Method for inducing immature oocytes and method for producing mature oocytes

Abstract

To provide simple methods for inducing immature oocytes from cells such as pluripotent stem cells capable of differentiating to oocytes in shorter culture period of time compared with conventional methods.SOLUTION: A method for inducing immature oocytes comprises introducing four genes consisting of FIGLA, NOBOX, LHX8 and TBPL2, or transcripts or expressed proteins thereof, into at least one type of cell selected from the group consisting of pluripotent stem cells, epiblast-like cells and primordial germ cells.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for inducing immature oocytes and a method for producing mature oocytes.

Background Art

[0002] In mammals, totipotency, defined as the ability of individual development from a single cell, is possessed by single cells as a special property. However, the mechanism for forming such totipotency has been facing difficulties even in fields with strong social demands such as infertility treatment. The reason is that the oogenesis process that progresses in the fetal ovary cannot be reproduced in vitro.

[0003] The inventors have previously developed an in vitro culture system for reconstructing oocytes from mouse pluripotent stem cells Specifically, mouse ES cells or iPS cells are induced to differentiate into primordial germ cell-like cells (PGCLCs) using a medium containing a humoral factor such as BMP4, and the obtained PGCL Cs are mixed with ovarian somatic cells to produce a reconstructed ovary. Next, in the reconstructed ovary, the period from P GCLCs to the formation of metaphase II oocytes is divided into three periods: the "in vitro differentiation period", the "in vitro growth period", and the "in vitro maturation period", and the optimal culture conditions for obtaining secondary follicles, oocyte nucleus stage oocytes, and metaphase II oocytes are established during each culture period (see, for example, Non-Patent Document 1, etc.).

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] However, in the method described in Non-Patent Document 1, it takes a period of about 3 to 4 weeks to obtain oocytes of primordial follicles from mouse pluripotent stem cells, and in primates and other large mammals, it is expected that more than 1 year of culture is required to produce oocytes from pluripotent stem cells in an in vitro culture system. In view of the above circumstances, the present invention has been made, and provides a method for simply inducing immature oocytes from cells having the ability to differentiate into oocytes, such as pluripotent stem cells, by culturing for a shorter period than before. Further, a method for simply producing mature oocytes from cells having the ability to differentiate into oocytes, such as pluripotent stem cells, by culturing for a shorter period than before is provided.

[0006]

Means for Solving the Problems

[0007] As a result of intensive studies to achieve the above object, the inventors have found that by introducing specific genes involved in the formation of oocytes into pluripotent stem cells and culturing them for a short period of about 5 to 10 days, it is possible to induce differentiation into immature oocytes, and thus have completed the present invention.

[0008] That is, the present invention includes the following aspects. The method for inducing immature oocytes according to the first aspect of the present invention is FIGLA, NOBOX, LH Four types of genes consisting of X8 and TBPL2, or their transcripts or expressed proteins are introduced into at least one cell selected from the group consisting of pluripotent stem cells, epiblast-like cells, and primordial germ cells. It includes introducing. The method for inducing immature oocytes according to the first aspect may include introducing four types of genes consisting of FIGLA, NOBOX, LHX8, and TBPL2 into the cells. The method for inducing immature oocytes according to the first aspect may further include introducing the gene of STAT3, or its transcript or expressed protein into the cells. The method for inducing immature oocytes according to the first aspect may further include introducing one or more genes selected from the group consisting of SOHLH1, SUB1, and DYNLL1, or their transcripts or expressed proteins into the cells. The method for inducing immature oocytes according to the first aspect may further include introducing three types of genes consisting of SOHLH1, SUB1, and DYNLL1 into the cells. The cells may be pluripotent stem cells. In the method for inducing immature oocytes according to the first aspect, the expression of the gene is controlled to be induced by the presence of an expression-inducing substance. After the introduction, the cells are proliferated. After the proliferation, the expression-inducing substance is added to the medium to induce the expression of the gene. The method for producing mature oocytes according to the second aspect of the present invention is Four types of genes consisting of FIGLA, NOBOX, LHX8, and TBPL2, or their transcripts or expressed proteins are selected from the group consisting of pluripotent stem cells and primordial germ cells. It includes introducing into at least one cell selected. The method for producing mature oocytes according to the second aspect of the present invention may include introducing four types of genes consisting of FIGLA, NOBOX, LHX8, and TBPL2 into the cells. The method for producing mature oocytes according to the second aspect of the present invention may further include introducing the gene of STAT3, or its transcript or expressed protein into the cells. The method for producing mature oocytes according to the second aspect of the present invention may further include introducing one or more genes selected from the group consisting of SOHLH1, SUB1, and DYNLL1, or their transcripts or expressed proteins into the cells. The method for producing mature oocytes according to the second aspect of the present invention may further include introducing three types of genes consisting of SOHLH1, SUB1, and DYNLL1 into the cells. The cells may be pluripotent stem cells.

[0009] In the method for producing mature oocytes according to the second aspect of the present invention, the expression of the gene is controlled to be induced by the presence of an expression-inducing substance. After the introduction, the cells are proliferated. After the proliferation, the expression-inducing substance is added to the medium to induce the expression of the gene. introducing it into at least one type of cell that can be introduced; co-culturing the cells after introduction and ovarian somatic cells; and includes The method for producing a mature oocyte according to the second aspect may further include introducing the gene of STAT3, or its transcript or the expressed protein into the cells.

Advantages of the Invention

[0010] According to the method for inducing immature oocytes of the above aspect, immature oocytes can be easily induced from cells having the ability to differentiate into oocytes such as pluripotent stem cells in a shorter culture period than before. According to the method for producing mature oocytes of the above aspect, a large number of mature oocytes can be easily produced from cells having the ability to differentiate into oocytes such as pluripotent stem cells in a shorter culture period than before. According to the method for producing mature oocytes of the above aspect, a large number of mature oocytes can be easily produced from cells having the ability to differentiate into oocytes such as pluripotent stem cells in a shorter culture period than before. According to the method for producing mature oocytes of the above aspect, a large number of mature oocytes can be easily produced from cells having the ability to differentiate into oocytes such as pluripotent stem cells in a shorter culture period than before. It can be done.

Brief Description of the Drawings

[0011]

Figure 1

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[0012] <Method for inducing immature oocytes> In one embodiment, the present invention provides a method for the detection of HIV-1-associated ... The four genes, or their transcripts or expressed proteins, were used to infect pluripotent stem cells, embryonic stem cells, and At least one cell selected from the group consisting of piblast-like cells and primordial germ cells (hereinafter referred to as These cells are sometimes collectively referred to as "cells capable of differentiating into oocytes" The present invention provides a method for deriving immature oocytes, comprising introducing a fertilized egg into the oocyte.

[0013] In the case of mice, the conventional method involves in vitro generation of immature oocytes from pluripotent stem cells. It takes a little less than a month to induce differentiation into the progenitor cells in vitro. It took about 11 days to induce differentiation from germ cells to immature oocytes. In contrast, in the method for inducing immature oocytes of this embodiment, differentiation of pluripotent stem cells or the like into oocytes is performed. The above four types of genes are introduced into cells with differentiation potential, and the cells are then cultured for a short period of time, typically between 5 and 10 days. By culturing, they can be induced to differentiate into immature oocytes. In the body, it takes more than nine months to induce differentiation from primordial germ cells to immature oocytes. However, by using the method for inducing immature oocytes according to the present embodiment, this period can be dramatically shortened. It is possible.

[0014] In addition, in the conventional method, pluripotent stem cells are induced to differentiate into PGCLCs, and the PGCLCs To induce further differentiation into immature oocytes, at least two steps with different culture conditions were performed. In contrast, the method for inducing immature oocytes of the present embodiment requires multiple steps. Potential stem cells can be directly induced to differentiate into immature oocytes.

[0015] In this specification, the term "immature oocyte" refers to a primary oocyte that has not undergone follicular growth. The immature oocyte does not necessarily have a follicular structure. In addition, as shown in the examples below, in the case of immature oocytes, it is not necessary to use an oocyte cell structure. Some maternal effect genes, such as the Stella gene and Padi6 gene, which are cell markers, are expressed. It is manifested. In addition, in this specification, the term "follicle" refers to a cell that contains an oocyte and the somatic cells (granulosa cells) surrounding it. It consists of a cytoplasmic membrane (cytoplasmic membrane and capsule cells). The method for inducing immature oocytes according to this embodiment will be described in detail below.

[0016] [Oocyte formation genes] Oocyte formation used to introduce cells capable of differentiating into oocytes, such as pluripotent stem cells The gene is a gene that the inventors have previously used to study the expression dynamics of genes in the oocyte lineage using RNA sequencing. These genes were identified through RNA-Seq analysis. Specific examples of such children include FIGLA, NOBOX, SOHLH1, LHX8, and SU. B1, STAT3, TBPL2, DYNLL1, etc. As shown in the Examples below, Four genes, namely FIGLA, NOBOX, LHX8, and TBPL2, are particularly important for inducing pluripotent stem cells into immature oocytes. Therefore, among the above-mentioned oocyte formation genes at least four types of genes consisting of FIGLA, NOBOX, LHX8, and TBPL2, or their transcripts or expressed proteins are introduced into cells, so that cells with the ability to differentiate into oocytes can be induced into immature oocytes.

[0017] In addition to the above four types of genes or their transcripts or expressed proteins, it is preferred to further introduce the STAT3 gene, or its transcript or expressed protein. More preferably, the STAT3 gene is further introduced. As shown in the examples described later, in addition to FIGLA, NOBOX, LHX8, and TBPL2, introducing STAT3 into cells can further improve the formation rate of oocytes.

[0018] In addition to the five genes of FIGLA, NOBOX, LHX8, TBPL2, and STAT3, or their transcripts or expressed proteins, it is further preferred to introduce one or more genes selected from the group consisting of SOHLH1, SUB1, and DYNLL1, or their transcripts or expressed proteins. More preferably, it is further preferred to introduce three genes consisting of SOHLH1, SUB1, and DYNLL1, or their transcripts or expressed proteins. As shown in the examples described later, by introducing the above eight types of genes, or their transcripts or expressed proteins into cells, cells with the ability to differentiate into oocytes can be more efficiently induced into immature oocytes.

[0019] The FIGLA gene regulates multiple oocyte-specific genes (including genes involved in folliculogenesis and genes encoding zona pellucida (ZP1, ZP2, and ZP3)) and encodes a basic helix-loop-helix (bHLH) transcription factor. The transcription factor FIG GLA binds to the E-box (5’-CAN NTG-3’) of the ZP (ZP1, ZP2, and ZP3) promoter. Diseases associated with FIGLA include, for example, premature ovarian insufficiency type 6), pseudohermaphroditism, etc. The gene ontology (GO) annotation of FIGLA includes sequence-specific DNA binding and protein dimerization activities. An important paralog of FIG LA is SCX. FIGLA is also known as Folliculogenes is Specific BHLH Transcription Factor, Fa ctor In The Germline Alpha, Folliculogene sis-Specific Basic Helix-Loop-Helix Prot ein, Transcription Factor FIGa, BHLHC8 (BHL Hc8), Folliculogenesis Specific Basic Hel ix-Loop-Helix, FIGALPHA (FIGalpha), POF6.

[0020] Information on the nucleotide sequence of oocyte formation genes such as the FIGLA gene, the nucleotide sequence of the mRNA of the gene , and the amino acid sequence of the protein encoded by the gene can be obtained from databases such as Genbank .

[0021] The nucleotide sequence of the human FIGLA gene is, for example, available in Genbank under "Gene ID: 3" ​It is disclosed as "44018". The nucleotide sequence of the mRNA of the human FIGLA gene is, for example, disclosed as the accession number NM_001004311 in Genbank. The amino acid sequence of human FIGLA is disclosed as the accession number NP_00 1004311 in Genbank.

[0022] The nucleotide sequence of the mouse FIGLA gene is disclosed, for example, as "Gene ID: 26910" in Genbank. The nucleotide sequence of the mRNA of the mouse FIGLA gene is disclosed, for example, as the accession number NM_012013 in Genbank. The amino acid sequence of mouse FIGLA is disclosed as the accession number NP_036 143 in Genbank.

[0023] The NOBOX gene encodes a transcription factor involved in oogenesis. Diseases associated with NOBOX include, for example, premature ovarian insufficiency (type 5), etc. The GO annotation of NOBOX includes DNA-binding transcription factor activity and DNA-binding specific to the promoter sequence near RNA polymerase II. Specifically, it preferably binds to nucleotide sequences such as "5'-TAATTG-3'", "5'-TAGTTG-3'", and "5'-TAATTA-3'". An important paralog of NOBOX is UNCX. NOBOX is also known as NOBOX Oogenesis Homeobox, Homeobox Protein N OBOX, Newborn Ovary Homeobox-Encoding Gen e, Newborn Ovary Homeobox-Encoding, TCAG_1 2042, OG-2 (OG2), OG2X, POF5. OBOX, Newborn Ovary Homeobox-Encoding Gen e, Newborn Ovary Homeobox-Encoding, TCAG_1 2042, OG-2 (OG2), OG2X, POF5.

[0024] The nucleotide sequence of the human NOBOX gene is disclosed, for example, as "Gene ID: 1 35935" in Genbank. The nucleotide sequence of the mRNA of the human NOBOX gene is, for example, disclosed as accession numbers NM_001080413 and XM_0011 34420 in Genbank. The amino acid sequence of human NOBOX is disclosed as accession numbers NP_001073882 and XP_001134420 in Genbank.

[0025] The nucleotide sequence of the mouse NOBOX gene is disclosed, for example, as "Gene ID: 18291" in Genbank. The nucleotide sequence of the mRNA of the mouse NOBOX gene is disclosed, for example, as accession number NM_130869 in Genbank. . The amino acid sequence of mouse NOBOX is disclosed as accession number NP_570 939 in Genbank.

[0026] The SOHLH1 gene is one of the gonad-specific transcription factors essential for spermatogenesis, oogenesis, and folliculogenesis, and encodes a basic helix-loop-helix (bHLH) transcription factor. SOHLH1 plays a role in regulating the differentiation of oocytes without affecting the first meiosis. Diseases associated with SOHLH1 include, for example, non-obstructive azoospermia, ovarian dysgenesis, etc. The GO annotation of SOHLH1 includes DNA-binding transcription factor activity and protein dimerization activity. An important paralog of the SOHLH1 gene is S ​Genesis Specific Basic Helix-Loop-Helix 1 、Spermatogenesis-And Oogenesis-Specific Basic Helix-Loop-Helix-Containing Protei n 1、Spermatogenesis Associated 27、C9orf1 57、NOHLH、TEB2、Chromosome 9 Open Reading Frame 157、Newborn Ovary Helix Loop Helix 、BA100C15.3、SPATA27、BHLHe80、SPGF32、ODG5 and is also called.

[0027] The nucleotide sequence of the human SOHLH1 gene is disclosed, for example, as "Gene ID: 402381" in Genbank. The nucleotide sequence of the mRNA of the human SOHLH1 gene is disclosed, for example, as Genbank accession numbers NM_001012415, XM_49 7082. The amino acid sequence of human SOHLH1 is disclosed as Genbank ac cession numbers NP_001012415, XP_497082 .

[0028] The nucleotide sequence of the mouse SOHLH1 gene is disclosed, for example, as "Gene ID :227631" in Genbank, and the nucleotide sequence of the mRNA of the mouse SOHLH1 gene is disclosed, for example, as Genbank accession numbers NM_001001714, XM_ 130180, and the amino acid sequence of mouse SOHLH1 is disclosed as Genban k accession numbers NP_001001714, XP_130180 .

[0029] LHX8 is a member of the LIM homeobox family of proteins and is involved in pattern formation and differentiation of various tissues. LIM homeobox family proteins contain, in addition to a DNA-binding homeodomain, two tandemly repeated cysteine-rich double zinc finger motifs known as LIM domains. LIM homeobox family proteins are transcription factors involved in tooth morphogenesis, oogenesis, and neuronal differentiation. Diseases associated with the LHX8 gene include, for example, cleft palate, odontoma, etc. LHX8 is also known as LIM Homeobox 8, LIM / Home obox Protein Lhx8, LIM-Homeodomain Protein Lhx8, LIM Homeobox Protein 8, LHX7.

[0030] The nucleotide sequence of the human LHX8 gene is disclosed, for example, as "Gene ID:43 1707" in Genbank. The nucleotide sequence of the mRNA of the human LHX8 gene is, for example , Genbank accession numbers NM_001001933, XM_086344 , NM_001256114, XM_017001316, XM_017001317 and is disclosed as such. The amino acid sequence of human LHX8 is Genbank accession numbers NP_001001933, XP_086344, NP_001243043, XP_ 016856805, XP_016856806 and is disclosed as such.

[0031] The nucleotide sequence of the mouse LHX8 gene is disclosed, for example, as "Gene ID:1 is disclosed as "6875". The nucleotide sequence of the mouse LHX8 gene mRNA is, for example, Genbank accession numbers NM_010713, XM_00650107 2, XM_017319470. The amino acid sequence of mouse LHX8 is Genbank accession numbers NP_034843, XP_006501135, XP_017174959.

[0032] The SUB1 gene is a gene encoding a transcriptional regulator. SUB1 functions in cooperation with TAF and acts as a coactivator that mediates the functional interaction between upstream activators and general transcriptional functions. Diseases associated with SUB1 include, for example, onychomycosis and the like. The GO annotation of SUB1 includes single-stranded DNA binding. SUB1 is also called SUB1 Homolog, Transcriptional Regula tor, Positive Cofactor 4, Activated RNA Po lymerase II Transcriptional Coactivator P15, PC4, P14, Activated RNA Polymerase II Transcription Cofactor 4, RPO2TC1, P15.

[0033] The nucleotide sequence of the human SUB1 gene is disclosed, for example, as "Gene ID:10 923" in Genbank. The nucleotide sequence of the human SUB1 gene mRNA is, for example, Genbank accession numbers NM_006713, XM_017008986, XM_017008987, XM_011513944. Human SU The amino acid sequence of B1 is disclosed as Genbank accession number NP_006704, XP_ 016864475, XP_016864476, XP_011512246. It has been disclosed.

[0034] The nucleotide sequence of the mouse SUB1 gene is disclosed, for example, as "Gene ID: 2 0024" in Genbank. The nucleotide sequence of the mRNA of the mouse SUB1 gene is, for example, Genbank accession numbers NM_011294, XM_00652004 2. The amino acid sequence of mouse SUB1 is disclosed as Genbank accession number NP_035424, XP_006520105.

[0035] STAT3 is a member of the STAT protein family. The STAT protein family responds to cytokines and growth factors such as interferon (IFN), epidermal growth factor (EGF), interleukin 5 (IL5), interleukin 6 (IL6), hepatocyte growth factor (HGF), leukemia inhibitory factor (LIF), bone morphogenetic protein 2 (BMP2), etc., is phosphorylated by receptor-associated kinases, and then forms homo- or heterodimers, translocates to the cell nucleus where they act as transcription factors, and play important roles in many cellular processes such as cell proliferation and apoptosis. Diseases associated with STAT3 include , for example, early-onset multi-organ autoimmune diseases, autosomal dominant hyper IgE syndrome, etc. The GO annotation of STAT3 includes DNA-binding transcription factor activity and sequence-specific DNA binding. An important paralog of the STAT3 gene is STAT1. ST AT3 is also known as Signal Transducer And Activator Of Transcription3, Acute-Phase Response Fact or, APRF, Signal Transducer And Activator Of Transcription 3, DNA-Binding Protein A PRF, ADMIO1, ADMIO, and HIES.

[0036] The nucleotide sequence of the human STAT3 gene is disclosed, for example, as "Gene ID:6 774" in Genbank. The nucleotide sequence of the mRNA of the human STAT3 gene is, for example , Genbank accession numbers NM_001369512, NM_001369 513, NM_001369514, NM_001369516, NM_0013695 17, NM_001369518, NM_001369519, NM_00136952 0, NM_003150, NM_139276, NM_213662, XM_01702 4973, XM_011525146, XM_011525145, XM_017024 972, XM_005257617, XM_005257616, XM_0170249 75, XM_024450896, XM_017024974, XM_01702497 6. The amino acid sequence of human STAT3 is Genbank accession number NP_001356441, NP_001356442, NP_0013564 43, NP_001356445, NP_001356446, NP_00135644 7, NP_001356448, NP_001356449, NP_003141, NP _644805, NP_998827, XP_016880462, XP_011523 448, XP_011523447, XP_016880461, XP_0052576 74, XP_005257673, XP_016880464, XP_02430666 4, XP_016880463, XP_016880465 are disclosed as such.

[0037] The nucleotide sequence of the mouse STAT3 gene is disclosed, for example, as "Gene ID: 20848" in Genbank. The nucleotide sequence of the mRNA of the mouse STAT3 gene is disclosed, for example, as accession numbers NM_011486, NM_213659 , NM_213660, XM_011248846, XM_017314401 in Genbank. The amino acid sequence of mouse STAT3 is accession number in Genbank NP_035616, NP_998824, NP_998825, XP_0112471 48, XP_017169890 are disclosed as such.

[0038] The TBPL2 gene is a gene encoding TATA box-binding protein-like 2. TBPL2 is a transcription factor that forms a complex with TAF3 to induce the differentiation of myoblasts into muscle cells. The complex replaces TFIID at specific promoters in the early stages of differentiation. Examples of diseases associated with TBPL2 include, for example, retinitis pigmentosa, etc. The GO annotation of TBPL2 includes DNA-binding transcription factor activity. The important paralog of the TBPL2 gene is TBP. TBPL2 is TATA-Box B inding Protein Like 2, TATA Box-Binding P inding Protein Like 2, TATA Box-Binding P ​Protein-Related Factor 3, TATA Box-Binding Protein-Like Protein 2, TBP-Related Fact or 3, TBP-Like Protein 2, TBP2, also known as TRF3.

[0039] The nucleotide sequence of the human TBPL2 gene is disclosed, for example, as "Gene ID:3 87332" in Genbank. The nucleotide sequence of the mRNA of the human TBPL2 gene is, for example, disclosed as accession number NM_199047 in Genbank. The amino acid sequence of human TBPL2 is disclosed as accession number NP_95024 8 in Genbank.

[0040] The nucleotide sequence of the mouse TBPL2 gene is disclosed, for example, as "Gene ID: 227606" in Genbank. The nucleotide sequence of the mRNA of the mouse TBPL2 gene is , for example, disclosed as accession numbers NM_001289689, NM_19 9059 in Genbank. The amino acid sequence of mouse TBPL2 is disclosed as accession numbers NP_001276618, NP_951014 in Genbank .

[0041] The DYNLL1 gene encodes a protein classified as a light chain among the proteins that make up cytoplasmic dynein, an enzyme complex with a molecular weight of approximately 1200 kDa. Examples of diseases associated with DYNLL1 include, for example, chronic intestinal venous insufficiency. The GO annotation of DYNLL1 includes protein homodimerization activity and protein domain specific binding. An important paralog of the DYNLL1 gene is DYNLL2. ​ DYNLL1 is Dynein Light Chain LC8-Type 1, Pr otein Inhibitor Of Neuronal Nitric Oxide Synthase, Dynein, Cytoplasmic, Light Pol ypeptide 1, Dynein Light Chain 1, Cytopla smic, 8 KDa Dynein Light Chain, DNCLC1, DNC L1, DLC1, DLC8, PIN, Cytoplasmic Dynein Ligh t Polypeptide, also known as Hdlc1 (HDLC1), LC8a, and LC8 do.

[0042] The nucleotide sequence of the human DYNLL1 gene can be found, for example, in Genbank under "Gene ID: The nucleotide sequence of the mRNA of the human DYNLL1 gene is disclosed as, for example, For example, Genbank accession numbers NM_001037494, NM_0010 The amino acid sequence of human DYNLL1 is disclosed as 37495 and NM_003746. Columns indicate Genbank accession numbers NP_001032583, NP_00103 2584, and is disclosed as NP_003737.

[0043] The nucleotide sequence of the mouse DYNLL1 gene can be found, for example, in Genbank under "Gene ID :56455". The nucleotide sequence of the mRNA of the mouse DYNLL1 gene is disclosed, for example, as Genbank accession number NM_019682. The amino acid sequence of mouse DYNLL1 is available in Genbank under the accession number NP_ It is disclosed as 062656.

[0044] [Cells capable of differentiating into oocytes] Cells capable of differentiating into oocytes used in the method for inducing immature oocytes of this embodiment These consist of pluripotent stem cells, epiblast-like cells (EpiLCs), and primordial germ cells. It is preferable that the cell is at least one type of cell selected from the group consisting of:

[0045] (pluripotent stem cells) As used herein, "pluripotent stem cells" refer to "autologous stem cells" that can proliferate while maintaining an undifferentiated state. It refers to undifferentiated cells that have the ability to self-renew and the ability to differentiate into all three germ layers. Pluripotent stem cells include, but are not limited to, induced pluripotent stem cells (iP S cells), embryonic stem cells (ES cells), embryonic germ cells (EG cells) derived from primordial germ cells , which are isolated during the establishment and culture of GS (Germline Stem) cells from testicular tissue. Multipotent GS cells (mGS cells), Mu isolated from bone marrow mesenchymal cells ES cells are derived from somatic cells by nuclear reprogramming. The pluripotent stem cells listed above can be obtained by known methods. Cut.

[0046] As used herein, "iPS cells" refers to cells that have been differentiated by introducing several genes into the somatic cells. This refers to cells that can be reprogrammed into cells of various tissues and organs by The iPS cells used in the method for inducing immature oocytes of this embodiment are obtained from an appropriate donor. The cells may be derived from primary cultured somatic cells collected from iPS cells can be induced to differentiate into any germ cell lineage. In principle, the somatic cells used for preparation can be derived from either ectodermal or endodermal germ cells. Cells from skin, hair, gums, blood, etc., which are less invasive and easier to collect, can be used in i These cells are suitable as somatic cells for use in preparing PS cells. This may be done according to a method known in the art. tion of germline-competent induced pluripotent stem cells.”, Nature, Vol. 448, p313-317, 2007.” (Reference 1), “Hamanaka S. et al., “Generation of germline- competent rat induced pluripotent stem cells.”, PLoS One, Vol. 6, Issue 7, e220 08, 2011. (Reference 2) and other known preparation methods can be used.

[0047] The ES cells used in the method for inducing immature oocytes of this embodiment can be obtained by a known method. For example, an inner cell mass can be collected from the blastocyst of a fertilized egg of a target animal, and the inner cell mass can be It can be established by culturing cell clusters on feeder cells derived from fibroblasts. In addition, early embryos created by nuclear transfer of somatic cell nuclei can be cultured. ES cells established by the method described below can also be used. ES cells were cultured without feeder cells in a 2i (2 inhibitor; PD032 5901 and CHIR99021), and LIF (Leukemia Inhibitor It can be maintained in serum-free medium supplemented with serum factor (Reference 3 : “Ying QL et al., ‘The ground state of embryonic stem cell self-renewal.’, N ature, Vol. 453, No. 7194, p519-523, 2008.”

[0048] (epiblast-like cells) In addition, in this specification, “epiblast-like cells (EpiLCs)” are pluripotent stem cells (for example, iPS cells, ES cells, etc.) differentiated under specific culture conditions, and have characteristics similar to those of the epiblast (the tissue that differentiates into primordial germ cells in vivo). The method for inducing the differentiation of EpiLCs from pluripotent stem cells (iPS cells or ES cells) can be carried out by referring to known methods, for example, Japanese Patent Application Laid-Open No. 2013-538038 (Reference 4), and “Hayashi K. et al., ‘Reconstitution of the mouse germ cell specification pathway in cultur e by pluripotent stem cells.’, Cell, Vol. 146, No. 4, p519-532, 2011.” (Reference 5), etc.

[0049] (primordial germ cells) Also, in this specification, “primordial germ cells” are cells that are expected to differentiate into germ cells and finally differentiate into eggs or sperm through meiosis. Primordial germ cells can be derived from the body or can be primordial germ cell-like cells (PG CLCs) induced to differentiate from pluripotent stem cells. When recovering primordial germ cells from the body, for example, they can be recovered together with the gonads from the fetuses of female mice (aged 11.5 days to 12.5 days) When collecting the gonads from a living body, they may be collected together with the mesonephros, or the mesonephros may be dissected or collected separately.

[0050] As described above, the "primordial germ cells" include primordial germ cell-like cells differentiated from pluripotent stem cells. The method of inducing the differentiation of PGCLCs from pluripotent stem cells (iPS cells or ES cells) can be carried out by a known method, for example, referring to the above-mentioned reference 5 and the like.

[0051] When using PGCLCs derived from pluripotent stem cells as primordial germ cells, it is preferable to remove undifferentiated cells from the induced pluripotent stem cell population in advance. Such a method is known. For example, by introducing a nucleic acid encoding a fusion protein in which Blimp1, which is a marker gene of primordial germ cells, is bound to a reporter protein into pluripotent stem cells, PGCLCs induced to differentiate from pluripotent stem cells and undifferentiated cells can be easily separated by fluorescence-activated cell sorting (FACS) method or the like. fluorescence-activated cell sorting (FACS) method

[0052] In addition, the "primordial germ cells" also include cells obtained by modifying the genes of primordial germ cells derived from a living body or primordial germ cell-like cells derived from pluripotent stem cells using genetic engineering techniques. As a method for modifying the genes of primordial germ cells derived from a living body and primordial germ cell-like cells derived from pluripotent stem cells, known genome editing methods such as the CRISPR system, the method using Transcription Activator-Like Effector Nucleases (TALEN), the method using zinc finger nucleases, and the homologous recombination method can be used to achieve the desired purpose. CRISPR system, Transcription Activator-Like Effector Nucleases (TALEN) method, zinc finger nuclease method, and known genome editing methods such as homologous recombination method are used to achieve the desired purpose. It is possible to introduce nucleic acids, vectors, etc. Examples of methods for introducing nucleic acids, vectors, etc. include for example, microinjection, electroporation, lipofection, nucleic acid introduction methods using viral vectors, and the like. In addition, the method for introducing foreign genes and foreign nucleic acid fragments is not limited to the methods listed above as long as the genetically modified primordial germ cells can differentiate into functional oocytes by the method of this embodiment . Note that genetic modification of primordial germ cells can be performed at an appropriate timing during the culture period of primordial germ cells. For example, in mice, it can be performed during the period from 11.5 days old to 12.5 days old . In addition, in the case of using primordial germ cell-like cells derived from pluripotent stem cells, the pluripotent stem cells before induction of differentiation into primordial germ cell-like cells can also be genetically modified by known methods . Among them, pluripotent stem cells are preferable as the cells having the ability to differentiate into oocytes used in the method for inducing immature oocytes of this embodiment. In particular, when ES cells are used, a large number of immature oocytes can be obtained due to their high proliferation ability . .

[0053] .

[0054] In addition, cells having the ability to differentiate into oocytes can be derived from mammals. Mammals include, but are not limited to, for example, humans, chimpanzees and other primates; dogs, cats, rabbits, horses, sheep, goats, cows, pigs, rats (including nude rats), mice (including nude mice and skid mice), hamsters, guinea pigs and other domestic animals, pet animals and laboratory animals, etc., and are not limited to these .

[0055] [Introduction process] In the method for inducing immature oocytes of this embodiment, the oocyte formation gene is a gene encoding pluripotent oocytes. At least one cell selected from the group consisting of stem cells, EpiLCs, and primordial germ cells It will be introduced.

[0056] In addition, instead of the oocyte formation gene, the mRNA that is its transcript or its expressed protein The mRNA of the oocyte formation gene and its expressed protein may be The mRNA and the mRNA consisting of the base sequence indicated by the Genbank accession number are An expressed protein consisting of the amino acid sequence can be used.

[0057] The method of introduction is not particularly limited, and may vary depending on the target cells and the type of material to be introduced (whether it is nucleic acid or not). The type of the substance can be appropriately selected depending on whether it is a substance or a protein.

[0058] The method for introducing the oocyte formation gene into the cells is not particularly limited, and known methods can be used. Specifically, for example, lipofection, microinjection, injection method, DEAE dextran method, gene gun method, electroporation method, Examples include the calcium phosphate method.

[0059] The method for introducing mRNA of an oocyte formation gene into cells is not particularly limited, and may be any of known methods. The above methods can be appropriately selected and used. Specifically, for example, Lipofectam ine (registered trademark) MessengerMAX (Life Technologies) Examples of such methods include a method using a commercially available RNA transfection reagent such as those manufactured by Eppendorf Pharmaceuticals.

[0060] The method for introducing the expressed protein of the oocyte formation gene into cells is not particularly limited. A known method can be appropriately selected and used. Specifically, for example, protein introduction A method using a reagent, a method using a protein transduction domain (PTD) fusion protein, Examples include the microinjection method.

[0061] Oocyte formation genes are expressed in the form of expression vectors to induce pluripotent stem cells, EpiLCs, and primordial cells. The gene may be introduced into at least one cell selected from the group consisting of germ cells and transiently expressed. In addition, oocyte formation genes are expressed in a group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells. The gene may be stably integrated into the chromosome of at least one selected cell type. Since oocyte formation genes can be expressed in pluripotent stem cells, EpiLCs, and and primordial germ cells. preferable.

[0062] Oocyte formation genes are expressed in the form of vectors to generate pluripotent stem cells, EpiLCs, and primordial germ cells. When the gene is introduced into at least one cell selected from the group consisting of oocyte formation genes, and a promoter that controls the expression of the base sequence of the oocyte formation gene. An expression vector can be used in which the bases of the oocyte formation genes are The base sequence is operably linked to a promoter. All of the genes to be used may be integrated into one expression vector, and these may be used in one type of In particular, from the viewpoint of transfer efficiency, it is necessary to integrate the genes into different vectors. It is preferred to incorporate all of the genes into one expression vector.

[0063] There are no particular limitations on the promoter, and it is possible to use any promoter that is active in the target cells. It may also be an expression-inducible promoter whose activity can be induced by a drug or the like.

[0064] Examples of promoters active in target cells include promoters that are active in almost all cells. The cytomegalovirus promoter (CMV promoter) has strong promoter activity. CMV early enhancer / chicken beta act in (CAG promoter), etc.

[0065] Inducible promoters are those that can artificially control promoter activity. , doxycycline-inducible promoter (TetO promoter), etc.

[0066] The expression vector contains, in addition to the nucleotide sequence of the oocyte formation gene and the promoter, Binds to enhancers, poly(A) addition signals, marker genes, replication origins, and replication origins The marker gene may contain a gene encoding a protein that controls replication. The term "gene" refers to a gene that enables the selection or selection of cells by introducing the marker gene into the cells. Specific examples of marker genes include drug resistance genes, Examples include photoprotein genes, luciferase genes, and chromoenzyme genes. They may be used alone or in combination of two or more. For example, puromycin resistance gene, geneticin resistance gene, neomycin resistance gene, Sex gene, tetracycline resistance gene, kanamycin resistance gene, zeocin resistance gene , hygromycin resistance gene, chloramphenicol resistance gene, etc. A specific example of a fluorescent protein gene is the green fluorescent protein (GFP) gene. Examples include the yellow fluorescent protein (YFP) gene and the red fluorescent protein (RFP) gene. Specific examples of the luciferase gene include the luciferase gene. Specific examples of the chromogenic enzyme gene include the β-galactosidase gene, Examples include the glucuronidase gene and alkaline phosphatase gene.

[0067] The expression vector into which the oocyte formation gene is incorporated is not particularly limited, and any known expression vector can be used. Examples of expression vectors that can be used include plasmid vectors, Examples include virus vectors.

[0068] The plasmid vector is derived from a group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells. There are no particular limitations on the plasmid vector, as long as it can be expressed in at least one selected cell type. For example, the plasmid vectors commonly used for mammalian cell expression are Examples of plasmid vectors for mammalian cell expression include: , pX459, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNA I / Neo and the like, but are not limited to these.

[0069] Examples of viral vectors include retroviral (including lentiviral) vectors. -, adenovirus vector, adeno-associated virus vector, Sendai virus vector , herpes virus vectors, vaccinia virus vectors, pox virus vectors , poliovirus vector, silbis virus vector, rhabdovirus vector, para Examples include paramyxovirus vectors, orthomyxovirus vectors, etc.

[0070] Among them, as the expression vector, a plasmid vector is preferable.

[0071] When integrating the oocyte formation gene into the chromosome, a known knock-in system can be used to perform it. As known knock-in systems, for example, CRISPR / Ca s system, Transcription Activator-Like Effec tor Nucleases (TALEN), zinc finger nucleases and other known genome editing methods are used to cut the chromosome and homologous recombination is performed using a donor vector for homologous recombination Examples include methods such as those using a transposon vector system.

[0072] The donor vector contains a base sequence adjacent to the target region as a homology arm. The donor vector can contain the base sequence of the oocyte formation gene (hereinafter sometimes referred to as the "knock-in sequence") between the 5' arm and the 3' arm. In addition, in order to stably express the oocyte formation gene, it is preferable to set the target region within the safe harbor region .

[0073] The donor vector may be a circular DNA vector (for example, a plasmid vector) , or a linear DNA vector. The donor vector may contain other sequences in addition to the homology arm and the knock in sequence. Examples of other sequences include, for example, marker genes, replication origins, and genes encoding proteins that bind to the replication origin and control replication Examples include children. Examples of the marker gene include the same ones as described above.

[0074] The method for introducing the donor vector is not particularly limited and can be appropriately selected according to the target cells. Examples of the method for introducing the donor vector into cells include, for example, the lipofection method, the microinjection method, the DEAE dextran method, the gene gun method, the electroporation method, the calcium phosphate method, and the like.

[0075] In the transposon vector system, as shown in the examples described later, a transposon vector incorporating the nucleotide sequence of the oocyte formation gene is introduced into cells, and by acting on the transposase, it can be easily incorporated into the chromosomes of the cells. Further, by acting on the transposase again, the nucleotide sequence of the above oocyte formation gene incorporated into the chromosome can be excised from the chromosome and removed without leaving a trace. Examples of the transposon include, for example, piggyBac (registered trademark), Sleeping Beauty, Tol II, mariner, and the like. The method for inducing immature oocytes in this embodiment may include any step in addition to the above introduction step. Examples of the optional step include, for example, a step of proliferating cells (proliferation step), a step of selecting cells into which the introduced oocyte formation gene, or its transcript or its expressed protein has been introduced (selection step), a step of culturing the cells after the introduction step in a state where the oocyte formation gene is expressed intracellularly or the expressed protein of the oocyte formation gene is present (culture step), and the like. Further, when introducing the oocyte formation gene in the introduction step, Examples of the transposon include, for example, piggyBac (registered trademark), Sleeping Beauty, Tol II, mariner, and the like.

[0076] The method for inducing immature oocytes in this embodiment may include any step in addition to the above introduction step. Examples of the optional step include, for example, a step of proliferating cells (proliferation step), a step of selecting cells into which the introduced oocyte formation gene, or its transcript or its expressed protein has been introduced (selection step), a step of culturing the cells after the introduction step in a state where the oocyte formation gene is expressed intracellularly or the expressed protein of the oocyte formation gene is present (culture step), and the like. Further, when introducing the oocyte formation gene in the introduction step, Examples of the optional step include, for example, a step of proliferating cells (proliferation step), a step of selecting cells into which the introduced oocyte formation gene, or its transcript or its expressed protein has been introduced (selection step), a step of culturing the cells after the introduction step in a state where the oocyte formation gene is expressed intracellularly or the expressed protein of the oocyte formation gene is present (culture step), and the like. Further, when introducing the oocyte formation gene in the introduction step, a step of culturing the cells after the introduction step in a state where the oocyte formation gene is expressed intracellularly or the expressed protein of the oocyte formation gene is present (culture step), and the like. Further, when introducing the oocyte formation gene in the introduction step, Examples of the optional step include, for example, a step of proliferating cells (proliferation step), a step of selecting cells into which the introduced oocyte formation gene, or its transcript or its expressed protein has been introduced (selection step), a step of culturing the cells after the introduction step in a state where the oocyte formation gene is expressed intracellularly or the expressed protein of the oocyte formation gene is present (culture step), and the like. Further, when introducing the oocyte formation gene in the introduction step, It includes a step of inducing the expression of the introduced oocyte-forming gene (expression induction step). This can be achieved.

[0077] [Proliferation step] In the proliferation step, in order to obtain a larger amount of immature oocytes, at least one type of cell selected from the group consisting of pluripotent stem cells, EpiLCs and primordial germ cells is proliferated.

[0078] In the proliferation step, for example, from the group consisting of pluripotent stem cells, EpiLCs and primordial germ cells At least one type of cell selected can be proliferated by culturing in a proliferation medium. As the proliferation medium, a known medium for culturing ES cells, iPS cells, EpiLCs, primordial germ cells, etc. can be used, but it is not limited thereto. ES cells, iPS cells Any medium suitable for culturing EpiLCs and primordial germ cells may be used . Specifically, as the proliferation medium, for example, as shown in the examples described later, 2i (2 inhibitor; PD0325901 and CHIR99021), and a serum-free medium supplemented with LIF( Leukemia Inhibitory Factor), etc. can be mentioned . .

[0079] As the culture conditions in the proliferation step, the known conditions for culturing at least one type of cell selected from the group consisting of pluripotent stem cells, EpiLCs and primordial germ cells can be used . Specifically, for example, the culture temperature can be set to about 30°C or higher and 37°C or lower. The culture period, in the case of mice, is not particularly limited, but for example, it can be set to about 1 day or more and 10 days or less, and can be set to about 3 days or more and 7 days or less. Also, those skilled in the art, depending on the origin . . A suitable culture period can be determined depending on the animal species.

[0080] The proliferation step may be performed before or after the introduction step. If the gene is expressed later, the expression timing of the gene is changed. In the presence of proteins, a group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells In order to initiate differentiation induction into immature oocytes from at least one type of cell selected from the above, Therefore, as will be described later in the expression induction process, When the expression is controlled so as to be induced by the presence of an expression inducer, A subsequent growth step can be carried out in the absence of an inducer.

[0081] In addition, when an oocyte formation gene is introduced in the form of a transient expression vector, or when an oocyte formation gene is introduced in the form of a transient expression vector, When introducing a transcript or an expressed protein of a cell formation gene, the cell growth factor is increased before the above-mentioned introduction step. It is preferable to carry out the process.

[0082] [Selection process] In the selection step, oocyte formation genes, or their transcripts or their expressed proteins are introduced. Select transfected cells.

[0083] In the selection step, for example, a reporter gene may be used to detect the expression of an oocyte formation gene or Cells into which the transcript or the expressed protein has been introduced can be selected. Specifically, for example, when an oocyte formation gene is introduced into a cell in the form of an expression vector, the By including a reporter gene in the expression vector, the expression of oocyte formation genes and A reporter gene is expressed together with or independently of the expression of the oocyte formation gene, Cells can be selected. When integrating into the chromosomes of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells, a construct in which a reporter gene is functionally linked upstream or downstream of the oocyte-forming gene can be integrated to express the reporter gene along with the expression of the oocyte-forming gene or independently of the expression of the oocyte-forming gene, thereby allowing cells to be selected. When introducing a transcript of the oocyte-forming gene, a construct in which a transcript of the reporter gene is functionally linked upstream or downstream of the transcript of the oocyte-forming gene is introduced into the cells, so that the reporter gene can be expressed along with the expression of the oocyte-forming gene to select cells. When introducing the expressed protein of the oocyte-forming gene into the cells, a fusion protein of the expressed protein of the oocyte-forming gene and the expressed protein of the reporter gene is introduced into the cells, so that the cells can be selected. As the reporter gene, those exemplified as the marker gene in the description of the above "introduction step" can be used.

[0084] [Expression induction step] In the above introduction step, when the expression of the oocyte-forming gene is controlled to be induced by the presence of an expression inducer, adding the expression inducer to the medium induces the expression of the oocyte-forming gene. When differentiation of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells into immature oocytes is induced, cell proliferation stops. Therefore, to obtain a larger amount of immature oocytes, before performing the expression induction step ​​​​​​​​​​​​It is preferable to carry out the propagation step after the oocyte formation gene is inserted into the expression vector. When the gene is introduced into cells in this form, the proliferation step, the introduction step, and the expression induction step should be carried out in this order. On the other hand, it is preferable to inject oocyte formation genes into pluripotent stem cells, EpiLCs, and primordial germ cells. When the vector is to be integrated into the chromosome of at least one type of cell selected from the group consisting of cells, the introduction step It is preferable to carry out the proliferation step and the expression induction step in this order.

[0085] The expression of an oocyte formation gene can be induced, for example, by inducing the oocyte formation gene with an inducible promoter. a promoter (e.g., the doxycycline-inducible promoter (TetO promoter)) The gene is introduced into cells in a specifically linked form, and an expression inducer (e.g., doxycycline) is added to the medium. Alternatively, for example, a method of expressing an oocyte formation gene by adding As shown in the Examples below, the ProteoTuner (registered trademark) system (Clontech) Specifically, a method using a gene encoding an oocyte formation gene upstream or downstream thereof can be used. Destabilizing Domain (DD, 12 kDa) ) is operably linked to a construct into cells, The fusion protein expressed by the construct is proteasome-soluble in the absence of an expression inducer. On the other hand, as an expression inducer, it is protected from degradation by the proteasome. The medium was supplemented with the small molecule compound Shield1 (a membrane-permeable small molecule compound, 750 Da) that protects against HIV-1. By adding the oocyte formation gene, it is possible to stably express and accumulate the gene in the cells. do.

[0086] The amount of the expression inducer to be added is determined so that the expression level of the oocyte formation gene reaches a desired level. It may be any concentration without special limitation. For example, when the expression inducer is doxycycline the concentration in the medium can be, for example, about 1 nM or more and 10 μM or less. When the expression inducer is Shield1, the concentration in the medium can be, for example, about 10 nM or more and 10 μM or less.

[0087] As the medium used in the expression induction step, those exemplified as the growth medium in the above growth step can be used.

[0088] [Culture step] In the culture step, the cells after the above introduction step are cultured in a state where the oocyte formation gene is expressed intracellularly or the expression protein of the oocyte formation gene is present.

[0089] As the culture conditions, it can be carried out under known conditions for culturing at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells. Specifically, for example, the culture temperature can be about 30°C or more and 37°C or less. The culture period can be, for example, about 1 day or more and 10 days or less, and can be about 3 days or more and 7 days or less in the case of mice.

[0090] As the medium used in the culture step, those exemplified as the growth medium in the above growth step can be used. When the expression of the oocyte formation gene is controlled to be induced by the presence of the expression inducer, the above expression inducer is added to the medium and the cells are cultured in a state where the oocyte formation gene is expressed intracellularly.

[0091] As a method for inducing immature oocytes in a preferred embodiment, the expression of the oocyte formation gene is It is controlled to be induced by the presence of an expression inducer and includes the following 1) to 5). 1) FIGLA, NOBOX, SOHLH1, LHX8, SUB1, STAT3, TBP Introducing one or more oocyte formation genes selected from the group consisting of L2 and DYNLL1 into the chromosome of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells (preferably pluripotent stem cells); 2) Propagating the cells after introduction; 3) Selecting the cells into which the oocyte formation gene has been introduced among the cells after propagation ; 4) Adding an expression inducer to the medium in the cells after selection to induce the expression of the oocyte formation gene ; 5) Culturing the cells after expression induction in a state where the oocyte formation gene is expressed intracellularly

[0092] In the method for inducing immature oocytes of the present embodiment, as described above, from the introduction of an oocyte formation gene, or its transcript or its expressed protein, in a short culture period of about 5 days or more and 10 days or less, at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells can be induced to differentiate into immature oocytes.

[0093] That at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells has been induced to differentiate into immature oocytes can be confirmed from the expression of known oocyte marker genes (e.g., Stella, etc.). Specifically, as shown in the examples described later, Stella, which is an oocyte marker gene, and a reporter protein ( for example, an improved cyan fluorescent protein (Enhanced cyan fluore rescence protein, ECFP)) ​​​A nuclear fragment encoding a fusion protein bound to the ECFP (E2 scent protein; ECFP) The acid is previously added to at least one selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells. By introducing the gene into the chromosome of one type of cell, it can be detected by the expression of Stella-ECFP. From the fluorescence emitted, it can be confirmed that differentiation into immature oocytes has been induced. Fluorescence-activated Stella-ECFP expression Undifferentiated cells were extracted from the differentiated cell population by cell sorting (FACS) etc. This allows cells in a poor state to be removed and immature oocytes to be easily isolated.

[0094] <Method for producing mature oocytes> In one embodiment, the present invention relates to FIGLA, NOBOX, SOHLH1, LHX8, One or more members selected from the group consisting of SUB1, STAT3, TBPL2 and DYNLL1 The gene, or its transcript or expressed protein, is then transferred to pluripotent stem cells, EpiLCs, and progenitor cells. and introducing the gene into at least one cell selected from the group consisting of germ cells. and co-culturing the cells and ovarian somatic cells.

[0095] In the method for producing a mature oocyte of this embodiment, the introduction of an oocyte formation gene (introduction step ) is the same as the introduction step described in the above "Method for inducing immature oocytes," so no explanation will be given here. love.

[0096] In this specification, the term "mature oocyte" refers to an egg in the metaphase of the second meiotic division. Also called secondary oocyte. Also called mature oocyte. As shown in the Examples below, the Npm2 gene, which is a marker for activated oocytes, was expressed in is performing.

[0097] [Agglomerate formation step] The method for producing mature oocytes according to this embodiment is based on the introduction step described in the above "Method for inducing oocytes". After that, at least one type of cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells, and ovarian somatic cells are co-cultured to form aggregates (agglomerate formation step ).

[0098] The ovarian somatic cells used in the agglomerate formation step are somatic cells collected from the ovary of a living body, and by co-culturing with at least one type of cell selected from the group consisting of the above pluripotent stem cells, EpiLCs, and primordial germ cells, they are cells that differentiate into granulosa cells or theca cells that make up ovarian follicles.

[0099] In addition, the method for collecting somatic cells from the ovary of a living body can be carried out according to the method described in the above reference 5.

[0100] Specifically, for example, the method for collecting somatic cells from the ovary is to surgically collect the ovary from a living body and dissociate the somatic cells that make up the ovary by trypsin treatment or the like. At this time, it is preferable to remove the germ cells inherent in the ovary derived from a living body. The method for removing the germ cells inherent in the ovary can be carried out by a known method. For example, the Magnetic activated cell sorting method using an anti-SSEA1 antibody or an anti-CD31 antibody can be used to remove the inherent germ cells. Here, the ovary is preferably derived from a fetus for collecting ovarian somatic cells. In the case of a mouse, for example, the gonad (ovary) derived from a mouse fetus at embryonic age (also referred to as "embryonic age") 12.5 days can be used. ting age Moreover, those skilled in the art can appropriately select the reproductive gonads (ovaries) at a preferable time based on the present disclosure and common general knowledge in the technical field, as well as the animal species from which they are derived. In the case of ,

[0101] , , , , , ,

[0102] , , , , , , , , , , , , ,

[0103] , , it is preferable to form an aggregate consisting of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction step, and ovarian somatic cells, by co-culturing them.

[0101] In the aggregate formation step, by co-culturing at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction step, and ovarian somatic cells. In the case of , , ,

[0102] , , , , , , , , , , , , ,

[0103] , , it is preferable to form an aggregate consisting of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction step, and ovarian somatic cells. In the case of , ,

[0102] , , , , , , , , , , , , ,

[0103] , , it is preferable to form an aggregate consisting of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction step, and ovarian somatic cells. In the case of ,

[0102] , , , , , , , , , , , , ,

[0103] , , it is preferable to form an aggregate consisting of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction step, and ovarian somatic cells.

[0102] In the case of , , , , , , , , , , ,

[0103] , , it is preferable to form an aggregate consisting of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction step, and ovarian somatic cells. In the case of , , , , , , , , , ,

[0103] , , a method for producing an aggregate consisting of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction step, and ovarian somatic cells is, for example, as shown in the examples described later, in S10 medium (StemPro (registered trademark)-34 SFM, manufactured by Life Technologies) supplemented with 10% fetal bovine serum (FCS), 150 μM ascorbic acid, 1× Glutamax, 1× penicillin / streptomycin, and 55 μM 2-mercaptoethanol, by mixing and aggregating at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction step, and ovarian somatic cells, and culturing them. When the expression of the oocyte formation gene is controlled to be induced by the presence of an expression inducer, the above expression inducer is added to the medium, and the cells are cultured in a state where the oocyte formation gene is expressed. It is preferable to use a low-attachment culture dish (for example, a cell low-adhesion U-bottom 96-well plate, etc.) for the culture. In the case of , , , , , , , , ,

[0103] , , a method for producing an aggregate consisting of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction step, and ovarian somatic cells is, for example, as shown in the examples described later, in S10 medium (StemPro (registered trademark)-34 SFM, manufactured by Life Technologies) supplemented with 10% fetal bovine serum (FCS), 150 μM ascorbic acid, 1× Glutamax, 1× penicillin / streptomycin, and 55 μM 2-mercaptoethanol, by mixing and aggregating at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction step, and ovarian somatic cells, and culturing them. When the expression of the oocyte formation gene is controlled to be induced by the presence of an expression inducer, the above expression inducer is added to the medium, and the cells are cultured in a state where the oocyte formation gene is expressed. It is preferable to use a low-attachment culture dish (for example, a cell low-adhesion U-bottom 96-well plate, etc.) for the culture. In the case of , , , , , , , ,

[0103] , , a method for producing an aggregate consisting of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction step, and ovarian somatic cells is, for example, as shown in the examples described later, in S10 medium (StemPro (registered trademark)-34 SFM, manufactured by Life Technologies) supplemented with 10% fetal bovine serum (FCS), 150 μM ascorbic acid, 1× Glutamax, 1× penicillin / streptomycin, and 55 μM 2-mercaptoethanol, by mixing and aggregating at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction step, and ovarian somatic cells, and culturing them. When the expression of the oocyte formation gene is controlled to be induced by the presence of an expression inducer, the above expression inducer is added to the medium, and the cells are cultured in a state where the oocyte formation gene is expressed. It is preferable to use a low-attachment culture dish (for example, a cell low-adhesion U-bottom 96-well plate, etc.) for the culture. In the case of , , , , , , ,

[0103] , , a method for producing an aggregate consisting of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction step, and ovarian somatic cells is, for example, as shown in the examples described later, in S10 medium (StemPro (registered trademark)-34 SFM, manufactured by Life Technologies) supplemented with 10% fetal bovine serum (FCS), 150 μM ascorbic acid, 1× Glutamax, 1× penicillin / streptomycin, and 55 μM 2-mercaptoethanol, by mixing and aggregating at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction step, and ovarian somatic cells, and culturing them. When the expression of the oocyte formation gene is controlled to be induced by the presence of an expression inducer, the above expression inducer is added to the medium, and the cells are cultured in a state where the oocyte formation gene is expressed. It is preferable to use a low-attachment culture dish (for example, a cell low-adhesion U-bottom 96-well plate, etc.) for the culture. In the case of , , , , , ,

[0103] , , a method for producing an aggregate consisting of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction step, and ovarian somatic cells is, for example, as shown in the examples described later, in S10 medium (StemPro (registered trademark)-34 SFM, manufactured by Life Technologies) supplemented with 10% fetal bovine serum (FCS), 150 μM ascorbic acid, 1× Glutamax, 1× penicillin / streptomycin, and 55 μM 2-mercaptoethanol, by mixing and aggregating at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction step, and ovarian somatic cells, and culturing them. When the expression of the oocyte formation gene is controlled to be induced by the presence of an expression inducer, the above expression inducer is added to the medium, and the cells are cultured in a state where the oocyte formation gene is expressed. It is preferable to use a low-attachment culture dish (for example, a cell low-adhesion U-bottom 96-well plate, etc.) for the culture. In the case of , , , , ,

[0103] , , a method for producing an aggregate consisting of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction step, and ovarian somatic cells is, for example, as shown in the examples described later, in S10 medium (StemPro (registered trademark)-34 SFM, manufactured by Life Technologies) supplemented with 10% fetal bovine serum (FCS), 150 μM ascorbic acid, 1× Glutamax, 1× penicillin / streptomycin, and 55 μM 2-mercaptoethanol, by mixing and aggregating at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction step, and ovarian somatic cells, and culturing them. When the expression of the oocyte formation gene is controlled to be induced by the presence of an expression inducer, the above expression inducer is added to the medium, and the cells are cultured in a state where the oocyte formation gene is expressed. It is preferable to use a low-attachment culture dish (for example, a cell low-adhesion U-bottom 96-well plate, etc.) for the culture. In the case of , , , ,

[0103] , , a method for producing an aggregate consisting of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction step, and ovarian somatic cells is, for example, as shown in the examples described later, in S10 medium (StemPro (registered trademark)-34 SFM, manufactured by Life Technologies) supplemented with 10% fetal bovine serum (FCS), 150 μM ascorbic acid, 1× Glutamax, 1× penicillin / streptomycin, and 55 μM 2-mercaptoethanol, by mixing and aggregating at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction step, and ovarian somatic cells, and culturing them. When the expression of the oocyte formation gene is controlled to be induced by the presence of an expression inducer, the above expression inducer is added to the medium, and the cells are cultured in a state where the oocyte formation gene is expressed. It is preferable to use a low-attachment culture dish (for example, a cell low-adhesion U-bottom 96-well plate, etc.) for the culture. In the case of , , ,

[0103] , , a method for producing an aggregate consisting of at least one cell selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells after the introduction step, and ovarian somatic cells is,​​​​​​​​ In addition, those skilled in the art will be able to determine the incubation time depending on the animal species. A preferable culture period can be set as appropriate.

[0104] Furthermore, after the introduction step, the cells are selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells. The ratio of at least one type of cell to be mixed with ovarian somatic cells is set to 100%. There is no limitation as long as cells are formed. For example, in the case of mice, At least one selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells Preferably, the ratio of the number of cells to the number of ovarian somatic cells is about 2:1.

[0105] Furthermore, after the introduction step, the cells are selected from the group consisting of pluripotent stem cells, EpiLCs, and primordial germ cells. At least one type of cell or ovarian somatic cells, or an ovary containing ovarian somatic cells, is cryopreserved. The cryopreservation method can be performed by a known method. For example, For example, the pluripotent stem cells after the introduction step are selected from the group consisting of EpiLCs and primordial germ cells. Cryopreservation of at least one type of cell or ovarian somatic cells was performed using 10% DMSO solution or a commercially available cryopreservative (Cell This can be done by slow freezing using Lubanker (registered trademark, etc.).

[0106] The ovarian somatic cells used in the method for producing mature oocytes of this embodiment are composed of aggregates. At least one selected from the group consisting of pluripotent stem cells, EpiLCs and primordial germ cells. Even if the cells are from the same species of mammal, they may be from a different species of mammal. However, it is preferable to use one derived from the same species of mammal. As for animals, the same as those exemplified in the explanation of the cells capable of differentiating into oocytes can be used. Examples include:

[0107] In the method for producing a mature oocyte of this embodiment, after the introduction step, the aggregate formation An optional step may be included before the step of "immature oocytes" described above. Examples of the steps include the proliferation step and selection step described in "Induction method".

[0108] [Culture process] Furthermore, the method for producing mature oocytes of this embodiment further comprises the steps of: may include:

[0109] In the culturing step, the aggregates formed after the aggregate forming step are transferred onto a collagen membrane and cultured. is preferred.

[0110] The culture conditions can be those known for culturing aggregates. Specifically, For example, the culture temperature can be set to about 30°C or higher and 37°C or lower. In the case of the above, the period can be between 7 and 35 days, and between 8 and 30 days. In addition, those skilled in the art can appropriately set a preferable culture period depending on the animal species from which the cells are derived. It can be determined.

[0111] The medium used in the culture step is one exemplified as the growth medium in the growth step. In addition, the expression of oocyte formation genes can be inhibited by the presence of an expression inducer. When the expression is regulated to be induced, the expression inducer is added to the medium, and the expression is induced intracellularly. The eggs are cultured in a state where oocyte formation genes are expressed.

[0112] After the culture process, the aggregates have a secondary follicle structure, and the oocytes are surrounded by multilayered granulosa cells. Theca follicularis, which surrounds the multilayered granulosa cells, ) is formed, and the theca cells ( Theca cells have luteinizing hormone receptors ne receptor in granulosa cells, and follicle stimulating hormone receptor in They express the stimulating hormone receptor.

[0113] Next, the follicles obtained in the culture step are cultured using the method described in Non-Patent Document 1. The process of obtaining mature oocytes is called the growth process. It can be divided into two stages:

[0114] [Growth process] In the growth step, the follicles obtained in the culture step are isolated into individual follicles and cultured in a growth medium. Cultivate.

[0115] The culture conditions can be as described in Non-Patent Document 1. For example, the culture temperature can be set to about 30°C or higher and 37°C or lower. In some cases, the period may be between 7 and 15 days, and between 8 and 13 days. In addition, a person skilled in the art can appropriately determine a preferable culture period depending on the animal species from which the cells are derived. It is possible.

[0116] As a growth medium, a medium having the composition described in Non-Patent Document 1 can be used. For example, in the case of mice, 5% fetal calf serum (FCS) was added for the first 2 days of culture. 2% polyvinylpyrrolidone (Sigma), 150 μM ascorbic acid, 1x GlutaMAX, 1x penicillin / streptomycin, 100 μM 2-mercaptoethanol butanoethanol, 55 μg / mL sodium pyruvate (Nacalai Tesque) ), 0.1 IU / mL follicle-stimulating hormone (Follistim (registered trademark), manufactured by MSD), 15ng / mL of BMP15 (Bone morphogenetic protein 15), and 15ng / mL of GDF9 (Growth Differentiation Factor 9). α-ME containing α-methyl-2 ... Then, on the second day after the start of the culture, the follicles are cultured using the above-mentioned composition of BMP15 and The medium was replaced with α-MEM without GDF9 and 0.1% Type IV collagenase (MP The follicles are then incubated in a 5% FCS-containing medium (manufactured by Biomedicals). After washing several times with α-MEM containing α-MEM, the follicles were cultured in the same medium as above, but excluding BMP15 and GDF9. The cells are cultured using α-MEM for 11 days from the start of culture.

[0117] After the growth process, the follicles have an antral follicle structure and cumulus oocyte complexes containing germinal vesicle-stage eggs. is formed.

[0118] [Maturity process] In the maturation step, the follicles obtained in the growth step are cultured using a maturation medium.

[0119] The culture conditions can be as described in Non-Patent Document 1. For example, the culture temperature can be set to about 30°C or higher and 37°C or lower. In some cases, the period may be between 7 and 15 days, and between 8 and 13 days. In addition, a person skilled in the art can appropriately determine a preferable culture period depending on the animal species from which the cells are derived. It can be done.

[0120] As the maturation medium, a medium having the composition described in Non-Patent Document 1 can be used. Specifically for example, in the case of a mouse, 5% FCS, 25 μg / mL of sodium pyruvate, 1× penicillin / streptomycin, 0.1 IU / mL of follicle-stimulating hormone, 4 ng of EGF (Epidermal Growth Factor), and 1.2 IU / mL of hCG (Human chorionic gonadotropin, abbreviation: gonado lopin, manufactured by ASKA Pharmaceutical Co., Ltd.)-containing α-MEM can be mentioned.

[0121] The follicles after the maturation process have matured into eggs (secondary oocytes) at the metaphase of the second meiosis. That the egg is a secondary oocyte at the metaphase of the second meiosis can be evaluated, for example, by visual observation using a microscope or the like with the release of the first polar body as an index.

[0122] The obtained mature oocytes (secondary oocytes) are suitably used for infertility treatment. That is, in one embodiment, the present invention provides an infertility treatment method using the mature oocytes obtained by the above method. In addition, the mature oocytes obtained by using the method for producing mature oocytes of the present embodiment are suitably used for efficient breeding of industrial animals and reproduction of rare animals. That is, in one embodiment the present invention provides a method for breeding industrial animals or a method for reproducing rare animals using the mature oocytes obtained by the above method. Note that as the animals to which the above applies, mammals are preferred. Examples of mammals include the same ones as those exemplified above. In addition, the mature oocytes obtained by using the method for producing mature oocytes of the present embodiment are infertility agents. This will be useful in investigating the causes and elucidating the mechanisms of menopausal diseases. [Example]

[0123] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples. do not have.

[0124] [Example 1] (Vector construction) The CAG promoter and destabilization domain (DD) were used as CAG-DD-hTFAP2C promoter. Reference 6: Kobayashi T et al., “Principles of early human development ent and germ cell program from conserved model systems.”, Nature, Vol. 546, No. 7658, p416-420, 2017.) and used conventionally. C vector (Reference 7: “Shimamoto S et al., “Hypoxia induces the dormant stat e in oocytes through expression of Foxo3”, PNAS, https: / / doi.org / 10.1073 / pnas. 1817223116, 2019.") to create the PB-CAG-DD vector. FIGLA, NOBOX, SOHLH1, LHX8, SUB1, STAT3, TBPL2 The cDNAs of the eight genes of DYNLL1 and DYNLL1 were cloned into the ovaries of 13.5-day-old female mice. The DNA was amplified and cloned using the Infusion HD Cloning Kit (Takara Bio). The cDNAs were amplified according to the manufacturer's instructions and cloned into the PB-CAG-DD vector. According to the protocol, PCR was performed using KOD Fx Neo or KOD Plus Neo DNA polymerase (manufactured by TOYOBO). (Performed by PCR using KOD Fx Neo or KOD Plus Neo DNA polymerase (manufactured by TOYOBO).

[0125] (Vector transfection) ES cells were pre-maintained in a serum-free medium supplemented with 2i and LIF without using feeder cells (see Reference 3 above). Also, to monitor the differentiation into immature and mature oocytes, a variant of yellow fluorescent protein (YFP), mVenus (membrane-targeted Venus), under the expression control of Blimp1, an important determinant of the germ cell lineage, a gene encoding an improved cyan fluorescent protein (Enhanced cyan fluorescent protein; ECFP) under the expression control of Stella, a germ cell and oocyte marker, and a gene encoding red fluorescent protein mCherry (membrane-targeted Cherry) under the expression control of Nucleoplasmin 2 (Npm2), an activated (mature) oocyte marker, were inserted into the chromosome of mouse ES cells (Blimp1-mVenus:Stella-ECFP:Npm2-mCherry (BVSCNmC)). The PB-CAG-DD vector containing the eight constructed genes was transfected simultaneously with the hyperactive PBase (hypBase) plasmid using Lipofectamine 2000. After puromycin selection for 5 days in a serum-free medium supplemented with 2i and LIF, single colonies were expanded. (See above reference 3). Also, to monitor the differentiation into immature and mature oocytes, a variant of yellow fluorescent protein (YFP), mVenus (membrane-targeted Venus), under the expression control of Blimp1, an important determinant of the germ cell lineage, a gene encoding an improved cyan fluorescent protein (Enhanced cyan fluorescent protein; ECFP) under the expression control of Stella, a germ cell and oocyte marker, and a gene encoding red fluorescent protein mCherry (membrane-targeted Cherry) under the expression control of Nucleoplasmin 2 (Npm2), an activated (mature) oocyte marker, were inserted into the chromosome of mouse ES cells (Blimp1-mVenus:Stella-ECFP:Npm2-mCherry (BVSCNmC)). The PB-CAG-DD vector containing the eight constructed genes was transfected simultaneously with the hyperactive PBase (hypBase) plasmid using Lipofectamine 2000. After puromycin selection for 5 days in a serum-free medium supplemented with 2i and LIF, single colonies were expanded. (Inducing differentiation into immature oocytes) (Inducing differentiation into immature oocytes) (Inducing differentiation into immature oocytes) (Inducing differentiation into immature oocytes) (Inducing differentiation into immature oocytes) (Inducing differentiation into immature oocytes) (Inducing differentiation into immature oocytes) (Inducing differentiation into immature oocytes) (Inducing differentiation into immature oocytes) (Inducing differentiation into immature oocytes) (Inducing differentiation into immature oocytes) (Inducing differentiation into immature oocytes) (Inducing differentiation into immature oocytes) (Inducing differentiation into immature oocytes)

[0126] (Inducing differentiation into immature oocytes) Then, 10% fetal calf serum (FCS), 150 μM ascorbic acid, 1× glutamate amax, 1x penicillin / streptomycin and 55 μM mercaptoethanol Supplemented S10 medium (StemPro®-34 SFM, Life Technologies) A medium containing 0.5 μM Shield1 (Clontech) was mixed with 10 μM Shield1 (Clontech) (hereafter, 1× oocyte differentiation induction medium (referred to as "oocyte differentiation induction medium") was dispensed into a low-attachment U-bottom 96-well plate. 10 5 ES cells were transferred and cultured for 5 days to induce differentiation into immature oocytes. The results were observed under a microscope (Carl Zeiss, model number: Zeiss LSM 700). The results are shown in Figure 1. In Figure 1, "oocyte formation genes" refer to genes including FIGLA, NOBOX , SOHLH1, LHX8, SUB1, STAT3, TBPL2, and DYNLL1. "Oocyte formation gene expression OFF" indicates the addition of a medium containing Shield1. These cells are the precursors of oocyte formation, and "oocyte formation gene expression ON" is indicated by the addition of medium containing Shield1. The cells were cultured for 5 days.

[0127] As can be seen from Figure 1, cells in which the expression of oocyte formation genes is OFF do not form colonies. The fluorescence of ECFP (Stella-ECFP) expressed under the control of Stella expression was In contrast, cells in which the expression of oocyte formation genes was ON were In this case, cells are separated from each other, and strong fluorescence of Stella-ECFP is detected. This suggests that differentiation into mature oocytes was induced.

[0128] (Mature oocyte production) After transfection, the cells were cultured for 5 days and selected with puromycin. ES cells of the colony (1×10 5 cells) were mixed with ovarian somatic cells derived from female mice at 12.5 days post-fertilization in the above oocyte differentiation induction medium to prepare an aggregate, which was cultured for 2 days 4 with 3×10 cells. The ovarian somatic cells were isolated from the ovaries of female mice at 12.5 days post-fertilization using the method described in Non-Patent Document 1 or the like in advance and used. Next, the aggregate was transferred onto a Transwell-COL membrane (manufactured by Coaster) and cultured in the above oocyte differentiation induction medium for 28 days. The expression of each marker in the aggregate on the 2nd, 6th, 8th, 10th, and 12th days from the start of the culture was observed under a confocal microscope (manufactured by Carl Zeiss, model number: Zeiss LSM 700), and the results are shown in FIG. 2. In FIG. 2, in the upper row, the oocytes were visualized by the fluorescence of Stella-ECFP, which is a germ cell and oocyte marker. In the lower row, the oocytes were visualized by the fluorescence of the red fluorescent protein mCherry (Npm2 -mCherry) expressed under the control of the expression of Nucleoplasmin 2 ( Npm2), which is an activated (mature) oocyte marker.

[0129] From FIG. 2, throughout the culture period, the fluorescence of Stella-ECFP was continuously observed in the cells. On the other hand, the fluorescence of Npm2-mCherry was observed on the 8th day from the start of the culture (see the arrow in the lower row of "Day8" in FIG. 2), indicating that the maturation of the oocytes was progressing.

[0130] Also, on the 28th day from the start of the culture, individual follicles were manually isolated using a sharpened tungsten needle. The isolated follicles had a secondary follicle structure. The follicles were described in Non-Patent Document 1 Under the culture conditions of the "in vitro growth period" and "in vitro maturation period" By culturing, the eggs matured through antral follicles into metaphase II meiotic eggs.

[0131] [Example 2] Using mouse iPS cells, we investigated the induction of differentiation into oocytes in the same way as ES cells. .

[0132] (Vector transfection) iPS cells were generated using the virus buster established in the paper of Non-Patent Document 1. The mouse BVSC iPS cells were used. The PB-CAG-DD vector was hypnotized with Lipofectamine 2000. In addition to the reactive PBase (hypBase) plasmid, mouse BVSC iPS cells were transfected and incubated in serum-free medium supplemented with 2i and LIF for 5 days. Single colonies were expanded after puromycin selection.

[0133] (Induction of differentiation into immature oocytes) The iPS cells transfected with the vector were cultured in the same manner as in Example 1. The cells were cultured for 5 days to induce differentiation into immature oocytes.

[0134] (Mature oocyte production) After transfection, the cells were cultured for 5 days and selected with puromycin. S cells (1×10 5 3 × 10 4 12.5 days after fertilization The cells were mixed with ovarian somatic cells from day-old female mice to form aggregates, which were then cultured for two days. The aggregate was transferred onto a Transwell-COL membrane (manufactured by Coaster), and the above oocytes were The cells were cultured in differentiation-inducing medium for 21 days. Stella-E cells in the aggregates on the 21st day of culture. The expression of CFP was measured using a confocal microscope (Carl Zeiss, model number: Zeiss LSM The results of observation under 700°C are shown in Figure 3. In Figure 3, the image on the left is a bright-field image, and the image on the right is a The side image shows the oocytes, which are identified by the fluorescence of Stella-ECFP, a germ cell and oocyte marker. This is a fluorescent image that visualizes the mother cells.

[0135] As shown in Figure 3, Stella-ECFP fluorescence was observed in cells on the 21st day after the start of culture. This suggests that iPS cells can be induced to differentiate into oocytes in the same way as ES cells. It was confirmed that this was the case.

[0136] [Example 3] (Identification of key factors in oocyte formation genes) To identify the genes that are important factors in oocyte formation, we used the genes shown in the left panel of Figure 4. A total of 26 types of genes were prepared using the same method as in Example 1, so as to obtain the above combinations of genes. The vector was then constructed. Then, mouse ES cells (Bli mp1-mVenus:Stella-ECFP:Npm2-mCherry(BVSC Each vector was transfected into NmC. The follicles were cultured for 5 days using the same method as in Example 1 to induce differentiation into immature oocytes. After transfection, the cells were cultured for 5 days and selected with puromycin. ES cells derived from cells (1 × 10 5 3 × 10 4 Individual reception The cells were mixed with ovarian somatic cells from a 12.5-day-old female mouse to form aggregates, which were then cultured for two days. The resulting aggregates were then cultured for 21 days using the same method as in Example 1. The number of oocytes formed from each cell line was counted. The area of blue fluorescent protein (CFP) expressed downstream of the gene was calculated from the fluorescence area of the oocyte. The area of the cells was calculated, and the results are shown in Figure 4.

[0137] From Figure 4, among the oocyte formation genes, FIGLA, NOBOX, LHX8 and TBP Four genes comprising L2 induce differentiation of pluripotent stem cells into immature oocytes and It was revealed that FIGLA, NOBOX, and L In addition to the four genes HX8 and TBPL2, the STAT3 gene was introduced. It was revealed that this further improved the efficiency of oocyte formation. [Industrial Applicability]

[0138] According to the method for inducing immature oocytes of this embodiment, it is possible to easily and efficiently in a shorter period of culture than conventional methods. It is possible to induce immature oocytes from cells that have the ability to differentiate into oocytes, such as competent stem cells. According to the method for producing mature oocytes of the above aspect, it is possible to easily produce many mature oocytes in a shorter period of culture than conventional methods. To produce large amounts of mature oocytes from cells that have the ability to differentiate into oocytes, such as oocyte-producing stem cells. can be done.

Claims

**Claim 1** An immature oocyte obtained by introducing a gene containing at least FIGLA, NOBOX, LHX8, TBPL2, and a reporter gene, or a transcript or expressed protein thereof, into pluripotent stem cells. **Claim 2** The immature oocyte according to claim 1, which expresses Stella. **Claim 3** The immature oocyte according to claim 1 or 2, wherein the gene further contains STAT3. **Claim 4** The immature oocyte according to any one of claims 1 to 3, wherein the gene further contains at least one selected from the group consisting of SOHLH1, SUB1, and DYNL1. **Claim 5** A mature oocyte obtained by co-culturing the immature oocyte according to any one of claims 1 to 4 and ovarian somatic cells. **Claim 6** The mature oocyte according to claim 5, which expresses Npm2.

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