Sox2, Klf4, and c-Myc genes into reporter mouse-derived fibroblasts having a neomycin resistance gene knocked in the Fbxl5 gene locus so as to enable the forced expression of the genes. Okita et al (2) have succeeded in establishment of an iPS cell ("Nanog iPS cell") almost equivalent to the embryonic stem (ES) cell in terms of gene expression and epigenetic modification by producing transgenic mice in which a green fluorescent protein (GFP) gene and a puromycin resistance gene have been incorporated into the Nanog gene locus, the expression of which is limited to pluripotent cells as compared with that of Fbxl5, forcing mouse-derived fibroblasts to express the above- mentioned, and selecting puromycin-resistant and GFP positive cells. Similarly, with the use of activation of Nanog (3_, 4) or Oct3/4 (3_) as an indicator, other groups have reproduced the establishment of iPS cells having a capability equivalent to ES cells. Although the Fbxl5, Nanog, and Oct3/4 are currently known as markers for detection (or selection) of iPS cells accordingly, the use of other markers for selection of iPS cells has never been reported.
[0007] The "ECAT gene" (ES cell associated transcript gene) as used herein
collectively refers to a series of genes specifically expressed specifically in totipotent cells such as ES cells. Regarding such ECAT gene, ES cell-specific expression of Oct3/4 (5), ECATl to ECATlO (6, 7), ECATl 5-1, ECAT 15-2 (7, 8), and ECAT 16 (8) has been reported.
[0009] 1. Takahashi, K. and Yamanaka, S., Cell, 126: 663-676 (2006)
[0010] 2. Okita, K. et al., Nature, 448: 313-317 (2007)
[0011] 3. Wernig, M. et al., Nature, 448: 318-324 (2007)
[0012] 4. Maherali, N. et al., Cell Stem Cell, 1 : 55-70 (2007)
[0013] 5. Takeda et al., Nucleic Acids Research, 20:4613-4620 (1992)
[0017]SUMMARY OF INVENTION
[0018] An object of the present invention is to provide a new marker for detection of an iPS cell, and another object of the invention is to provide a method for detection of an iPS cell using the marker.
[0019] Through analysis of the EST database using Digital Differential Display, we have made discoveries regarding ES cell associated transcript 11 (hereinafter referred to as "ECATl 1," which is also referred as "Lltdl"). As a result of the expression analysis of the ECATl 1 gene, they have confirmed that the gene is an ECAT gene because of its expression, which is almost limited to undifferentiated stem cells in mice and humans.
[0020] Next, we have produced an ECATl 1 homozygous mutation knock-in mouse (ECATI l deficient mouse) having EGFP and puromycin resistance genes knocked-in to the ECATl 1 gene locus. By analyzing ECATl 1 homozygous mutantation (i.e., deletion)-having ES cells which have been established from the mouse, we have
concluded that the ES cells have proliferation and differentiation potencies, which are not different from those of wild-type ES cells, and have no abnormalities in functions generally regarded as characteristics of ES cells. As described above, it has now been revealed that ECATl 1 has no important functions in maintenance of the
[0021]undifferentiation and pluripotency of ES cells. As a result, from the view point that homozygous mutation (i.e., deletion)-having cells can be used, it has now been suggested that the ECATl 1 is useful as a marker for ES cells or iPS cells.
[0022] Next, we introduced four genes (Oct3/4, Klf4, Sox2, and c-Myc) or three genes (Oct3/4, Klf4, and Sox2) into ECATl 1 -deficient mouse embryonic fibroblasts (MEF) through retrovirus, thereby establishing an iPS cell. As a result, GFP-positive colonies exhibiting ES cell-like morphology were obtained and so it has now been revealed that iPS cells can be detected using the expression of the ECATl 1 gene as an indicator. Furthermore, it has now been revealed that, as compared with the case of selecting Nanog expression ("Nanog-EGFP") as an indicator, a greater number of GFP positive cells can be detected at an earlier time by selection of the expression of ECATl 1 ("ECATl 1-EGFP") as an indicator.
[0023] As described above, it has now been revealed that, according to the present invention, iPS cells can be detected (or selected) using the expression of ECATl 1 (or the promoter activity of ECATl 1) as an indicator. Similarly with the use of the ECATl 1 expression as an indicator, screening for a substance capable of nuclear- reprogramming a somatic cell, screening for a substance capable of maintaining a pluripotent stem cell, and the like can also be carried out.
[0024] The present invention has been completed based on those findings.
[0025] Specifically, the present invention is as described below.
[0026] (1) A method for detection of an induced pluripotent stem cell (an iPS cell), comprising the following steps of:
[0027] (a) bringing a somatic cell comprising a DNA in which a marker gene is present at a position wherein the expression of the marker gene is controlled by an ECATl 1 gene promoter, into contact with a nuclear reprogramming substance; and
(b) detecting the expression of the marker gene following the step (a).
[0028] (2) The method according to (1), wherein the marker gene is a fluorescent protein gene, a luminescent enzyme gene, a chromogenic enzyme gene, a drug resistance gene, or a combination thereof, or an endogenous ECATl 1 gene.
[0029] (3) The method according to (2), whereby the expression of the endogenous ECATl 1 gene is detected by an RT-PCR or ELISA method.
[0030] (4) The method according to any one of (1) to (3), wherein the somatic cell is a mouse- or human-derived somatic cell.
[0031] (5) The method according to any one of (1) to (4), wherein the nuclear reprogramming substance comprises at least one substance selected from the group consisting of Oct3/4, Sox2, Klf4, c-Myc, Nanog, and Lin28, and nucleic acids encoding the same.
[0032] (6) The method according to (5), wherein the nuclear reprogramming substances are Oct3/4, Klf4, and Sox2, or nucleic acids encoding the same.
[0033] (7) The method according to (5), wherein the nuclear reprogramming substances are Oct3/4, Klf4, Sox2, and c-Myc, or nucleic acids encoding the same.
[0034] (8) An iPS cell, which is selected by the method according to any one of (1) to (7).
[0035](9) The iPS cell according to (8), wherein an exogenous marker gene is incorporated into the chromosome.
[0036] (10) A marker for detection of an iPS cell, comprising a polynucleotide capable of specifically bind to an ECATl 1 gene.
[0037] (11) The marker according to (10), wherein the ECATl 1 gene is a gene comprising the nucleotide sequence of SEQ ID NO: 1 or 3.
[0038] (12) The marker according to (10) or (1 1), wherein the polynucleotide capable of specifically binding to an ECATl 1 gene is a polynucleotide comprising at least 15 continuous nucleotides in the nucleotide sequence of SEQ ID NO: 1 or 3 and/or a polynucleotide complementary thereto.
[0039] (13) The marker according to any one of (10) to (12), which is used as a probe or a primer in detection of an iPS cell.
[0040] (14) A marker for detection of an iPS cell, comprising an antibody that specifically
recognizes ECAT 11.
[0041] (15) The marker according to (14), wherein ECATl 1 is a protein comprising the amino acid sequence of SEQ ID NO: 2 or 4.
[0042] (16) A method for screening for a somatic cell nuclear-reprogramming substance comprising the following steps of:
[0043] (a) bringing a somatic cell comprising a DNA in which a marker gene is present at a position wherein the expression of the marker gene is controlled by an ECATl 1 gene promoter, into contact with a test substance; and
[0044] (b) detecting the expression of the marker gene following the step (a).
[0045] (17) The method according to (16), wherein the marker gene is a fluorescent protein gene, a luminescent enzyme gene, a chromogenic enzyme gene, a drug resistance gene, or a combination of thereof, or an endogenous ECATl 1 gene.
[0046] (18) The method according to (17), wherein the expression of the endogenous ECATl 1 gene is detected by an RT-PCR or ELISA method.
[0047] (19) The method according to any one of (16) to (18), wherein the somatic cell is a mouse- or human-derived somatic cell.
[0048] (20) A marker for screening for a somatic cell nuclear-reprogramming substance comprising a polynucleotide capable of specifically binding to an ECATl 1 gene.
[0049] (21) The marker according to (20), wherein the ECATl 1 gene is a gene comprising the nucleotide sequence of SEQ ID NO: 1 or 3.
[0050] (22) The marker according to (20) or (21), wherein the polynucleotide capable of specifically binding to an ECATI l gene is a polynucleotide comprising at least 15 continuous nucleotides in the nucleotide sequence of SEQ ID NO: 1 or 3 and/or a polynucleotide complementary thereto.
[0051] (23) The marker according to any one of (20) to (22), which is used as a probe or primer in screening for a somatic cell nuclear-reprogramming substance.
[0052] (24) A marker for screening for a somatic cell nuclear-reprogramming substance comprising an antibody that specifically recognizes ECATI l .
[0053] (25) The marker according to (24), wherein the ECATl 1 is a protein comprising the
amino acid sequence of SEQ ID NO: 2 or 4.
[0054] (26) A method for screening for a substance capable of maintaining the undifferentiation and pluripotency of a pluripotent stem cell, comprising the following steps of:
[0055] (a) bringing a pluripotent stem cell comprising a DNA in which a marker gene is present at a position where the expression of the marker gene is controlled by an ECATl 1 gene promoter, into contact with a test substance in a medium in which the undifferentiation and pluripotency of the pluripotent stem cell cannot be maintained; and
[0056] (b) detecting the expression of the marker gene following the step (a).
[0057] (27) The method according to (26), wherein the marker gene is a fluorescent protein gene, a luminescent enzyme gene, a chromogenic enzyme gene, a drug resistance gene, or a combination thereof, or an endogenous ECATl 1 gene.
[0058] (28) The method according to (27), wherein the expression of the endogenous ECATl 1 gene is detected by an RT-PCR or ELISA method.
[0059] (29) The method according to any one of (26) to (28), wherein the pluripotent stem cell is an iPS cell or ES cell.
[0060] (30) The method according to (29), wherein the iPS cell or ES cell is a mouse- or human-derived cell.
[0061] (31) A marker for screening for a substance capable of maintaining the undifferentiation and pluripotency of a pluripotent stem cell, comprising a polynucleotide capable of specifically binding to an ECATl 1 gene.
[0062] (32) The marker according to (31), wherein the ECATI l gene is a gene comprising the nucleotide sequence of SEQ ID NO: 1 or 3.
[0063] (33) The marker according to (31) or (32), wherein the polynucleotide capable of specifically binding to an ECATl 1 gene is a polynucleotide comprising at least 15 continuous nucleotides in the nucleotide sequence of SEQ ID NO: 1 or 3 and/or a polynucleotide complementary thereto.
[0064] (34) The marker according to any one of (31) to (33), which is used as a probe or a primer in screening for a substance capable of maintaining the undifferentiation and pluripotency of a pluripotent stem cell.
(35) A marker for screening for a substance capable of maintaining the undifferentiation and pluripotency of a pluripotent stem cell, comprising an antibody that specifically recognizes ECAT 11.
[0065] (36) The marker according to (35), wherein the ECATl 1 is a protein comprising the amino acid sequence of SEQ ID NO: 2 or 4.
[0066] (37) A method for detection of an ES cell, comprising a step of detecting the expression of an endogenous ECATI l gene.
[0067] (38) The detection method according to (37), wherein the expression of the endogenous ECATl 1 gene is detected by an RT-PCR or ELISA method.
[0068] (39) A marker for detection of an ES cell, comprising a polynucleotide capable of specifically binding to an ECATl 1 gene.
[0069] (40) The marker according to (39), wherein the ECATl 1 gene is a gene comprising the nucleotide sequence of SEQ ID NO: 1 or 3.
[0070] (41) The marker according to (39) or (40), wherein the polynucleotide capable of specifically binding to an ECATI l gene is a polynucleotide comprising at least 15 continuous nucleotides in the nucleotide sequence of SEQ ID NO: 1 or 3 and/or a polynucleotide complementary thereto.
[0071] (42) The marker according to any one of (39) to (41), which is used as a probe or a primer in detection of an ES cell.
[0072] (43) A marker for detection of an ES cell, comprising an antibody that specifically recognizes ECAT 11.
[0073] (44) The marker according to (43), wherein the ECATl 1 is a protein comprising the a ammiinnno Λ aCcΛiΛd C sPeΠqHuPeΠnΓcPe n off S SFEOQ I TDD N NOO-: 29 n orr 44.
[0074]BRIEF DESCRIPTION OF DRAWINGS
[0075] Fig. 1 shows photographs showing the results of RT-PCR analysis by which the expression of mouse and human ECATl 1 genes was examined (Figs. IA and IB, respectively). In the figure, "MGl .19" and "RF8" are ES cell lines, and "NCR-G3" is an embryonic carcinoma.
Fig. 2 shows the construction of an ECATl 1 targeting vector.
[0076] Fig. 3 is the graph showing proliferation potencies of ECAT 11 -homozygous deficient ES cells (ECATl 1 NULL) and heterozygous deficient ES cells (ECATl 1 HETERO)Tn the figure, "RF8WT" and "#6202" are Wild Type ES cell lines.
[0077] Fig. 4 shows the images of the histological staining (hematotoxin-eosin staining) of teratomas obtained by subcutaneously injecting the ECATl 1 homozygous deficient ES cell (ECATl 1-/-) and the ECATl 1 heterozygous deficient ES cell
[0078](ECATl 1+/-) into the backs of nude mice. The histological images shown herein are, from the top, panoramic images, ectodermal images, mesodermal images, and endodermal images.
[0079] Fig. 5 shows the merges of a phase-contrast image of cells and a GFP positive colony image when ECATl 1 homozygous deficient ES cells (#620-6, #757-1, #757-2) and ECATl 1 heterozygous deficient ES cells (#706-1, #749-1) were cultured in the presence of LIF ("L+"), in the absence of LIF ("L-"), or in the presence of retinoic acid ("RA").
[0080] Fig. 6 shows the photographs showing cells at day 6 (Fig. 6A) and at day 10 (Fig. 6B) after infection, which are: cells (ECATl 1 -/-Limbs 3VIEFs) obtained from limbs-derived ECATl 1 deficient MEF by iPS induction using 4 genes; and cells (Nanog-EGFP MEFs) obtained as controls from Nanog-EGFP heterozygous MEFs by iPS induction using 4 genes. In the figure, "Phase" indicates a phase-contrast image, "EGFP" indicates a GFP positive colony image, and "Merge" indicates a merge of the phase-contrast image and the GFP positive colony image. "RFP" indicates a negative control excited with another fluorescence (by intrinsic fluorescence detection).
[0081] Fig. 7A is the graph showing the number of GFP positive colonies obtained from limbs- or body-derived ECATl 1 deficient (homozygous or heterozygous) MEFs by iPS induction using 4 genes and GFP positive colonies obtained as controls by iPS induction using 4 genes from Nanog-EGFP heterozygous MEFs. Fig. 7B is the graph showing the results obtained by performing similar iPS induction using 3 genes.
[0082] Fig. 8 (top panel) is the graph showing the number of colonies obtained from
limbs- or body-derived ECATl 1 deficient (homozygous or heterozygous) MEFs by iPS induction using 4 genes ("4F") or 3 genes ("3F") and colonies obtained from Nanog- EGFP heterozygous MEFs ("Ng-EGFP") as controls by iPS induction using 4 genes. In the top panel, "non ES-like" indicates a non ES-like colony, "ES-like" indicates an ES-like colony, "mixed" indicates mixed colonies of said cells, and "Too tight colonies" indicates colonies showing strong aggregation as observed. Also, Fig. 8 (bottom panel) shows the photographs showing morphologies of non ES-like colonies and ES-like colonies (the marge of the phase-contrast image and of the GFP positive colony image).
[0083]In the bottom panel, "686C-5" and "686C-9" indicate Nanog-EGFP heterozygous MEFs-derived cells, and the remaining 4 photographs ("686E-11", "686H-8", "686L-7", and "6860-3") show ECATl 1-EGFP homozygous deficient MEFs-derived cells.
[0084]DETAILED DESCRIPTION OF INVENTION
[0085] The term "gene" as used herein may refer not only to cDNA (mRNA), but also to genomic DNA, depending on the technical content.
[0086] The terms "nucleic acid" and "polynucleotide" as used herein are intended to include both DNA and RNA.
[0087] Examples of the term "antibody" as used herein include a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single-chain antibody, or portions of the above antibodies having antigen-binding properties, such as a Fab fragment or a fragment generated from a Fab expression library.
[0088] Examples of the term "ECATl 1 gene" as used herein include a mouse ECATl 1 gene comprising the nucleotide sequence of SEQ ID NO: 1, a human ECATl 1 gene comprising the nucleotide sequence of SEQ ID NO: 3, and a gene comprising a nucleotide sequence analogous to these nucleotide sequences. The mouse ECATl 1 gene is registered in GenBank under Accession Nos. NM OO 1081202 and AB211064. Also, the human ECATl 1 gene is registered in GenBank under Accession Nos.
[0089]NM O 19079 and AB211065.
[0090] Examples of the above-described "gene comprising a nucleotide sequence
analogous to..." include genes comprising a nucleotide sequence having high identity with the nucleotide sequence shown in SEQ ID NO: 1 or 3 and genes capable of hybridizing to the complementary strand of the nucleotide sequence shown in SEQ ID NO: 1 or 3 under stringent conditions.
[0091] Example of the "genes comprising a nucleotide sequence having high identity" are genes comprising a nucleotide sequence having 70% or more, preferably 80% or more, more preferably 90% or more, particularly preferably 95% or more, such as 97% or more, 98% or more, or 99% or more, identity with the nucleotide sequence shown in SEQ ID NO: 1 or 3. The term "identity" as used herein for sequences refers to a percentage (%) of identical nucleotides (or identical amino acid residues) to the total number of nucleotides (or total number of amino acid residues) containing, if any, the number of gaps, when two nucleotide sequences (or amino acid sequences) are aligned to achieve the maximum match with or without introduction of gaps. The sequence identity can be determined using a known algorithm, such as XBLAST, NBLAST, or Gapped BLAST (e.g., Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87: 2264-2268; Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90: 5873-5877; Altshul et al., 1997, Nucleic Acids Res. 25 : 3389-3402). Also, by using such algorithms, one can access to a public GenBank database (e.g., NCBI (U.S.A.)) and search for a nucleotide sequence (or an amino acid sequence) homologous to a desired gene (or protein).
[0092] Also, the "stringent conditions" used in the term "genes capable of hybridizing under stringent conditions to the complementary strand of the nucleotide sequence shown in SEQ ID NO: 1 or 3" refers to conditions for hybridization and washing that can be controlled by appropriately changing a temperature, salt concentration and the like during hybridization reaction or washing, a salt concentration, and the like, and that can be set depending on a desired sequence identity such that nucleic acids having a sequence identity of 50-70% or more, preferably 80% or more, or further preferably 90% or more, can maintain hybridization. Generally, hybridization conditions are set at a temperature about 5-10°C lower than the melting temperature (Tm) at which two
nucleic acids achieve 50% hybridization, and at an ionic strength and a pH, which are used. Stringent hybridization conditions may be low stringency conditions, moderately stringent conditions, or highly stringent conditions. Preferably, moderately stringent conditions or highly stringent conditions are employed. Examples of such conditions include, but are not limited to, conditions comprising hybridization with 1-6 x SSC at 45-68°C, followed by one or more times of washing with 0.1-0.2 x SSC and 0.1-0.2% SDS at 50-680C. Here, the "1 x SSC" refers to a solution containing 150 mM NaCl and 15 mM sodium citrate, pH 7.0.
[0093] Examples of the "ECATl 1" as used herein include a mouse ECATl 1 protein comprising the amino acid sequence of SEQ ID NO: 2, a human ECATl 1 protein comprising the amino acid sequence of SEQ ID NO: 4, and a protein comprising an amino acid sequence analogous to these amino acid sequences.
[0094] An example of the "protein comprising an amino acid sequence analogous to..." is a protein encoded by "a gene comprising a nucleotide sequence analogous to that of the ECATl 1 gene as recited in the present invention," and specifically a protein comprising an amino acid sequence having 70% or more, preferably 80% or more, more preferably 90% or more, or particularly preferably 95% or more identity with the amino acid sequence of SEQ ID NO: 2 or 4. The "identity" is as defined above.
[0095] Herein, the same names are used herein for nuclear reprogramming substance genes, such as symbols of Oct, Nanog, Sox, KIf, Myc and Lin for example, regardless of types of animals, unless otherwise specified. Although these specific gene names are generally used as the mouse gene names, they are used herein not only for mouse- derived genes but also for human- or other animal-derived genes, unless otherwise specified. In addition, Oct3/4 is also named Oct3, Oct4, or POU5F1, indicating the same transcription factor. Herein, the "Oct3/4" may be used collectively.
[0096] The method for detection of an iPS cell, screening method, and the like of the present invention as described below are principally described in WO2005/080598 and WO 2006/035741. The present invention can be implemented using such descriptions.
(1) Method for detection of iPS cell
[0097] The present invention provides a method for detection of an iPS cell comprising the following steps of:
[0098] (a) bringing a somatic cell comprising a DNA in which a marker gene is present at a position wherein the expression of the marker gene is controlled by an ECATl 1 gene promoter, into contact with a nuclear reprogramming substance; and
[0099] (b) detecting the expression of the marker gene following the step (a).
[0100] Here, the "ECATl 1 gene" may refer to an ECATl 1 gene from any animal species such as mouse, rat, monkey, and human, as described above. Preferable examples include a mouse-derived ECATl 1 gene and a human-derived ECATl 1 gene.
[0101] The "marker gene" refers to any gene that enables detection, screening, or selection of cells through introduction of the marker gene into cells. Examples of the marker gene include fluorescent protein genes, luminescent enzyme genes, chromogenic enzyme genes, drug resistance genes, and combinations thereof. Another example is an endogenous ECATl 1 gene.
[0102] Specifically, the fluorescent protein genes include a GFP (green fluorescent protein) gene, an YFP (yellow fluorescent protein) gene, an RFP (red fluorescent protein) gene, and an aequorin gene. Cells expressing such fluorescent protein genes can be detected by fluorescence microscopy. Also, such cells can be separated and selected using a cell sorter or the like with the use of differences in fluorescence intensity. Moreover, such cells can be selected by subjecting cells to limiting dilution at a concentration of one or less cell per well, culturing and growing the cells, and then detecting fluorescence-emitting cells (or wells) under fluorescence microscope.
[0103]Alternatively, colonies, which are formed on a medium such as soft agar medium, may be selected under fluorescence microscope for example.
[0104] An example of the luminescent enzyme genes is a luciferase gene. The cells expressing the luminescent enzyme gene can be detected by adding a luminescent substrate and then measuring the amount of luminescence using a luminescence spectrometer. Alternatively, such cells can be selected by subjecting cells to limiting
dilution at a concentration of one or less cell per well, culturing and growing the cells, collecting some cells from each well, adding a luminescent substrate, and then measuring the luminescence using a luminescence spectrometer.
[0105] Examples of the chromogenic enzyme genes include a β-glycosidase gene, a β- glucuronidase gene, an alkaline phosphatase gene, and a secreted alkaline phosphatase (SEAP) gene. Cells expressing the chromogenic enzyme genes can be detected by adding a chromogenic substrate and then observing the presence or absence of color development. Also, such cells can be selected by subjecting cells to limiting dilution at a concentration of one or less cell per well, culturing and growing the cell, collecting some cells from each well, adding a chromogenic substrate, and then observing the color development.
[0106] Examples of the drug resistance genes include a puromycin resistance gene (puro), a neomycin resistance gene (neo), a tetracycline resistance gene (tet), a kanamycin resistance gene (km), a zeocin resistance gene (zeo), and a hygromycin resistance gene (hygro). Cells are cultured on a medium (referred to as a selection medium) containing each drug, so that only cells expressing an introduced drug resistance gene can survive. Thus, cells containing a drug resistance gene can be easily selected by culturing the cells on a selection medium.
[0107] Combinations of these marker genes include fusion forms, tandem forms, and the like. An example thereof is the β geo gene, which is a fusion gene of neomycin resistance gene (neo) and β-galactosidase gene (β-gal).
[0108] All of the marker genes as described above are known in the art. Vectors containing such marker genes are available from Invitrogen Corporation, GE
[0109]HEALTHCARE BIOSCIENCE, Promega, MBL (Medical and Biological Laboratories, Japan), and the like.
[0110] When the marker gene is an endogenous ECATl 1 gene, the promoter activity of the ECATl 1 gene can be measured by detecting the expression (transcription or translation) of the endogenous ECATl 1 gene in cells. Examples of the detection method include a method for detection of an ECATl 1 mRNA and a method for detection
of an ECATl 1 protein. As the former, an RT-PCR method is known well, and as the latter, an ELISA method is known well. These methods are specifically described in § (1-6) and § (1-7) below.
[0111] Of the above-described marker genes, particularly preferable marker genes are a fluorescent protein gene, a drug resistance gene, and a gene containing such drug resistance gene, in view of easy selection of cells.
[0112] As used herein, the "somatic cells" refers to all animal cells (preferably, cells of mammals including humans) excluding germ-line cells such as ova and oocytes, totipotent cells, and ES cells. Examples of somatic cells as used herein include, but are not limited to, fetal somatic cells, neonate somatic cells, and mature somatic cells. Further examples include primary cultured cells, passaged cells, and established cell lines, and also include tissue stem cells and tissue precursor cells. Specific examples of somatic cells include, but are not limited to, (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells, (2) tissue precursor cells, and (3) differentiated cells such as lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (e.g., skin cells), hair follicle cells, hepatocytes, gastric mucosal cells, enterocytes, splenocytes, pancreatic cells (e.g., pancreatic exocrine cells), brain cells, pneumocytes, renal cells, and skin cells. When iPS cells are used for treatment of disease, it is desired to use somatic cells isolated from a patient. For example, somatic cells involved in diseases, or somatic cells utilizable for treatment of diseases, can be used.
[0113] In the method for detection of an iPS cell of the present invention, somatic cells comprising a DNA in which a marker gene is present at a position wherein the expression of the marker gene is controlled by an ECATl 1 gene promoter are used.
[0114] The term "promoter" as used herein refers to a region that controls gene expression or transcription and is intended to include "promoter region" or "promoter and enhancer regions."
[0115] Many methods for allowing a marker gene to be present at a position wherein the expression of the marker gene is controlled by an ECATl 1 gene promoter are
known. The marker gene may be allowed to be present using any of methods known in the art, which methods are principally divided into the method by which a marker gene is allowed to be present using an individual organism (e.g., mouse) (see § 1.1 below), and the method by which a marker gene is allowed to be present at the cell level without using any individual organism (see § 1.2 below).
[0116](1-1) Method for allowing marker gene to be present using individual organism (e.g., mouse)
[0117] When a marker gene is allowed to be present using an individual organism (such as mouse), the marker gene is allowed to be present at a position wherein the expression of the marker gene is controlled by an ECATl 1 gene promoter. In this case, the ECATl 1 gene itself of the individual organism may be present in a form capable of being expressed, or alternatively, the ECATI l gene may be present in a disrupted form. Because our studies revealed that the ECATl 1 gene was not essential for maintaining the properties of undifferentiation and pluripotency of ES cells, the ECATI l gene may be present in its disrupted form.
[0118] A gene promoter is generally present in a region upstream of exon 1. So, it is desirable that a marker gene is allowed to be present in a region downstream of the initiation site of exon 1 of the ECATl 1 gene, so that the expression of the marker gene is controlled by the ECATl 1 gene promoter. In this case, a marker gene may be allowed to be present at any position as long as such position is downstream of the initiation site of the exon 1.
[0119](1-1 -a) A case where ECATl 1 gene is disrupted
[0120] As the method for disruption of an ECATl 1 gene, any method known in the art may be employed. The most often employed technique is a knock-in method, which comprises disrupting the ECATl 1 gene as a target by homologous recombination using a vector, which comprises a marker gene and allows homologous recombination to occur at any position of the ECATl 1 gene (hereinafter, referred to as targeting vector), and
knocking-in the marker gene at the position of the disrupted gene, instead.
[0121]Preferred examples of the methods for detection of an iPS cell of the present invention include: a detection method comprising the following steps of:
[0122] (a) bringing a somatic cell comprising a gene resulting from knock-in of a gene comprising a drug resistance gene to an endogenous ECATl 1 gene, into contact with a nuclear reprogramming substance; and
[0123] (b) examining the presence or absence of living cells in a selection medium following the step (a); and
[0124]a detection method comprising the following steps of:
[0125] (a)' bringing a somatic cell containing a gene in which a marker gene comprising a fluorescent protein coding gene has been knocked-in at an endogenous ECATl 1 gene, into contact with a nuclear reprogramming substance; and
[0126] (b)' examining the presence or absence of fluorescent protein-expressing cells following the step (a)'.
[0127] Various methods for knock-in of a marker gene are known. Of these methods, the promoter trap method can preferably be used. This method comprises inserting a targeting vector that lacks a promoter into the genome via homologous recombination. When homologous recombination takes place correctly, a marker gene is expressed by the endogenous promoter (i.e., ECATl 1 gene promoter). An example of the method for allowing a marker gene to be present at a position wherein the expression of the marker gene is controlled by an ECATl 1 gene promoter by using the promoter trap method, is described below.
[0128] First, the genomic sequence of an ECAT l 1 gene, which is required for targeting, is determined. When the genomic sequence is already known based on the Mouse Genome Resources (http://www.ncbi.nlm.nih.gov/genome/guide/mouse/) etc., which is a public database, it can be sequenced using the sequence information. When the genomic sequence is not known, a genomic clone containing a desired genomic region of the ECATl 1 gene may be isolated, or alternatively, a genomic nucleotide sequence may be determined by screening a genomic library that is available by persons
skilled in the art by PCR or the like using portions of the ECATl 1 gene as primers. Examples of such genomic library to be used include a mouse BAC (bacterial artificial chromosome) library (Invitrogen) and a PAC (Pl -derived artificial chromosome) library (Invitrogen).
[0129] Next, based on the above-identified genomic DNA sequence of the ECATl 1 gene, a genomic region (of the ECATl 1 gene) to be substituted with a marker gene (hereinafter, referred to as ECATl 1 gene genomic region A) is determined. A 5' region (51 arm) and a 31 region (3' arm) flanking the genomic region A of the ECATl 1 gene are amplified by PCR or the like using a genomic DNA as a template. Here, an example of a genomic DNA to be used as a template is a genomic DNA of a mouse BAC clone (e.g., RP24-326M13, sold by BACPAC) containing the ECATl 1 gene. PCR primers can be designed based on the sequence of the above-described genomic DNA of the ECATl 1 gene. The thus amplified 5' arm and 3' arm are inserted into each side flanking a marker gene cassette in a promoter-trap targeting vector. Examples of the promoter- trap targeting vector to be used include pBSSK(-)-IRES-β geo containing an IRES (internal ribosome entry site)-β geo (which is the fusion of β-galactosidase gene and neomycin resistance gene) cassette (Mountford P. et al., Proc. Natl. Sci. U. S. A, 91 : 4303-4307 (1994)) and similar vectors containing an IRES-Hygro (hygromycin resistance gene) cassette. Here, the IRES-Hygro cassette can be prepared by
[0130]substituting a β geo portion of the IRES-β geo cassette with Hygro (Invitrogen), for example. Another example is a pBS-GIP-PHF-NBl (Okita et al., Nature, 448: 313-317 (2007)) having an EGFP-IRES-Puro-FRT-Hyg cassette.
[0131] Next, the thus constructed targeting vector is digested with a restriction enzyme for linearization, and then the resultant is introduced into ES cells by
[0132]electroporation or the like.
[0133] Examples of ES cells to be used for introduction include RF8 cell line (Meiner, V. et al., Proc. Natl. Acad. Sci. U.S.A., 93: 14041-14046 (1996)), JI cell line (Li, E. et al., Cell, 69: 915-926 (1992)), CGR8 cell line (Nichols, J. et al., Development, 110: 1341-1348 (1990)), and MGl .19 cell line (Gassmann, M. et al., Proc. Natl. Acad. Sci.,
U.S.A., 92: 1292-1296 (1995)), as well as commercially available mouse ES cell line 129SV (R-CMTI-IA), mouse ES cell line C57/BL6 (NO.R-CMTI-2A), mouse ES cell line DBA-I (No.R-CMTI-3A), and mouse ES cell line Pluri Stem B6-White (Cat. No. R- SCROI lA) (DS Pharma Biomedical CO., LTD.) and the like.
[0134] A targeting vector can be introduced into ES cells by an electroporation method (see Meiner, V. et al., Proc. Natl. Acad. Sci. U.S.A., 93: 14041-14046 (1996), for example), a calcium phosphate method, a DEAE-dextran method, a method using a lipid for gene transfer (Lipofectamine, Lipofectin; Invitrogen), or the like.
[0135]Subsequently, ES cells in which the targeting vector has been introduced can be selected based on the properties of a marker gene (e.g., a drug resistance gene) used herein. It may be confirmed that homologous recombination takes place correctly in the thus selected ES cells by Southern blot or the like using a portion of the ECATl 1 gene as a probe. ES cells can be prepared as described above, heterozygously comprising a gene resulting from knock-in of a marker gene to the ECATl 1 gene (as a genomic gene.
[0136] Any medium known in the art may be used for culturing ES cells. For example, the medium for RF8 cell has the following composition: 15% FBS, 0.1 mM Non Essential Amino Acids (GIBCO BRL), 2 mM L-glutamine, 50 U/ml penicillin- streptomycin, and 0.1 1 mM 2-ME (GIBCO BRL) in Dulbecco's Modified Eagle Medium (DMEM). Also, a commercially available pre-prepared medium can also be used.
[0137] When feeder cells are used for culturing ES cells, as such feeder cells, fibroblasts prepared by a conventional technique from mouse embryos or a fibroblast- derived STO cell line (Meiner, V. et al., Proc. Natl. Acad. Sci. U.S.A., 93: 14041-14046 (1996)) may be used. Also, a commercially available product may be used herein. Feeder cells are desirably used for culturing ES cells after that the proliferation thereof is stopped by treatment with mitomycin C.
[0138] Also, when no feeder cells are used for culturing ES cells, LIF (Leukemia Inhibitory Factor) can be added, followed by culturing the ES cells. Used as LIF is mouse recombinant LIF, rat recombinant LIF (e.g., Chemicon), or the like.
[0139] Next, ES cells containing the above-described targeting vector are introduced
into mice, so that knockout mice (which are marker gene knock-in mice) are produced. A method for producing the marker gene knock-in mouse is known in the art.
[0140]Specifically, the above ES cells are injected into mouse (e.g., C57BL/6) blastocysts and then the blastocysts are implanted in the uteri of pseudopregnant female mice (e.g., ICR), so that chimeric mice are produced. Subsequently, heterozygous mutant mice containing a hetereozygously knocked-in marker gene are produced by crossing the chimeric mice with normal mice (e.g., C57BL/6). By crossing between heterozygous mutant mice, homozygous mutant mice having the homozygously knocked-in marker gene are obtained.
[0141] Regarding the production of knock-in mice, see publications about ECAT3 knock-in mouse (Tokuzawa, Y., et al., Molecular and Cellular Biology, 23(8): 2699- 2708 (2003)), ECAT4 knock-in mouse (Mitsui, K., et al., Cell, 113: 631-642 (2003)), and ECAT5 knock-in mouse (Takahashi, K., K. Mitsui, and S. Yamanaka, Nature, 423 (6939): p541-545 (2003), Japanese Patent Publication (Kokai) No. 2003-265166 A), for example.
[0142] Examples of somatic cells used in the method for detection of an iPS cell of the present invention include somatic cells isolated from the above-described
[0143]heterozygous knock-in mice and somatic cells isolated from homozygous knock-in mice.
[0144](1-1-b) A case where ECAT l 1 gene is not disrupted
[0145] Alternatively, a marker gene can also be allowed to be present at a position wherein the expression of the marker gene is controlled by an ECATl 1 gene promoter, without disrupting the ECATl 1 gene. An example of such a technique is a technique using transgenic non-human animals that are produced by introducing a BAC vector, a PAC vector, or the like (comprising a DNA in which a marker gene is present at a position wherein the expression of the marker gene is controlled by the ECATl 1 gene promoter) into mouse individual, rat individual, or the like. An example of such a technique using a BAC vector is as described below.
[0146] A BAC clone ("ECATl 1 gene-containing BAC clone") to be used containing
an ECATl 1 gene and a promoter region thereof can be isolated and identified based on the sequence information of the ECATI l gene, as described in §1-1 -a above. Also, a commercially available product (e.g., RP24-326M13, sold by BACPAC) may also be used.
[0147] In the ECATl 1 gene-containing BAC clone, a marker gene can be incorporated into an appropriate site between the ORF of the ECATl 1 gene and a promoter region of the ECATl 1 gene. For example, a marker gene can be incorporated into an appropriate site adjacent to the 5' side of the ORF of the ECATl 1 gene. This can be easily performed using Red/ET Recombination (Gene Bridges), for example.
[0148] A method for producing transgenic animals in which the BAC vector (hereinafter, the vector may also be referred to as "marker gene-containing BAC vector," in which a marker gene is present at a position wherein the expression of the marker gene is controlled by the ECATl 1 gene promoter, as described above) has been introduced, is known. For example, such transgenic animals can be produced by procedures as described in Experimental Medicine, Supplement, "New Genetic
[0149]Engineering Handbook (Shin Idenshi Ko-gaku Handbook) Revised, Third Edition" (YODOSHA, Japan, 1999) (Jp..)
[0150] Hereinafter, the production of transgenic animals, such as transgenic mice, is described below.
[0151] A method for introducing a gene into a mouse fertilized egg is not particularly limited. A gene can be introduced by microinjection or electroporation for example. After introduction, the thus obtained egg cells are cultured and then implanted in the oviduct of surrogate mother mice. Subsequently, the implanted mice are allowed to give birth, and then desired mouse offsprings are selected from the obtained offsprings. This selection can be carried out by examining mouse offspring-derived DNAs for the presence or absence of the transferred gene by dot blot hybridization, PCR or the like.
[0152] Heterozygous transgenic mice (which heterozygously contain the transferred gene) are produced by crossing the above-described mouse offsprings with wild-type mice. Thus, transgenic mice homozygously comprising the BAC vector comprising a
marker gene can be obtained through crossing of heterozygous mice.
[0153] In the detection method of the present invention, both somatic cells isolated from the above heterozygous transgenic mice and somatic cells isolated from
[0154]homozygous transgenic mice can be used. Preferably, homozygous transgenic mice- derived somatic cells are used.
[0155] Somatic cells that are isolated from the above-mentioned knock-in mice or transgenic mice may be any cells which do not express a marker gene or which express a marker gene at a low level. Examples of the somatic cells include cells other than totipotent cells such as ES cells. Specific examples include: (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and sperm stem cells; (2) tissue precursor cells; or (3) differentiated cells such as lymphocytes, epithelial cells, muscle cells, and fibroblasts.
[0156](1-2) Method for allowing a marker gene to be present at the cell level without using animal individual
[0157] Various methods are known for allowing a marker gene to be present at a position wherein the expression of the marker gene is controlled by an ECATl 1 gene promoter within cells without using individuals. The marker gene may be allowed to be present at said position by using any method known in the art. A general example of the method is a method for introducing a vector comprising a marker gene into cells.
[0158] Cells used for gene transfer may be somatic cells or ES cells. Somatic cells may be derived from any animal species, such as mouse, human, and monkey. Other examples of somatic cells may be primary cultured cells or established cell lines. For example, the primary cultured cells include, but are not limited to, mouse embryonic fibroblasts (MEFs), bone marrow-derived mesenchymal stem cells, or sperm stem cells, and established cell lines such as NIH3T3. As ES cells, in addition to the above-listed mouse ES cells, human or monkey ES cells can also be used. Examples of human ES cells include KhES-I, KhES-2, and KhES-3 (Stem Cell Research Center, Institute for Frontier Medical Sciences, Kyoto University). Examples of monkey ES cells include
cynomolgus monkey ES cells (ASAHI TECHNO GLASS CO., LTD.). When such ES cells are used for the detection method of the present invention, the properties of undifferentiation and pluripotency of the ES cells should be removed or cancelled before use.
[0159] As the method for introduction of a vector into cells, general introduction methods, but being appropriate for host cells, may be used. Specific examples thereof include a calcium phosphate method, a DEAE-dextran method, an electroporation method, and a method using lipids for gene transfer (Lipofectamine, Lipofectin;
[0160]Invitrogen) and the like.
[0161] Examples of vectors used for introduction include vectors that enable cloning of at most about 300-kb DNA, such as a BAC vector, a PAC vector, a plasmid vector, and a targeting vector described in §1-1 above. Some methods for preparing somatic cells in which a marker gene is present at a position where the expression of the marker gene is controlled by an ECATl 1 gene promoter, by using each vector are described below.
[0162](1-2-a) A case where BAC vector or PAC vector is used
[0163] With the use of a BAC or PAC vector comprising an ECATl 1 gene promoter, a marker gene is allowed to be present at a position where the expression of the marker gene is controlled by an ECATl 1 gene promoter. Examples of the use of BAC vector are described below.
[0164] The BAC clone (hereinafter, referred to as ECATI l gene-comprising BAC clone) comprising an ECATl 1 gene promoter can be isolated and identified based on the sequence information of the ECATI l gene, as described in § 1-1 above. Otherwise, commercially available products (e.g., RP24-326M13 : BACPAC) may be used.
[0165] In the ECATl 1 gene-containing BAC clone, a portion of the ECATl 1 gene can be easily substituted with a marker gene using Red/ET Recombination (Gene Bridges), for example. The ECATl 1 gene promoter is generally present in a region upstream of exon 1 of the ECATl 1 gene. Therefore, in order that a marker gene is controlled by an
ECATl 1 gene promoter in terms of expression, the marker gene is desirably present in a region downstream of the exon 1 initiation site of the ECATl 1 gene. In this case, a marker gene may be present at any position on the ECATl 1 gene, as long as it is present downstream of the exon 1 initiation site.
[0166] The above-constructed BAC vector, in which a marker gene is present at a position where the expression of the marker gene is controlled by an ECATl 1 gene promoter, is introduced into somatic cells, so that the somatic cells for the detection method of the present invention can be prepared. In addition, to enable easy selection of said cells (in which the BAC vector has been introduced) in a selective medium, a gene comprising a drug resistance gene (hereinafter, referred to as a second drug resistance gene) are preferably inserted in the BAC vector. In this case, to enable expression in somatic cells, the addition of a promoter that is expressed in a somatic cell onto the 5'-side or the 3'-side of the second drug resistance gene is required. Moreover, the second drug resistance gene may be a same type of drug resistant gene as or different type of drug resistant gene from that of a marker gene present at a position where the expression of the marker gene is controlled by the ECATl 1 gene promoter. The second drug resistance gene is desirably a different type of drug resistance gene.
[0167]When the second drug resistance gene of the same type as described above is used, a
[0169] .$■ loxP or FRT sequence is previously added to each side of the second drug resistance gene, cells in which a BAC vector has been introduced are selected in a selective medium, and then the second drug resistance gene can be cleaved using recombinase Cre or FLP.
[0170] Unlike the above, when the second drug resistance gene is not inserted into a BAC vector, a second expression vector comprising the second drug resistance gene is co-transfected with the above BAC vector, followed by selection with a selective medium. In this case, transfection is desirably carried out using a BAC vector in an extremely excessive amount compared with that of the second expression vector.
[0171] When the above BAC vector in which a marker gene is present at a position where the expression of the marker gene is controlled by the ECATl 1 gene promoter is
introduced into ES cells, ES cells expressing the introduced marker gene can be selected based on the properties of the marker gene used. Subsequently, the ES cells are differentiated into somatic cells, thereby preparing somatic cells to be used for screening of the present invention. ES cells can be differentiated under culture conditions where no feeder cells are present. Hence, somatic cells obtained by causing differentiation under such conditions or somatic cells obtained by causing differentiation using an inducer of differentiation known in the art, such as retinoic acid, can be used for screening of the present invention. Examples of somatic cells differentiated from ES cells include tissue stem cells, tissue precursor cells, and somatic cells (e.g., nerve cells, skin corneocytes, cardiac muscle cells, skeletal muscle cells, blood cells, islet cells, and pigment cells).
[0172](1-2-b) A case where plasmid vector having no promoter is used
[0173] Cells used for the detection method of the present invention can be prepared by transformation of cells with a plasmid vector constructed by inserting a fusion gene of ECATl 1 gene promoter and marker gene into the vector having no promoter.
[0174] Examples of the vector include plasmid vectors, such as pBluescript (Stratagene) and pCR2.1 (Invitrogen), with no promoter.
[0175] Examples of the ECATl 1 gene promoter include an about 1-kb promoter and preferably an about 2-kb promoter, which are located upstream of the transcription initiation site of the gene.
[0176] Each ECATl 1 gene promoter can be identified by a technique or the like, which comprises: (i) a step of determining the 5 '-end by general methods, such as a 5'- RACE method (e.g., performed using a 5'full Race Core Kit (Takara Bio Inc.) or the like), an oligo-cap method, and Sl primer mapping; and (ii) a step of obtaining the 5'- upstream region using a Genome Walker Kit (Clontech) or the like and then measuring the promoter activity of the thus obtained upstream region. A marker gene is fused to the 3 '-side of the identified ECATl 1 gene promoter, which is subsequently inserted into the above-described plasmid vector, thereby resulting in the plasmid vector in which the
marker gene is present at a position where the expression of the marker gene is controlled by the ECATl 1 gene promoter.
[0177] The above-obtained vector is introduced into somatic cells or ES cells in a manner similar to that in § 1-2-a above, so that somatic cells for screening of the present invention can be prepared.
[0178](1-2-c) A case where targeting vector is used
[0179] Somatic cells used for screening of the present invention can also be prepared by introducing the targeting vector described in § 1-1 above into somatic cells or ES cells.
[0180] When the above-described targeting vector is introduced into somatic cells, for easy selection of cells in which the vector has been introduced in a selective medium, a gene comprising a drug resistance gene (i.e., a second drug resistance gene) is allowed to be present on the targeting vector in a manner similar to that in § 1-2-a above, or alternatively the targeting vector and a second expression vector containing the second drug resistance gene are co-transfected. It is more preferable to use somatic cells obtained by selection using a selective medium for screening of the present invention. In this case, transfection is desirably carried out using the targeting vector in an extremely excessive amount compared with that of the second expression vector.
[0181] When the above targeting vector is introduced into ES cells, cells expressing an introduced marker gene can be selected based on the properties of the marker gene exisiting on the targeting vector (For the preparation method, see Tokuzawa, Y., et al., Molecular and Cellular Biology, 23(8): 2699-2708 (2003), for example). A method for induction of from ES cells to somatic cells is similar to that in § 1-2-a above.
[0182] Somatic cells used for the method for detection of an iPS cell are desirably human somatic cells containing a vector in which a marker gene is inserted, so that the gene is present at a position where the expression of the marker gene is controlled by an ECATl 1 gene promoter, when treatment or disease models for humans are considered. Specifically, somatic cells prepared as described below are used.
First, somatic cells from patients are prepared by isolating the cells from humans, for example. Examples of such somatic cells include somatic cells involved in disease and somatic cells utilized for treatment of diseases. Any vectors described in § 1-2 above are introduced into the human somatic cells. Specifically, it is desirable to introduce a BAC vector (in which a marker gene is present downstream of the ECATl 1 gene promoter) or a PAC vector. A nuclear reprogramming substance is added to cells in which the BAC vector has been introduced, so that iPS cells appear. The iPS cells are selected depending on the properties of the marker gene used. For example, when a drug resistance gene is used as a marker gene, selection is carried out using a selection medium after addition of a nuclear reprogramming substance, whereby iPS cells can be easily selected using drug resistance as an indicator.
[0183] When a marker gene is an endogenous ECATl 1 gene, the endogenous ECATl 1 gene is already present at a position where the expression of the marker gene is controlled by the ECATl 1 gene promoter, so that no genetic manipulation described above is required and the somatic cells can be directly used. Such detection of promoter activity using an endogenous ECATl 1 gene can be effectively used for cells (e.g., human cells) that are genetically engineered with difficulty.
[0184] In step (a) above, somatic cells prepared as described above are brought into contact with a nuclear reprogramming substance.
[0185](1-3) Nuclear reprogramming substance
[0186] The term "nuclear reprogramming substance" in the present invention refers to a substance that enables induction and conversion of differentiated cells into
[0187]undifferentiated and, particularly, pluripotent cells. Examples of the nuclear reprogramming substance that can be used in the present invention are as described below, but are not limited thereto.
[0188] Specifically, examples of the nuclear reprogramming substance include one or more types of protein or nucleic acid selected from the group consisting of an Oct family member, a KIf family member, a Sox family member, a Myc family member, a Lin
family member, and Nanog (WO2007/69666; Science, 2007, 318: 1917-1920).
[0189]Specific examples of these family members and combinations thereof are as listed below:
[0190] (a) a combination of 2 types of nuclear reprogramming substances, comprising an Oct family member and a Sox family member;
[0191] (b) a combination of 3 types of nuclear reprogramming substances, comprising an Oct family member, a KIf family member, and a Sox family member;
[0192] (c) a combination of 4 types of nuclear reprogramming substances, comprising an Oct family member, a Sox family member, a Lin family member, and Nanog;
[0193] (d) a combination of 4 types of nuclear reprogramming substances, comprising an Oct family member, a KIf family member, a Sox family member, and a Myc family member;
[0194](e) 1 type of nuclear reprogramming substance, comprising an Oct family member;
[0195](f) a combination of 2 types of nuclear reprogramming substances, comprising an Oct family member and a KIf family member;
[0196] (g) a combination of 2 types of nuclear reprogramming substances, comprising an Oct family member and Nanog; and,
[0197] (h) a combination of 3 types of nuclear reprogramming substances, comprising an Oct family member, a KIf family member, and a Myc family member.
[0198] A particularly preferred nuclear reprogramming substance among the above family members is Oct3/4, Klf4, Sox2, c-Myc, Lin28, or Nanog.
[0199] Furthermore, in addition to the above-described nuclear reprogramming substances, one or more nuclear reprogramming substances selected from the group consisting of Fbxl5, ERas, ECAT15-2, Tell, and β-catenin may be combined therewith, and/or one or more nuclear reprogramming substances selected from the group consisting of ECATl, Esgl, Dnmt3L, ECAT8, GdO, Mybl2, ECAT15-1, Fthll7, Sall4, Rexl, UTFl, Stella, Stat3, and Grb2 may also be combined therewith. These combinations are specifically explained in WO2007/69666.
[0200] GenBank accession numbers (humans, mice, and rats) for the sequences of the above nuclear reprogramming substances such as Oct3/4, Nanog, Lin28, Lin28b,
ECATl, ECAT2 (also referred to as ESGl), ECAT3 (also referred to as Fbxl5), ECAT5 (also referred to as Eras), ECAT7, ECAT8, ECAT9 (also referred to as Gdf3), ECATlO (also referred to as Soxl5), ECAT15-1 (also referred to as Dppa4), ECAT15-2 (also referred to as Dppa2), Fthll7, Sall4, Rexl (also referred to as Zfp42), Utfl, Tell, Stella (also referred to as Dppa3), β-catenin (also referred to as Ctnnbl), Stat3, Grb2, c-Myc, Soxl, Sox2, Sox3, Sox4, Soxl l, Mybl2, N-Myc, L-Myc, Klfl, Klf2, Klf4, Klf5
[0201](WO2007/069666), FoxD3, ZNF206, Mybl2, and Otx2 (WO2008/118820) are as shown below. The amino acid sequence and the nucleotide sequence of each of these substances are available by accessing GenBank (NCBI, U.S.A.).
[0202] Regarding Oct3/4, the sequences of human Oct3/4, mouse Oct3/4, and rat Oct3/4, for example, are registered as NM 203289 or NM 002701, NM 013633, and NM OO 1009178, respectively.
[0203] Regarding Nanog, the sequences of human Nanog, mouse Nanog, and rat Nanog, for example, are registered as NM 024865, NM 028016, and NM OOl 100781, respectively.
[0204] Regarding Lin28, the sequences of human Lin28, mouse Lin28, and rat Lin28, for example, are registered as NM 024674, NM_145833, and NM OOl 109269, respectively.
[0205] As a factor analogous to Lin28, Lin28b belonging to the same Lin family is known. Regarding Lin28b, the sequences of human Lin28b and mouse Lin28b, for example, are registered as NM OO 1004317 and NM OO 1031772, respectively.
[0206] Regarding ECATl, the sequences of human ECATl and mouse ECATl are registered as AB211062 and AB21 1060, respectively.
[0207] Regarding ECAT2, the sequences of human ECAT2 and mouse ECAT2 are registered as NM_001025290 and NM 025274, respectively.
[0208] Regarding ECAT3, the sequences of human ECAT3 and mouse ECAT3 are registered as NM_152676 and NM_015798, respectively.
[0209] Regarding ECAT5, the sequences of human ECAT5 and mouse ECAT5 are registered as NM_181532 and NM_181548, respectively.
Regarding ECAT7, the sequences of human ECAT7 and mouse ECAT7 are registered as NM 013369 and NM 019448, respectively.
[0210] Regarding ECAT8, the sequences of human ECAT8 and mouse ECAT8 are registered as AB211063 and AB211061, respectively.
[0211] Regarding ECAT9, the sequences of human ECAT9 and mouse ECAT9 are registered as NM 020634 and NM 008108, respectively.
[0212] Regarding ECATlO, the sequences of human ECATlO and mouse ECATlO are registered as NM 006942 and NM 009235, respectively.
[0213] Regarding ECAT15-1, the sequences of human ECAT15-1 and mouse
[0214]ECAT 15-1 are registered as NM_018189 and NM 028610, respectively.
[0215] Regarding ECAT 15-2, the sequences of human ECAT 15-2 and mouse
[0216]ECAT 15-2 are registered as NM 138815 and NM 028615, respectively.
[0217] Regarding Fthll7, the sequences of human Fthl 17 and mouse Fthll7 are registered as NM 031894 and NM_031261, respectively.
[0218] Regarding Sal 14, the sequences of human Sal 14 and mouse Sal 14 are registered as NM_020436 and NM_175303, respectively.
[0219] Regarding Rexl, the sequences of human Rexl and mouse Rexl are registered as NM_174900 and NM_009556, respectively.
[0220] Regarding Utfl, the sequences of human Utfl and mouse Utfl are registered as NM_003577 and NM_009482, respectively.
[0221] Regarding Tell, the sequences of human Tell and mouse Tell are registered as NM_021966 and NM_009337, respectively.
[0222] Regarding Stella, the sequences of human Stella and mouse Stella are registered as NM l 99286 and NM 139218, respectively.
[0223] Regarding β-catenin, the sequences of human β-catenin and mouse β-catenin are registered as NM OO 1904 and NM_007614, respectively.
[0224] Regarding Stat3, the sequences of human Stat3 and mouse Stat3 are registered as NM_139276 and NM_213659, respectively.
[0225] Regarding Grb2, the sequences of human Grb2 and mouse Grb2 are registered
as NM 002086 and NM 008163, respectively.
[0226] Regarding FoxD3, the sequences of human FoxD3 and mouse FoxD3 are registered as NM 012183 and NM 010425, respectively.
[0227] Regarding ZNF206, the sequences of human ZNF206 and mouse ZNF206 are registered as NM 032805 and NM OO 1033425, respectively.
[0228] Regarding Mybl2, the sequences of human Mybl2 and mouse Mybl2 are registered as NM 002466 and NM 008652, respectively.
[0229] Regarding Otx2, the sequences of human Otx2 and mouse Otx2 are registered as NM 172337 and NM l 44841, respectively.
[0230] Regarding c-Myc, the sequences of human c-Myc and mouse c-Myc are registered as NM 002467 and NM 010849, respectively.
[0231] Regarding N-Myc, the sequences of human N-Myc and mouse N-Myc are registered as NM 005378 and NM 008709, respectively.
[0232] Regarding L-Myc, the sequences of human L-Myc and mouse L-Myc are registered as NM_001033081and NM 008506, respectively.
[0233] Regarding Soxl, the sequences of human Soxl and mouse Soxl are registered as NM 005986 and NM_009233, respectively.
[0234] Regarding Sox2, the sequences of human Sox2 and mouse Sox2 are registered as NM_003106 and NM_011443, respectively.
[0235] Regarding Sox3, the sequences of human Sox3 and mouse Sox3 are registered as NM 005634 and NM_009237, respectively.
[0236] Regarding Sox4, the sequences of human Sox4 and mouse Sox4 are registered as NM 003107 and NM 009238, respectively.
[0237] Regarding Soxl l, the sequences of human Soxl l and mouse Soxl l are registered as NM 003108 and NM 009234, respectively.
[0238] Regarding Mybl2, the sequences of human Mybl2 and mouse Mybl2 are registered as NM 002466 and NM 008652, respectively.
[0239] Regarding KIf 1, the sequences of human KIf 1 and mouse KIf 1 are registered as NM 006563 and NM_010635, respectively.
Regarding Klf2, the sequences of human Klf2 and mouse Klf2 are registered as NM O 16270 and NM 008452, respectively.
[0240] Regarding Klf4, the sequences of human Klf4 and mouse Klf4 are registered as NM 004235 and NM_010637, respectively.
[0241] Regarding Klf5, the sequences of human Klf5 and mouse Klf5 are registered as NM_001730 and NM_009769, respectively.
[0242] Examples of a method that can be used for introducing nuclear reprogramming substances into cells include, but are not limited to, known techniques, such as
[0243]microinjection, liposome, lipofection, electroporation, a calcium phosphate method, viral infection, a membrane permeable peptide vector, and membrane permeable peptide-modified liposome.
[0244] When nuclear reprogramming substances are nucleic acids, vectors (e.g., viral vectors, plasmids, artificial chromosome vectors, or episome vectors), transposons, and the like can be used.
[0245] Examples of viral vectors include a retrovirus vector, a lentivirus vector (Cell, 126, pp. 663-676, 2006; Cell, 131, pp. 861-872, 2007; Science, 318, pp. 1917-1920, 2007), an adenovirus vector (Science, 322, 945-949, 2008), an adeno-associated virus vector, and a Sendai virus vector. Also, examples of artificial chromosome vectors include a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), and a bacterial artificial chromosome (BAC or PAC). As a plasmid, a plasmid for
[0246]mammalian cells can be used herein (Science, 322: 949-953, 2008). Regarding an episome vector, an episome vector described in Science, 324: 797-800, 2009 can be used, for example.
[0247] A vector as used herein may contain regulatory sequences, such as promoter, enhancer, ribosome binding sequence, terminator, and polyadenylation site, so that nuclear reprogramming substances can be expressed. The vector may further contain a selection marker sequence such as a drug resistance gene (e.g., kanamycin resistance gene, an ampicillin resistance gene, and a puromycin resistance gene), a thymidine kinase gene, or a diphtheria toxin gene, and a reporter gene sequence such as a green fluorescent protein (GFP),
β -glucuronidase (GUS), or FLAG.
[0248] Regarding transposon, piggy Bac transposon (Nature, 458: 766-770, 2009;
[0249]Nature, 458: 771-775, 2009) can be exemplified.
[0250] Further examples of a method for introducing proteins as nuclear reprogramming substances into cells include a method using polyarginine (Cell Stem Cell, 4:381-384 (2009)) and a method using a commercially available reagent for protein introduction. As the reagent for protein introduction, cationic lipid-based BioPOTER Protein Delivery Reagent (Gene Therapy Systems), Pro-Ject™ Protein Transfection Reagent (PIERCE), and ProVectin (IMGENEX), lipid-based Profect-1 (Targeting Systems), membrane permeable peptide-based Penetrain Peptide (Q biogene) and Chariot Kit (Active Motif), and GenomONE (ISHIHARA SANGYO KAISHA, LTD) using HVJ envelope
[0251](inactivated Sendai virus) are commercially available. Protein introduction can be carried out according to protocols attached to these reagents.
[0252](1-4) Substance for improving efficiency for establishment of iPS cell
[0253] In addition to the above nuclear reprogramming substances, a substance for improving efficiency for establishment of iPS cells may be further introduced into cells, if necessary. Examples of such substance for improving efficiency for establishment of iPS cells include histone deacetylase (HDAC) inhibitors [e.g., low-molecular-weight inhibitors such as valproic acid (VPA) (Nat. Biotechnol., 26(7): 795-797 (2008)), Trichostatin A, sodium butyrate, MC 1293, and M344 and nucleic acid expression inhibitors such as siRNA and shRNA against HDAC (e.g., HDACl siRNA Smartpool® (Millipore) and HuSH 29mer shRNA Constructs against HDACl (OriGene))], DNA methyl transferase inhibitors (e.g., 5'-azacytidine) (Nat. Biotechnol., 26(7): 795-797 (2008)), G9a histone methyltransferase inhibitors [e.g., low-molecular-weight inhibitors such as BIX-01294 (Cell Stem Cell, 2: 525-528 (2008)) and nucleic acid expression inhibitors such as siRNA and shRNA against G9a (e.g., G9a siRNA (human) (Santa Cruz Biotechnology))], L-channel calcium agonist (e.g., Bayk8644) (Cell Stem Cell, 3, 568-574 (2008)), p53 inhibitors (e.g., siRNA and shRNA against p53 (Cell Stem Cell, 3,
475-479 (2008)), UTFl (Cell Stem Cell, 3, 475-479 (2008)), Wnt Signaling (e.g., soluble Wnt3a) (Cell Stem Cell, 3, 132-135 (2008)), and 2Ϊ/LIF (2i is an inhibitor for mitogen-activated protein kinase signaling and glycogen synthase kinase-3, PIoS Biology, 6 (10), 2237-2247 (2008)).
[0254](1-5) Detection of marker gene expression
[0255] In the method for detection of an iPS cell of the present invention, the expression of a marker gene is detected following step (a) above.
[0256] Medium and procedures for culture for induction of iPS cells are exemplified as follows.
[0257] Examples of a culture medium include, but are not limited to, (1) a DMEM, DMEM/F12, or DME medium containing 10-15% FBS (these media may further appropriately contain LIF (leukemia inhibitory factor), penicillin/streptomycin, puromycin, L-glutamine, nonessential amino acids, β-mercaptoethanol, and the like), (2) a medium for ES cell culture containing bFGF or SCF, such as a medium for mouse ES cell culture (e.g., TX-WES medium(Thromb-X)), and a medium for primate ES cell culture (e.g., a medium for primate (human &monkey) ES cells, ReproCELL, Kyoto, Japan).
[0258] An example of culture procedures is as follows. Somatic cells are brought into contact with nuclear reprogramming substances on a DMEM or DMEM/F12 medium containing 10% FBS at 37°C in the presence of 5% CO2 and are cultured for about 4 to 7 days. Subsequently, the cells are reseeded on feeder cells (e.g., mitomycin C-treated STO cells or SNL cells). About 10 days after contact between the somatic cells and the reprogramming factors or DNAs encoding the reprogramming factors, cells are cultured in a bFGF-containing medium for primate ES cell culture. About 30-45 days or more after the contact, iPS cell-like colonies can be formed. The culture procedures are appropriate for induction of primate iPS cells such as human iPS cells.
[0259] Alternatively, cells may be cultured using a DMEM medium containing 10% FBS (which may further appropriately contain LIF, penicillin/streptomycin, puromycin,
L-glutamine, nonessential amino acids, β-mercaptoethanol, and the like) on feeder cells (e.g., mitomycin C-treated STO cells or SNL cells) at 37°C in the presence of 5% CO2. After about 25-30 days or more, iPS cell-like colonies can be formed. This culture procedure is appropriate for induction of rodent iPS cells such as mouse iPS cells.
[0260] During the above culture, medium exchange with fresh medium is performed once a day from day 2 after the start of culture. In addition, the number of somatic cells to be used for nuclear reprogramming is not limited, but ranges from
[0261]approximately 5* 103 to approximately 5χ 106 cells per culture dish (100 cm2).
[0262] When a gene containing a drug resistance gene is used as a marker gene, cells expressing the marker gene can be selected by culturing the cells in a medium (selective medium) containing the relevant drug. Also, cells expressing the marker gene can be detected when the marker gene is a fluorescent protein gene, through observation with a fluorescence microscope, by adding a luminescent substrate in the case of a luminescent enzyme gene, or adding a chromogenic substrate in the case of a chromogenic enzyme gene. When a fluorescent protein gene, a luminescent enzyme gene, or a chromogenic enzyme gene is used as a marker gene, the relevant cells can be selected (separated) using a cell sorter, a limiting dilution method, a soft agar colony method, or the like.
[0263] When a marker gene is an endogenous ECATl 1 gene, ECATl 1 gene expression can be detected by an existing method such as a reverse transcription- polymerase chain reaction (RT-PCR) method, a quantitative PCR method, a microarray method, a hybridization method, enzyme-linked immunosorbent assay (ELISA), fluorescent antibody techniques, radioimmunoassay, or a blot method. Hereafter, markers used for detection of an endogenous ECATl 1 gene, and methods for detection of ECATl 1 gene expression using such markers are described.
[0264](1-6) Marker for detection of iPS cell
[0265](1-6-1) Polynucleotide
[0266] The present invention provides a marker for detection of an iPS cell, comprising a polynucleotide capable of specifically binding to an ECATl 1 gene.
Specific examples of the "ECATl 1 gene" include a mouse ECATl 1 gene comprising the nucleotide sequence of SEQ ID NO: 1 and a human ECATI l gene comprising the nucleotide sequence of SEQ ID NO: 3. Also, the above term
[0267]"polynucleotide capable of specifically binding to" refers to a polynucleotide with a specific length, which is capable of distinguishing the ECATl 1 gene from other genes and binding to the ECATl 1 gene. Examples of such bond (binding) include
[0268]noncovalent bonds such as a hydrogen bond, a hydrophobic bond, and a physical bond (Van der Waals force).
[0269] Specifically, the marker for detection of an iPS cell of the present invention can distinguish one gene from other genes and specify the gene, when it has generally a length of 15 nucleotides. Accordingly, a specific example of the marker of the present invention is a marker for detection of an iPS cell, which contains a polynucleotide containing at least 15 continuous nucleotides in the nucleotide sequence of SEQ ID NO: 1 or 3 and/or a polynucleotide complementary to the polynucleotide.
[0270] The above forward- strand polynucleotide and complementary-strand (reverse- strand) polynucleotide may be separately used as markers in a single-strand form or a double-strand form. The above polynucleotides that are labeled for detection are also included in the examples of the polynucleotide of the present invention.
[0271] The marker for detection of an iPS cell of the present invention may be, specifically, a polynucleotide comprising the nucleotide sequence (full-length sequence) of an ECATI l gene or a polynucleotide comprising a complementary sequence thereof. As long as the marker selectively (specifically) recognizes the ECATl 1 gene or a polynucleotide from the gene, it may be a polynucleotide comprising the above full- length sequence or a partial sequence of the complementary sequence thereof. In this case, an example of such partial sequence is that of a polynucleotide having a nucleotide length of at least 15 continuous nucleotides arbitrarily selected from the above full- length sequence or the nucleotide sequence of the complementary sequence thereof.
[0272] In addition, as used herein, the term "selectively (or specifically) recognizes" means, but is not limited to: that in a Northern blot method for example, the ECATl 1
gene or a polynucleotide from the gene can be specifically detected; or that in an RT- PCR method, the ECATl 1 gene or a polynucleotide from the gene is specifically generated, as long as persons skilled in the art can determine that the detected product or generated product is derived from the ECATl 1 gene.
[0273] The marker of the present invention can be designed using Oligo (ver.6) (Molecular Biology Insights) or vector NTI (Infomax), for example, based on the nucleotide sequence of the mouse ECATI l gene shown in SEQ ID NO: 1 and the nucleotide sequence of the human ECAT I l gene shown in SEQ ID NO: 3.
[0274]Specifically, candidate sequences of primers or probes, which can be designed on the basis of the nucleotide sequences of the above-described ECATI l genes using Oligo or vector NTI software, or sequences each partially containing at least said sequence can be used as primers or probes.
[0275] The marker of the present invention may be any marker having at least 15 continuous nucleotides in length, as described above. Specifically, the length can be appropriately selected and determined depending on the applications of the marker.
[0276](1-6-2) Polynucleotide as probe or primer
[0277] Whether or not test cells are iPS cells is detected by evaluating the presence or absence of the expression of or the expression level of the ECATl 1 gene in test cells. In this case, the above marker of the present invention can be used as a primer for specifically recognizing and amplifying RNA resulting from the expression of the ECATl 1 gene or a polynucleotide from the RNA or a probe for specifically detecting the RNA or a polynucleotide from the RNA.
[0278] When the marker of the present invention is used as a primer for detection of an iPS cell, an example of such marker is a marker having a nucleotide length generally ranging from 15 bp to 100 bp, preferably ranging from 15 bp to 50 bp, and more preferably ranging from 15 bp to 35 bp. Another preferable example is a marker having a nucleotide length ranging from 20 bp to 35 bp. When the marker is used as a detection probe, an example of the marker is a marker having a nucleotide length
generally ranging from 15 bp to the full-length sequence, preferably ranging from 15 bp to 1 kb, and more preferably ranging from 100 bp to 1 kb.
[0279] The marker of the present invention can be used as a primer or a probe in conventional methods for specifically detecting a specific gene, such as Northern blot, RT-PCR, in situ hybridization, and DNA chip. As a sample to be measured, total RNA prepared from test cells according to conventional methods may be used. Furthermore, various polynucleotides prepared based on the RNA may also be used. Also, cells may be directly used as samples to be measured.
[0281] The present invention provides a marker for detection of an iPS cell, which contains art antibody that specifically recognizes ECATl 1. Specific examples of such antibody include an antibody that can specifically recognize mouse ECATI l comprising the amino acid sequence of SEQ ID NO: 2 and an antibody that can specifically recognize human ECATl 1 comprising the amino acid sequence of SEQ ID NO: 4.
[0282] The form of the antibody of the present invention is not particularly limited and may be a polyclonal antibody or monoclonal antibody, which can be prepared using the protein of the present invention as an immunizing antigen. Moreover, an antibody having an antigen binding ability for a (poly)peptide comprising generally at least 8 continuous amino acids, preferably at least 15 continuous amino acids, and more preferably at least 20 continuous amino acids in the amino acid sequence of the protein of the present invention is also included in the examples of the antibody of the present invention.
[0283] Methods for producing such antibodies are known. The antibody of the present invention can also be produced according to these conventional methods (Current Protocol in Molecular Biology, Chapter 1 1.12-11.13 (2000)). Specifically, when the antibody of the present invention is a polyclonal antibody, a non-human animal such as a rabbit is immunized with the protein of the present invention expressed by and purified from Escherichia coli or the like according to conventional methods or
an oligo peptide synthesized having a partial amino acid sequence of the protein of the present invention according to conventional methods. Thus, the polyclonal antibody can be obtained from the serum of the immunized animal according to conventional methods. Meanwhile, when the antibody is a monoclonal antibody, a non-human animal such as a mouse is immunized with the protein of the present invention expressed by and purified from Escherichia coli or the like according to conventional methods or an oligopeptide having a partial amino acid sequence of the protein. The monoclonal antibody can be obtained from hybridoma cells prepared by fusing the thus obtained spleen cells and myeloma cells (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 1 1.4-11.11).
[0284] The ECATl 1 protein used as an immunizing antigen for preparation of an antibody may be obtained by DNA cloning, construction of each plasmid, transfection into a host, culture of a transformant, and subsequent collection of the protein from a culture product based on the gene sequence information (SEQ ID NOS: 1 and 3) provided by the present invention. These procedures can be carried out according to methods known in the art or methods described in documents (Molecular Cloning, T. Maniatis et al., CSH Laboratory (1983), DNA Cloning, DM. Glover, IRL PRESS (1985)), for example.
[0285] Specifically, a recombinant DNA (expression vector) that enables expression of a gene encoding ECATl 1 in desired host cells is prepared, the DNA is introduced into host cells for transformation, the transformant is cultured, and then a target protein is collected from the thus obtained culture product, so that the protein as an immunizing antigen for production of the antibody of the present invention can be obtained. Also, these partial peptides of the protein of the present invention can be produced by general chemical synthesis methods (e.g., peptide synthesis) based on the amino acid sequence information (SEQ ID NOS: 2 and 4) provided by the present invention.
[0286] The antibody of the present invention may be prepared using an oligo peptide having a partial amino acid sequence of ECATl 1. Such oligo (poly)peptide used for production of the antibody desirably has the immunogenicity similar to those of
ECATl 1. A preferable example thereof is an oligo (poly)peptide having said immu nog eni city and comprising at least 8 continuous amino acids, preferably at least 15 continuous amino acids, more preferably at least 20 continuous amino acids in the amino acid sequence of ECATI l.
[0287] An antibody against such oligo (poly)peptide can also be produced by enhancing immunological responses with the use of various adjuvants depending on hosts. Examples of such adjuvant include, but are not limited to, Freund's adjuvant, and mineral gel such as aluminium hydroxide, as well as surface active substances such as lysolecithin, pluronic polyol, polyanion, peptide, oil emulsion, keyhole limpet hemocyanin and dinitrophenol, and human adjuvants such as BCG (bacillus Caluuette Guecin) and Corynebacterium-parvum.
[0288] The antibody of the present invention is useful as a probe for detection of the presence or the absence of the expression of ECATl 1 in test cells.
[0289](1-7) Method for detection of iPS cell using the polynucleotide of ECATl 1 and antibody
[0290] The present invention provides a method for detection of an iPS cell using the above marker.
[0291] Specifically, the method for detection of an iPS cell of the present invention is to determine whether or not test cells are iPS cells by measuring the gene expression level of the ECATl 1 gene contained in test cells and the protein level of ECATl 1 from the gene.
[0292] The detection method of the present invention is specifically carried out as follows.
[0293](1-7-1) A case where RNA is used as subject to be measured
[0294] When RNA is used as a subject to be measured, iPS cells can be detected specifically by the method comprising the following steps of:
[0295](a) binding a test cell-derived RNA or a complementary polynucleotide transcribed therefrom, to the marker (which is a polynucleotide capable of specifically binding to
the ECATl 1 gene) of the present invention;
[0296] (b) measuring the test cell-derived RNA, to which the marker has been bound, or a complementary polynucleotide transcribed from the RNA, using the above marker as an indicator; and
[0297] (c) determining whether or not the test cell is an iPS cell based on the results measured in the step (b).
[0298] When RNA is used as a subject to be measured, the detection method of the present invention is carried out by detecting and measuring the expression level of the ECATl 1 gene in the RNA. Specifically, the detection method can be carried out by a known method, such as Northern blot, RT-PCR, DNA chip analysis, or in situ hybridization analysis, using the markers of the present invention comprising the above polynucleotides as primers or probes.
[0299] When the Northern blot method is used, the presence or the absence of the expression of or the expression level of the ECATl 1 gene in RNA can be detected and measured using the marker of the present invention as a probe. A specific example thereof comprises labeling the marker of the present invention (or complementary strand) with a radioisotope (RI), e.g.,3 2 P or3 3 P, a fluorescent substance, or the like, performing hybridization of the labeled marker to RNA from test cells transferred to a nylon membrane or the like according to conventional methods, and then detecting the thus formed double strand of the marker and RNA through detection of signals from the label (RI or fluorescent substance) for the marker using X-ray film or the like, or detecting and measuring the same using a radiation detector, a fluorescence detector, or the like. Also, detection and measurement can be carried out using Multi Bio Imager STORM (GE HEALTHCARE BIOSCIENCE), for example.
[0300] When the RT-PCR method is used, the presence or the absence of the expression of or the expression level of the ECATl 1 gene in RNA can be detected and measured using the markers of the present invention as primers. A specific example of the method comprises preparing cDNA from test cell-derived RNA according to conventional methods, performing hybridization of the cDNA to the markers of the
present invention (a pair of primers), so that a region of the ECATl 1 gene can be amplified using the cDNA as a template, carrying out the PCR method according to conventional methods, and then detecting the thus obtained amplified double-stranded DNA. Further examples of methods for detection of the thus amplified double- stranded DNA, which can be employed, include: a method that comprises detecting by ethidium bromide staining after agarose gel electrophoresis; a method that comprises detecting labeled double-stranded DNA produced by the above PCR using primers pre- labeled with RI or a fluorescent substance; and a detection method that comprises transferring the produced double-stranded DNA to a nylon membrane or the like according to conventional methods, performing hybridization of the labeled marker of the present invention as a probe to the DNA, and then performing detection. Currently, because many reagents for RT-PCR are soled from makers, RT-PCR can be performed according to instructions appended to those reagents.
[0301] When DNA chip analysis is employed, an example thereof is a detection method that comprises preparing a DNA chip in which the marker of the present invention as a DNA probe (either single strand or double strand) attached to a surface, performing hybridization of the DNA probe to cRNA prepared from test cell-derived RNA according to conventional methods, binding the formed double strand of DNA and cRNA to the labeled probe prepared from the marker of the present invention, and then performing detection.
[0302](1-7-2) A case where protein is used as subject to be measured
[0303] When a protein is used as a subject to be measured, the method for detection of an iPS cell according to the present invention can be carried out by detecting the ECATl 1 protein in test cells. Specifically, the detection can be carried out by the method comprising the following steps of:
[0304] (a) binding a test cell-derived protein to the marker of the present invention (i.e., an antibody that specifically recognizes ECATI l);
[0305] (b) measuring the test cell-derived protein to which the marker has been bound, by using
the marker as an indicator; and
[0306] (c) determining whether or not the test cell is an iPS cell based on the results measured in step (b).
[0307] More specifically, another example is a method that comprises detecting and quantitatively determining the protein of the present invention by a known method, such as Western blot or immunohistological staining, using the antibody-related marker of the present invention, and test cells as a sample.
[0308] The Western blot can be carried out by: after the use of the marker of the present invention as a primary antibody and then the use of a secondary antibody (the antibody binding to the primary antibody) labeled with a radioisotope such as125I, a fluorescent substance such as a fluorescamine derivative or a rhodamine derivative, an enzyme such as horseradish peroxidase (HRP), or the like, detecting signals from the radioisotope, the fluorescent substance, or the like of the obtained labeled compound using an X-ray film or the like; or detecting and measuring such signals using a radiation meter, a fluorescence detector, or the like. Also, detection and measurement can be carried out using Multi Bio Imager STORM (GE HEALTHCARE BIOSCIENCE) or the like.
[0309](1-8) Determination of iPS cell
[0310] It can be confirmed that cells expressing the marker gene detected by the methods described in § 1-1 to § 1-7 above retain functions as iPS cells by examining: (1) whether or not the cells express undifferentiated marker genes such as Oct3/4 and Nanog; (2) whether or not the cells are differentiated in vitro by stimulation with retinoic acid or the like; (3) whether or not teratomas are formed after transplantation thereof into nude mice; (4) whether or not chimeric mice are born as a result of injection of the cells into mouse blastocysts; (5) whether or not chimeric mice produce offsprings; and the like.
[0311](1-9) iPS cell
The present invention provides iPS cells selected by the above method for detection of an iPS cell of the present invention. An iPS cell wherein a DNA in which a foreign marker gene is present at a position wherein the expression of the marker gene is controlled by an ECATl 1 gene promoter is incorporated in the chromosome is a new, unknown iPS cell. The present invention also provides such new iPS cell.
[0312](2) Method for screening for somatic cell nuclear reprogramming substance
[0313] The present invention also provides a method for screening for a somatic cell nuclear reprogramming substance, comprising the following steps of:
[0314] (a) bringing a somatic cell containing a DNA in which a marker gene is present at a position wherein the expression of the marker gene is controlled by an ECATl 1 gene promoter, into contact with a test substance (or a substance to be tested); and
[0315] (b) detecting the expression of the marker gene following the step (a).
[0316] When the test substance is a nuclear reprogramming substance, iPS cells are induced from somatic cells. Thus, through detection of an iPS cell based on the expression of the marker gene located downstream of the ECATl 1 gene promoter, a substance capable of nuclear-reprogramming a somatic cell can be screened for.
[0317] All of (i) a method for preparing a somatic cell containing a DNA in which a marker gene is present at a position wherein the expression of the marker gene is controlled by an ECATl 1 gene promoter, (ii) a method for bringing a test substance into contact, and (iii) a method for detecting the expression of the marker gene, are as described in § 1 "Method for detection of iPS cell" above.
[0318] The term "test substance" as used herein is not limited, but refers to a nucleic acid, a peptide, a protein, an organic compound, an inorganic compound, or a mixture thereof Screening of the present invention is carried out specifically by bringing such test substance into contact with the above somatic cells. Specific examples of such test substance include cell extracts, gene libraries (genomic libraies or cDNA libraries), RNAi libraries, antisense nucleic acids, genes (genomic DNA, cDNA, or mRNA), proteins, peptides, low molecular weight compounds, high molecular weight
compounds, and natural compounds. More specific examples thereof include ES cells, eggs, cell extracts (extracted fractions) of ES cells or eggs, ES cell-derived or egg- derived cDNA libraries, genomic libraries, and protein libraries, or growth factors.
[0319] Origins of cDNA libraries, protein libraries or cell extracts (e.g., an organic compound or an inorganic compound) are preferably undifferentiated cells, such as ES cells or eggs.
[0320] These test substances are brought into contact with somatic cells in such a form that they can be incorporated into the somatic cells. For example, when a test sample is a nucleic acid (e.g., a cDNA library), the nucleic acid is introduced into somatic cells using calcium phosphate, DEAE-dextran, lipid for gene transfer, electric pulses, or the like.
[0321] Conditions for bringing somatic cells into contact with test substances are not particularly limited, as long as they are culture conditions (e.g., temperature, pH, and medium composition) appropriate for incorporation of the test substances without causing cell death.
[0322] Specifically, cell culture is carried out under culture conditions for ES cells before, during, or after the contact between somatic cells and test substances. Any method known in the art can be employed for culturing ES cells. An example of the medium for RF8 cells has the following composition: 15% FBS, 0.1 mM Non Essential Amino Acids (GIBCO BRL), 2 mM L-glutamine, 50 U/ml penicillin-streptomycin, and 0.11 mM 2-ME (GIBCO BRL) in Dulbecco's Modified Eagle Medium (DMEM).
[0323]Commercially available media, which are already prepared, may also be used.
[0324] When feeder cells are used for culturing ES cells, used as the feeder cells may be fibroblasts prepared from mouse embryos according to a conventional method or fibroblast-derived STO cell lines (Meiner, V. et al., Proc. Natl. Acad. Sci. U.S.A., 93: 14041-14046 (1996)). Commercially available products may also be used as the feeder cells. Feeder cells are desirably used for culturing ES cells after the proliferation of the feeder cells is stopped by treatment with mitomycin C.
[0325] Also, when the above feeder cells are not used for culturing ES cells, LIF
(Leukemia Inhibitory Factor) is added and then cells can be cultured. Examples of LIF include mouse recombinant LIF and rat recombinant LIF (e.g., Chemicon).
[0326] The days for culture under the above conditions for ES cell culture can be varied appropriately depending on the state of cells or the like, preferably ranging from about 1 day to 3 days.
[0327] When cells expressing a marker gene are detected (including a case of an increased gene expression level) compared with a case in which no test substance has been added, the test sample (or the substance to be tested) used is selected as the candidate of a substance capable of nuclear-reprogramming a somatic cell.
[0328] The above-described screening procedures can be repeated multiple times, where needed. For example, when a mixture, such as cDNA library or cell extract, is used in the first screening, the mixture is further subdivided (or fractionated), and then a similar screening is repeated on and after the second screening. Finally, a candidate for somatic cell nuclear reprogramming factor can be selected.
[0329] It can be confirmed that cells expressing a marker gene (obtained by addition of somatic cell nuclear reprogramming (candidate) substances selected by screening in the present invention) retain the functions of iPS cells by examining: (1) whether or not the cells express undifferentiated marker genes such as Oct3/4 and Nanog; (2) whether or not the cells are differentiated in vitro by stimulation with retinoic acid or the like; (3) whether or not teratomas are formed after transplantation thereof into nude mice; (4) whether or not chimeric mice are born as a result of injection into mouse blastocyts; (5) whether or not chimeric mice produce offsprings; and the like.
[0330](3) Method for screening for substance maintaining undifferentiation and pluripotency of pluripotent stem cell
[0331] The present invention provides a method for screening for a substance capable of maintaining the undifferentiation and pluripotency of pluripotent stem cells, comprising the following steps of:
[0332](a) bringing a pluripotent stem cell containing a DNA in which a marker gene is present
at a position wherein the expression of the marker gene is controlled by an ECATl 1 gene promoter, into contact with a test substance in a medium where the
[0333]undifferentiation and pluripotency of the pluripotent stem cells cannot be maintained; and
[0334] (b) detecting the expression of the marker gene following the step (a).
[0335] According to the system (or method) of the present invention, ES-like state can be easily monitored by expression of a marker gene having drug resistance or the like. For example, a test substance is added under culture conditions where pluripotent stem cells are unable to maintain the undifferentiation potency and pluripotency, and then the presence or the absence of cells expressing the marker gene is examined, so that a (candidate) substance maintaining the undifferentiation and pluripotency of the pluripotent stem cells can be easily screened for.
[0336] When pluripotent stem cells in which a marker gene is, present at a position wherein the expression of the marker gene is controlled by an ECATl 1 gene promoter are cultured in a medium where the undifferentiation potency and pluripotency cannot be maintained, the expression of the marker gene disappears or decreases. On the other hand, when a substance maintaining the undifferentiation and pluripotency of ES cells is present in the above medium, the expression of the marker gene is maintained. With the use of such properties, a (candidate) substance capable of maintaining the
[0337]undifferentiation and pluripotency of pluripotent stem cells can be easily screened for.
[0338] All of (i) the method for preparing pluripotent stem cells containing a DNA in which a marker gene is present at a position wherein the expression of the marker gene is controlled by an ECATl 1 gene promoter, (ii) the method for bringing a test substance into contact, (iii) the method for detection of the expression of a marker gene are as described in § 1 "Method for detection of iPS cell" above.
[0339] Examples of pluripotent stem cells used in the screening step (a) above include iPS cells and ES cells. Preferable examples thereof include mouse- or human-derived iPS cells and ES cells. The pluripotent stem cells may be any cells, as long as they are pluripotent stem cells containing a DNA in which a marker gene is present at a position
wherein the expression of the marker gene is controlled by an ECATl 1 gene promoter. Specific examples thereof include knock-in-mouse-derived ES cells as described in § 1- 1-a above, transgenic-mouse-derived ES cells as described in § 1-1 -b above, ES cells comprising a BAC vector or a PAC vector as described in § 1-2-a above, ES cells comprising a plasmid vector as described in § 1-2-b above, and ES cells comprising a targeting vector as described in § 1-2-c above.
[0340] The term "medium where the undifferentiation and pluripotency of ES cells cannot be maintained" as used herein (particularly, in the screening step (a) above) may be any medium, as long as it is a medium where the state of cells as iPS cells or ES cells cannot be maintained or a medium where the undifferentiation state cannot be maintained. For example, it is known that in the case of mouse ES cells, serum or feeder cells are essential for maintaining the undifferentiation and pluripotency of mouse ES cells at low density. Hence, culture conditions for the ES cells, from which "serum," "feeder cell," or both of them are excluded, can be employed, for example. Since feeder cells are essential for maintaining human ES cells (for maintaining the undifferentiation and pluripotency of the same), culture conditions for human ES cells, from which "feeder cell" is excluded, can be employed, for example.
[0341] Specifically, the culture conditions for ES cells as previously described (Meiner, V. L., et al., Proc. Natl. Acad. Sci. U.S.A., 93 (24): pl4041-14046 (1996)) from which "serum," "feeder cell," or both of them, are excluded, can be employed, for example.
[0342] Step (a) above is carried out by bringing pluripotent stem cells into contact with test substances in a medium where the undifferentiation and pluripotency of the pluripotent stem cells cannot be maintained. Test substances are brought into contact with the pluripotent stem cells before, during, or after the transfer of the pluripotent stem cells to a medium where undifferentiation and pluripotency cannot be maintained.
[0343] Examples of test substances (or test samples) used in the screening include, but are not limited to, nucleic acids, peptides, proteins, organic compounds, inorganic compounds, or mixtures thereof. The screening of the present invention is specifically
carried out by bringing these test substances into contact with pluripotent stem cells. Examples of the test substance include cell-secreted products, serum, cell extracts, gene (genomic or cDNA) libraries, RNAi libraries, nucleic acids (genomic DNA, cDNA, or mRNA), antisense nucleic acids, low molecular weight compounds, high molecular weight compounds, proteins, peptides, and natural compounds. Specific examples thereof include animal serum or fractions thereof, and feeder cell-secreted products or fractions thereof.
[0344] These test substances (or test samples) are brought into contact with somatic cells, in such a form that they can be incorporated into somatic cells. For example, when a test substance is a nucleic acid (e.g., cDNA library), it is introduced into somatic cells using calcium phosphate, DEAE-dextran, or lipid for gene transfer.
[0345] When a DNA containing a drug resistance gene is used as a marker gene, the selection is carried out in a medium (selective medium) containing the drug. The drug may be contained in such medium at the time of or after contact between pluripotent stem cells and test substances. Moreover, in the presence of test substances, the drug may be contained in a medium after culture in a medium where the undifferentiation and pluripotency of pluripotent stem cells cannot be maintained.
[0346] After step (a) above, the presence or the absence of cells expressing the marker gene is examined. When the presence of such cells expressing the marker gene is confirmed, the test sample (or test substance) used is selected as a candidate substance capable of maintaining the undifferentiation and pluripotency of pluripotent stem cells.
[0347] The above screening can be repeated multiple times, where needed. For example, when a mixture such as a feeder cell-secreted product or serum is used in the first screening, the mixture is further subdivided (or fractionated), and then a similar screening is repeated on and after the second screening. Finally, candidate substances capable of maintaining the undifferentiation and pluripotency of pluripotent stem cells can be selected.
[0348] Whether or not a (candidate) substance capable of maintaining the
[0349]undifferentiation and pluripotency of ES cells, which is selected by the screening of the
present invention, indeed maintains the undifferentiation and pluripotency of pluripotent stem cells can be confirmed by culturing iPS cells or ES cells under culture conditions where the candidate substance is added to a medium in which the undifferentiation and pluripotency of ES cells cannot be maintained and then examining various abilities of these cells. Specifically, this can be confirmed by examining: (1) whether or not iPS cells or ES cells cultured under the above culture conditions express undifferentiated marker genes such as Oct3/4 and Nanog; (2) whether or not the iPS cells or ES cells are differentiated in vitro by stimulation with retinoic acid or the like; (3) whether or not teratomas are formed after transplantation of the iPS cells or ES cells into nude mice; (4) whether or not chimeric mice are born as a result of injection into mouse blastocyts; (5) whether or not chimeric mice produce offsprings; and the like.
[0350](4) Method for detection of ES cell
[0351] The present invention provides a method for detection of an ES cell, comprising a step of detecting the expression of an endogenous ECATl 1 gene.
[0352] It has now been revealed that the expression of the ECATl 1 gene of the present invention is almost entirely limited to undifferentiated cells. Hence, the use of (1) a polynucleotide capable of specifically binding to the ECATl 1 gene or (2) an antibody that specifically recognizes ECATl 1 as a marker for detection of an ES cell enables detection of an ES cell. Specifically, an ES cell can be detected according to § 1-7 and § 1-8 above.
[0354] The present invention will next be described in detail by way of Examples, which should not be construed as limiting the scope of the present invention.
[0355]Example 1 Expression analysis of EC AT 1 1 gene
[0356] Expression analysis of the ECATl 1 gene was carried out by RT-PCR using Rever Tra Ace kit (Takara), as a reverse transcriptase, and PCR enzyme (Takara EX Taq
HS kit). The results are shown in Fig. 1. Specific expression of the gene was observed in undifferentiated ES cells (RF8) in mice. As a result of increasing the number of cycles, expression in the testes and ovaries was also confirmed, but no expression was observed in body tissues. Also, in humans, specific expression was observed in NCR-G3 (embryonic carcinoma) cells, which were neoplastic cells.
[0357]Hence, as a result of increasing the number of cycles, expression was slightly confirmed in the testes and placenta. As described above, the expression of the ECATl 1 gene was almost entirely limited to undifferentiated cells, so that the gene was confirmed to be an ECAT gene.
[0358]Example 2 Production of ECATl 1 homozygous deficient mouse
[0359] The coding region (from exon 3 to exon 5) of the ECATl 1 gene was substituted with a fusion gene cassette of enhanced GFP (EGFP) and a puromycin resistance gene, so as to knock-out the ECATl 1 gene. Simultaneously with this procedure, ECATl 1 homozygous deficient knock-in mice (ECATl 1EGFP/EGFP mice) were produced, for which the expression of the ECATl 1 gene can be monitored by excitation light of EGFP.
[0360]1) Construction of ECATl 1 targeting vector
[0361] A full length about 188-kbp BAC clone (RP24-326M13), containing ECATI l, was purchased from BACPAC. The BAC clone was altered using a Escherichia coli Red/ET recombinant enzyme to construct an ECATl 1 targeting vector. For negative selection, a DTA gene encoding a diphtheria toxin A chain was incorporated into an ECATl 1 upstream region. Recombination procedures were carried out according to RED/ET RECOMBINATION protocols (GENE BRIDGES). The construction of the targeting vector is shown in Fig. 2.
[0362] Specifically, the following two regions were subjected to recombination using Red/ET.
(I) ECATI l coding region
[0363] A region in the ECATl 1 exon 3 ranging from the initiation codon to exon 5 was substituted with an EGFP-IRES-Puro-FRT-Hyg (EGFP-IP -FRT-Hyg) cassette.
[0364]First, 50-bp homologous recombination regions were set upstream of the initiation codon and downstream of EX0N5, respectively. Next, 20-bp primer regions were designed for amplification of a cassette portion from pB S-GIP -PHF-NBl (Okita et al., Nature, 448: 313-317 (2007)) and then connected to the upstream homologous recombination region and the downstream homologous recombination region,
[0365]respectively. Primer sequences collectively with a total length of 70 bp (sense primer: TTC TGA CGC TCT CGC CAG GAA AAG GTG CGC TTT GTG TCA GTG TAT CCA CC TCG CCA CCA TGG TGA GCA AG (SEQ ID NO: 5), antisense primer: AGG TTC TCT GGA AAA TAA GCA GGG ACA TTA AGT AAA CCA ATG ACA AAT GC AGT TTA TGG CGG GCG TCC TG (SEQ ID NO: 6)) were prepared. An
[0366]approximately 4.3-kbp fragment comprising an EGFP-IP-FRT-Hyg cassette having ECATl 1 upstream and downstream homologous regions was prepared using the primers.
[0367]The product was introduced into DHlOβ containing BAC according to the above protocols for recombination. Screening was carried out to identify BAC clones in which recombination had taken place. Subsequently, the clones were introduced into Escherichia coli 294Flpe strain expressing Flpe, so that hygromycin gene for selection was subjected to the flip-out technique. The intermediate product was designated mod2RP24-326M13.
[0368](2) ECAT l 1 upstream region
[0369] Next, mod2RP24-326M13 was subjected to a procedure of inserting a DTA cassette to an approximately 8.7-kbp site upstream of the ECATl lgene. Specifically, the procedures for designing homologous portions and for recombination were carried out in a manner similar to those in (1) above (sense primer: GAT GTG TGC CAC CGT GCC CAG CAA CCC ACT CTT CTA TTA ATT AAT TAA CT AGC GCG CAA TTA ACC CTC AC (SEQ ID NO: 7); antisense primer: GGT GCA CAC ACA TAT GTG
CAG GTA AAT ACA TAT TTA ATT AAT TAA CTA GT GCG CGC GTA ATA CGA CTC AC (SEQ ID NO: 8)). The cassette portion was amplified using a tandem sequence of PGK-Hyg-DTA.
[0370]2) Production of ECATl 1 homozygous deficient mouse
[0371] The ECATI l targeting vector constructed in 1) above was digested overnight with BspTI for linearization, and then the linearized product was introduced by electroporation into RF8ES cell line. By selection in a medium supplemented with puromycin, clone candidates, in which homologous recombination had taken place, were narrowed down. Furthermore, it was confirmed by PCR and Southern blotting that homologous recombination had taken place correctly. It was also confirmed that no random integration had taken place. By injecting the homologous recombinant ES cells into the blastocysts of mice (C57BL/6), chimeric mice were obtained, from which ECAT 11 heterozygous mutant mice (ECAT 11EGFP/WT mice) were established. Wild- type, ECATl 1 heterozygous mutant, and ECATl 1 homozygous mutant (ECATl iEGFP/EGFP mice) mice were produced by crossing ECATl 1 heterozygous mutant mice. All of these mice were born in accordance with the Mendelian inheritance.
[0372]Example 3 Establishment of ECATl 1 deficient ES cell from mouse blastocyst and ability evaluation, and expression profiling for ES
[0373] Delayed blastocysts were collected after crossing of ECAT I l heterozygous mutant mice. Wild-type, ECATI l heterozygous mutant, and ECATI l homozygous mutant (deficient) ES cells were established. ES cells were established in a manner similar (resulting in almost no differences) to the establishment of blastocysts of any genotype.
[0374] The thus obtained ECATI l deficient ES cells (ECAT 11EGFP/EGFP ES cells) had proliferation potency (Fig. 3) and differentiation potency based on teratoma formation (Fig. 4) equivalent to those of wild-type ES cells. Hence, it was determined that the ECATI l deficient ES cells had no abnormalities in terms of functions generally
considered to be characteristics of ES cells. It was thus revealed that ECATl 1 had no important functions in maintenance of the undifferentiation and pluripotency of ES cells.
[0375] Also, alternate follow-up was carried out for ECATl 1 by EGFP expression. Expression took place continuously from the blastocystic stage to the ES cell stage. However, when the induction of differentiation was carried out under conditions involving addition of retinoic acid or conditions involving removal of LIF, expression disappeared (Fig. 5). Hence, it was demonstrated that ECATl 1 expression takes place uniquely in an undifferentiated state.
[0376]Example 4 Confirmation of ECATl 1 localization in mouse embryo and confirmation of lack of abnormalities in mouse
[0377] Wild-type, ECATI l heterozygous mutant, and ECATI l homozygous mutant embryos were collected after crossing of ECATI l heterozygous mutant mice.
[0378]Expression from E6.5 to E 17.5 was observed. In the case of ECATl 1 heterozygous mutant and homozygous mutant embryos, expression took place throughout the embryos until E8.5, but disappeared once from around ElO to around El 1.5. However, expression was observed again in limbs, mandibles, and spinal borders on and after E12.5.
[0379] Also, individual mice obtained by crossing ECATl 1 heterozygous mutant mice or ECATI l homozygous mutant mice were raised and observed. However, no abnormalities were found in appearance, behavior, or fertility. Small mice were observed infrequently, but they accounted for 5% or less of the mice obtained by crossing.
[0380]Example 5 Establishment of MEFs from ECATl 1 deficient mouse
[0381] On day 14.5 after crossing of ECATI l deficient mice, fetal mice were collected. After transaction of heads, all the internal organs under the pharynges were removed. The resulting parts were divided into limbs and body, and they were each fragmented into small portions using an ophthalmic knife. The fragments were treated
with trypsin, filtered, seeded on 100-mm dishes, and then cultured in 10% FBS/DMEM. Limbs-derived and body-derived mouse embryonic fibroblasts (MEFs) established on about day 4 after the initiation of culture were collected and then cryopreserved.
[0382]Example 6 Establishment of iPS cell using ECATl 1 deficient MEFs
[0383] Limbs-derived ECATI l deficient MEFs, body-derived ECATI l deficient MEFs, and control Nanog-EGFP heterozygous MEFs (Okita et al., Nature, 448:313- 317(2007)) were seeded at 1.0 x 105 cells per well on gelatin-coated 6-well dishes and then culture was initiated using 10% DMEM. On the next day, 4 genes (Oct3/4, Klf4, Sox2, and c-Myc) from mice or 3 genes (Oct3/4, Klf4, and Sox2) from mice were introduced using a retrovirus. Cells were collected 4 days after viral infection and then reseeded on MSTO cells at 3 stages of concentration (5 x 102, 5 x 103, and 5 x 104 cells/6-well dish). On the day following reseeding, culture was initiated in a medium for ES cells (DMEM (Nacarai tesque) supplemented with LIF, which had been prepared by adding 15% fetal calf serum, 2 mM L-glutamine (Invitrogen), 100 μM nonessential amino acid (Invitrogen), 100 μM 2-mercaptoethanol (Invitrogen), 50 U/mL penicillin (Invitrogen), and 50 mg/mL streptomycin (Invitrogen)). On day 3 after reseeding (7 days after infection), drug selection was initiated in a medium prepared by adding 2 μg/ml puromycin to a medium for ES cells.
[0384] On and after day 6 after infection, the excitation light from EGFP knocked-in at the ECATl 1 locus could be confirmed in limbs-derived ECATl 1 deficient MEFs for which iPS induction had been performed using 4 genes (Fig. 6). However, EGFP excitation light was not confirmed on day 6 after infection, but it was confirmed on and after day 10 after infection in Nanog-EGFP heterozygous MEFs for which iPS induction had been carried out using 4 genes (Fig. 6). As described above, it was revealed that reprogramming could be detected at an earlier stage by selection using ECATl 1 expression (ECATl 1 -EGFP) as an indicator than in the case where the selection had been carried out using Nanog expression (Nanog-EGFP) as an indicator.
[0385] Next, after retroviral infection, the number of colonies of mouse ES-like cells
(into which the 4 aforementioned genes had been introduced) that had appeared by day 18, as well as the number of colonies of mouse ES-like cells (into which the 3 aforementioned genes had been introduced) that had appeared by day 21, were counted. The results are shown in Fig. 7. The numbers of colonies were equivalent to the results obtained by conventionally employed Nanog-EGFP selection. Instead, as described above, the reprogramming could be detected at an earlier stage than in the case in which Nanog-EGFP had been used as an indicator. Hence, greater numbers of iPS-like colonies could be detected.
[0386] Culture of the collected colonies was continued. Nanog-EGFP selection was carried out for cells at passage 4, and ECATl 1-EGFP selection was carried out for cells at passage 3, in order to examine the number and morphology of ES-like cells. The results are shown in Fig. 8. Greater numbers of iPS-like colonies could be detected as a result of ECATl 1-EGFP selection compared with Nanog-EGFP selection. From morphological observations, ES-like morphology similar to that in cells subjected to Nanog-EGFP selection was observed. Hence, it could be confirmed that cells selected using ECATl 1 expression as an indicator were iPS cells.
