Method for preparing embryo enriched with primordial germ cells

Abstract

The present disclosure provides a method for preparing an embryo enriched with primordial germ cells, the method comprising: introducing a nucleotide, which encodes Dnd1 and Nanos, into an early embryo derived from a non-human vertebrate; and culturing the early embryo into which the Dnd1 and Nanos nucleotides have been introduced.

Description

Method for preparing embryos enriched for primordial germ cells

[0001] The present invention relates to the field of biotechnology. More specifically, the present invention relates to a method for preparing an embryo enriched in primordial germ cells. The present invention relates to a method for producing a transgenic non-human vertebrate, a method for producing a germline chimeric non-human vertebrate, or a method for producing a donor non-human vertebrate. The present invention relates to an embryo enriched in primordial germ cells.

[0002] The available natural aquatic resources have been at a low level in recent years. Various aquaculture methods have been developed to artificially secure aquatic resources. It is not rational to cultivate broodstock for all fish species used for food and establish seed production techniques, including reproductive control. If a fish species with established aquaculture methods could be used as broodstock (host fish species) to produce gametes of the target fish species for seed production (donor fish species), the effort required to develop production techniques could be reduced. This type of production method is also known as broodstock production. A broodstock production technique has been developed in which gametes from a large, commercially valuable donor fish species, such as tuna, are produced in a closely related species (host fish species, e.g., mackerel), which is easy to raise and inexpensive to raise.

[0003] The borrowed-litter production technique involves isolating primordial germ cells (PGCs) or germline stem cells, which are the source of gametes, from the gonads of donor species embryos or their adult counterparts, transplanting these cells into a surrogate parent (host fish species), and allowing them to produce functional gametes of the donor fish species. PGCs formed during early embryonic development are useful as donor cells because they can easily be conferred useful genetic characteristics using chromosome manipulation techniques (techniques for controlling the number and combination of chromosomes). Furthermore, transplanting a single PGC into a host fish species is useful because functional gametes of the donor fish species can be continuously produced in the host fish species (Non-Patent Document 1).

[0004] A method for increasing the number of PGCs in embryos has been developed in medaka fish, in which mRNA encoding dead end1 (Dnd1), a factor constituting germ plasm (germ granule factor), is introduced into fertilized eggs at the one-cell stage (Non-Patent Document 2). Non-Patent Document 2 reports that when Dnd1 is expressed throughout the cells at the early stage of development, the number of PGCs increases by approximately 1.5 to 2 times.

[0005] Even when the method of Non-Patent Document 2 was used on medaka, which belongs to the order Scarabaeidae, and zebrafish, which belongs to the order Cypriniformes and is a distant lineage, the number of PGCs did not increase (Non-Patent Document 3).

[0006] Saito et al. Xenogenesis in teleost fish through generation of germ-line chimeras by single primary germ cell transformation. Biology of Reproduction 78, 159-166 (2008) Hong et al. Dnd is a critical specifier of primordial germ cells in the medaka fish. Stem Cell Reports 6, 411-421 (2016) Weidinger et al. dead end, a novel vertebrate germ plasma component, is required for zebrafish primary germ cell migration and survival. Current Biology 13, 1429-1434 (2003)

[0007] Only about 10 to 40 primordial germ cells are formed per embryo. For example, in the field of litter production, there is a need for a method to significantly increase the number of primordial germ cells per embryo.

[0008] In embryos, not all cells expressing Dnd1, a germ cell-specific RNA-binding protein, differentiate into primordial germ cells. Therefore, the present inventors believed that another factor, in addition to Dnd1, is involved in differentiation into primordial germ cells. The present inventors found that introducing Nanos3, another germ granule factor, into early embryos in addition to Dnd1 significantly increases the number of primordial germ cells per embryo. The present inventors also found that co-introduction of Dnd1 and Nanos3 significantly increases the number of primordial germ cells per embryo not only in medaka fish but also in zebrafish, which are lineage-distant from medaka fish. Based on these findings, the present inventors have invented a versatile method that can significantly increase the number of primordial germ cells per embryo.

[0009] The present disclosure provides the following inventions: [Item 1] A method for preparing an embryo enriched in primordial germ cells, comprising introducing Dnd1 and Nanos into an early embryo derived from a non-human vertebrate and culturing the early embryo into which the Dnd1 and Nanos have been introduced. [Item 2] The method according to Item 1, wherein the Dnd1 and Nanos are proteins or nucleotides encoding them. [Item 3] The method according to Item 1, wherein, when the Dnd1 and Nanos are RNA encoding them, they contain a polyA sequence at their 3' ends, and when the Dnd1 and Nanos are DNA encoding them, they contain an exogenous polyadenylation sequence. [Item 4] The method according to any one of Item 1 to 3, further comprising introducing a gametogenesis factor into the early embryo. [Item 5] The method according to Item 4, wherein the gametogenesis factor is at least one selected from the group consisting of Daz, Dazl, Boule, meioC, Ythdc2, and Rbm46. [Item 6] The method of any one of Items 1 to 5, wherein the early embryo is an embryo at the 1-cell stage to 64-cell stage. [Item 7] The method of any one of Items 1 to 6, wherein the non-human vertebrate is an animal belonging to the fish class. [Item 8] The method of any one of Items 1 to 7, further comprising modifying genes of an early embryo derived from the non-human vertebrate. [Item 9] A method for producing a transgenic non-human vertebrate, comprising introducing Dnd1 and Nanos into an early embryo derived from a non-human vertebrate and modifying genes of the early embryo; and culturing the early embryo into which Dnd1 and Nanos have been introduced and whose genes have been modified to generate a transgenic non-human vertebrate, wherein the early embryo is an embryo at the 4-cell stage to 64-cell stage, and the transgenic non-human vertebrate contains gene-modified primordial germ cells in its gonadal region.[Item 10] A method for producing a germline chimeric non-human vertebrate, comprising: collecting donor primordial germ cells from an embryo enriched in primordial germ cells prepared by the method of any one of Items 1 to 8; injecting the collected donor primordial germ cells into a host embryo derived from a non-human vertebrate; and culturing the host embryo into which the donor primordial germ cells have been injected, wherein the germline chimeric non-human vertebrate contains the donor primordial germ cells in its gonadal region, and the donor non-human vertebrate that provided the donor primordial germ cells and the host non-human vertebrate that provided the host embryo are closely related. [Item 11] A method for producing a donor non-human vertebrate, comprising mating germline chimeric non-human vertebrates produced by the method of Items 10, wherein the germline chimeric non-human vertebrate contains the donor primordial germ cells in its gonadal region, and the donor non-human vertebrate contains the donor primordial germ cells in its gonadal region. [Item 12] The method of Item 10 or 11, wherein the donor non-human vertebrate and the host non-human vertebrate belong to fish, and the host non-human vertebrate and the donor non-human vertebrate are a combination of mackerel and tuna, a combination of salmon and rainbow trout, a combination of tiger pufferfish and grass pufferfish, or a combination of goldfish and koi carp. [Item 13] An embryo derived from a non-human vertebrate in which primordial germ cells have been enriched, the embryo being an embryo from the blastula stage onwards, and the number of primordial germ cells in the embryo is at least three times greater than the number of primordial germ cells in an embryo of the same developmental stage derived from a non-human vertebrate of the same species as the embryo. [Item 14] An embryo enriched in primordial germ cells prepared by the method of any of Items 1 to 8, or a non-human vertebrate produced by the method of any of Items 7 to 12.

[0010] Figure 1 is a flow chart showing the procedure for synthesizing Nanos3-SV40pA mRNA and Dnd1-SV40pA mRNA. Figure 2A is a schematic diagram showing the procedure for producing germline chimeric medaka. Figure 2B is a microscopic image of a donor embryo into which Dnd1 / Nanos3 were co-introduced. Figure 2C is a microscopic image of a host embryo into which donor primordial germ cells collected from a donor embryo were introduced. Figure 2D is a microscopic image of a germline chimeric medaka that developed from a host embryo containing donor primordial germ cells in its gonad region. Figure 3A is a schematic diagram showing the introduction of genes into one-cell stage embryos to knock down endogenous Dnd1 and / or Nanos3. The left side of Figure 3B is a schematic diagram showing the co-introduction of nucleotides encoding Dnd1 and / or Nanos3 into a single cell of a 16-cell stage embryo. The right side of Figure 3B is a microscopic image of a 16-cell embryo expressing BuckyBall:EGFP. Figure 3C is a fluorescent image showing the gonad region (arrowhead) of a control medaka. Figure 3D is a fluorescent image showing the gonad region (arrowhead) of a medaka developed from an embryo co-introduced with Dnd1 / Nanos3. Figure 3E is a fluorescent image showing the gonad region (arrowhead) of a medaka developed from an embryo introduced with Dnd1. Figure 3F is a fluorescent image showing the gonad region (arrowhead) of a medaka developed from an embryo introduced with Nanos3. Figure 4 shows microscopic images examining the expression of a series of germ granule factors. Figure 5A is a microscopic image of a donor embryo (DN-OE) overexpressing Dnd1 and Nanos3. Figure 5B is a microscopic image of a host embryo injected with donor primordial germ cells derived from a DN-OE embryo. Figure 5C is a microscopic image of a host embryo (control embryo) injected with control donor primordial germ cells. Figure 5D is a microscopic image of a host embryo (DN-chimeric embryo) injected with donor primordial germ cells derived from a DN-OE embryo. Figure 5E is a microscopic image of a chimeric medaka fish developed from a DN-chimeric embryo. Figure 5F is a microscopic image showing donor-derived primordial germ cells in the gonad region of a chimeric medaka fish developed from a DN-chimeric embryo. Figure 6 is a fluorescent microscopic image of a medaka embryo. The medaka embryo is a next-generation medaka embryo developed from a host embryo injected with donor primordial germ cells derived from a DN-OE donor embryo.Olvas-EGFP (green) indicates germ cells, and sox9b-DsRED (orange) indicates notochord and chondrocytes. The lower left image is an enlarged image of the area surrounded by a white frame in the center of the fluorescent image. The arrow in the lower left image indicates germ cells (green fluorescence) formed in the germ region. Figure 7A is a fluorescent microscope image of a medaka embryo co-introduced with medaka-derived Dnd1 / Nanos3. Figure 7B is a fluorescent microscope image of a medaka embryo co-introduced with medaka-derived Dnd1 / Nanos2. Figure 7C is a fluorescent microscope image of a medaka embryo co-introduced with zebrafish-derived Dnd1 / Nanos3 (Zdnd1 + Znanos3). The area surrounded by a dotted line indicates the gonadal region enriched in primordial germ cells.

[0011] (Method for preparing an embryo enriched in primordial germ cells) A first aspect of the present disclosure provides a method for preparing an embryo enriched in primordial germ cells, comprising introducing Dnd1 and Nanos into an early embryo derived from a non-human vertebrate; and culturing the early embryo into which Dnd1 and Nanos have been introduced. An embryo enriched in primordial germ cells is prepared by the method according to the first aspect. Accordingly, another aspect of the present disclosure provides an embryo enriched in primordial germ cells prepared by the method according to the first aspect. Another aspect of the present disclosure provides an embryo derived from a non-human vertebrate and enriched in primordial germ cells, the embryo being at the blastula stage or later, and the number of primordial germ cells in the embryo is at least three times greater than the number of primordial germ cells in an embryo at the same developmental stage derived from the same non-human vertebrate species as the embryo.

[0012] The term "primordial germ cells" refers to cells that differentiate into germ cells. Primordial germ cells can undergo meiosis and ultimately differentiate into eggs or sperm. Primordial germ cells can be identified, for example, based on known primordial germ cell markers. Known germ cell markers may be, for example, markers described in NATURE COMMUNICATIONS (2019) 10:3054 (the disclosure of which is incorporated herein by reference). Primordial germ cells can be identified, for example, based on the expression of known germ granule factors. Known germ granule factors may be, for example, vasa, piwil1, piwil2, dazl, tdrd1, tdrd9, or tdrd12. Primordial germ cells express, for example, at least one gene selected from the group consisting of vasa, piwil1, piwil2, dazl, tdrd1, tdrd9, and tdrd12.

[0013] Primordial germ cells can be identified, for example, according to known methods. Known methods include introducing nucleotides comprising a promoter of the aforementioned germ cell marker or germ granule factor operably linked to a fluorescent protein into an early embryo derived from a non-human vertebrate, and identifying primordial germ cells based on the fluorescence from the fluorescent protein. Known methods include introducing mRNA comprising the 3' untranslated region (UTR) of the aforementioned germ cell marker or germ granule factor and a fluorescent protein into an early embryo derived from a non-human vertebrate, and identifying primordial germ cells based on the fluorescence from the fluorescent protein. The fluorescent protein may be, for example, green fluorescent protein, red fluorescent protein, yellow fluorescent protein, or blue fluorescent protein. The term "untranslated region" refers to a region of an mRNA adjacent to the coding region that is not translated into a protein.

[0014] The primordial germ cells according to this embodiment are induced to differentiate by introducing Dnd1 and Nanos into early embryos of non-human vertebrates. The term "early embryo" includes embryos at the 1-cell stage to the 64-cell stage. Early embryos are, for example, embryos at the 1-cell stage to the 32-cell stage. Early embryos are obtained from sperm and eggs, for example, by natural mating or artificial insemination. Early embryos at the 2-cell stage or later can be obtained, for example, by culturing fertilized eggs.

[0015] The term "embryonic" includes a cell or cell mass in a vertebrate developmental stage after the fertilized egg. An embryo may be, for example, a one-cell stage embryo (also called a "zygote"). An embryo may be, for example, a developed individual (e.g., a juvenile, a fry, a chick, or a fetus). In one example, an embryo does not include a developed individual. The term "morula" includes embryos from the 64-cell stage to the blastula stage. A morula is an embryo containing, for example, 65 to 500 cells. The term "blastula" refers to an embryo containing cell layers surrounding a fluid-filled cavity called the blastocoel. The term "blastula," also called a gastrula, refers to an embryo in which the gastrula has formed. In a blastula, differentiation of, for example, the ectoderm, mesoderm, and endoderm is observed. Differentiation of the ectoderm, mesoderm, and endoderm can be examined, for example, by their layer structure or using known markers. The term "neurula" refers to an embryo from the time when the neural plate is formed to the time when the neural tube is completed. The neural plate is formed on the dorsal side of the ectoderm and is a structure that becomes the primordium of the central nervous system. The term "pharyngula" refers to an embryo at the embryonic development stage in which the pharyngeal arches are visible. The pharyngeal arches have a pillar-like protruding shape and refer to structures that differentiate into the head and neck.

[0016] A morula, blastula, blastula, neurula, or pharyngeal embryo can be obtained, for example, by culturing a fertilized egg. Each of the early embryos, morula, blastula, neurula, and pharyngeal embryo, can be identified, for example, by observing or detecting the structure of the embryo (e.g., the number of cells; the alveolar cavity; the structure of the archenteron; the layer structure of the ectoderm, mesoderm, endoderm, etc.; the neural plate; the pharyngeal arches) or the expression of a specific marker under a microscope.

[0017] Dnd1 and Nanos introduced into the early embryo of a non-human vertebrate may be in the form of protein or nucleotide, or a combination thereof. Dnd1 and Nanos may both be proteins, or nucleotides encoding both, or a combination thereof. Either Dnd1 or Nanos may be a protein and the other may be nucleotides encoding it. Since the effects of Dnd1 and Nanos in the developed embryo can be reduced or negligible, Dnd1 and Nanos are preferably both proteins, or mRNA encoding both, or a combination thereof. Since sustained action of Dnd1 and Nanos in the developing embryo can be expected, Dnd1 and Nanos are preferably DNA encoding them.

[0018] The term "Dnd1" is synonymous with "dead end 1" and is an RNA-binding protein conserved among vertebrates. Dnd1, in the methods of the present disclosure, includes wild-type Dnd1 derived from an animal of the same or different species as the non-human vertebrate that provided the early embryo into which it is introduced. The different species of animal is, for example, an animal of the same phylum or subphylum as the non-human vertebrate that provided the early embryo into which Dnd1 is introduced, preferably an animal of the same class or subclass, the same order or suborder, the same family or subfamily, or the same genus or subgenus. Sequence information for the amino acid sequence of Dnd1 and the nucleotide sequence encoding Dnd1 can be obtained from databank sites provided by public institutions. For example, the nucleotide sequence encoding medaka Dnd1 can be obtained from the National Center for Biotechnology Information (NCBI) under Gene ID: 100302723. For example, the nucleotide sequence encoding bluefin tuna Dnd1 can be obtained from NCBI under Gene ID: 121902261.

[0019] The amino acid sequences of Ddn1 and Nanos3 derived from medaka, zebrafish, and tuna are shown below.

[0020] Nucleotides encoding Dnd1 of a given species can be prepared, for example, from cells of that species (e.g., ovarian cells) by RT-PCR using reverse transcriptase. When the nucleotides encoding Dnd1 are DNA, they can be synthesized using known genetic engineering techniques. When the nucleotides encoding Dnd1 are RNA, they can be prepared by in vitro transcription of the DNA encoding Dnd1. Dnd1 protein of a given species is commercially available or can be prepared according to known methods. These known methods include isolating and purifying (e.g., HPLC) the Dnd1 protein from a natural source containing the Dnd1 protein. Dnd1 protein can be prepared according to genetic engineering techniques. These genetic engineering techniques include, for example, constructing a plasmid incorporating nucleotides encoding Dnd1 into an expression vector, introducing the plasmid into host cells, and culturing the host cells to express the Dnd1. The expressed Dnd1 can be obtained by isolation and purification from the host cells or culture supernatant.

[0021] As long as Dnd1 retains the function of this embodiment, it may have an amino acid sequence that differs from the amino acid sequence of wild-type Dnd1 of the non-human vertebrate that provided the early embryo into which Dnd1 is introduced. Dnd1 that retains the function of this embodiment and contains an amino acid sequence that differs from that of the wild-type Dnd1 is also referred to as a "Dnd1 variant." A Dnd1 variant, for example, has at least 70% (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) sequence identity with the amino acid sequence of the RNA-binding domain of the wild-type Dnd1. A Dnd1 variant, for example, has at least 50% (e.g., 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 150% or more, or 200% or more) of the RNA-binding ability of wild-type Dnd1. A Dnd1 variant, for example, has at least 70% (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) sequence identity with the amino acid sequence of the RNA-binding domain in wild-type Dnd1, and has at least 50% (e.g., 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 150% or more, or 200% or more) of the RNA-binding ability of wild-type Dnd1. Dnd1 variants include wild-type Dnd1 derived from animals of a different species than the non-human vertebrate that provided the early embryo into which it is introduced. Dnd1 variants include, for example, wild-type Dnd1 derived from animals of a different species than the non-human vertebrate that provided the early embryo into which Dnd1 and Nanos are introduced.

[0022] The RNA-binding domain in Dnd1 binds to RNA and comprises an amino acid sequence conserved among vertebrates. The RNA-binding domain in Dnd1 is known and is disclosed, for example, in Gene 558 (2015) 118-125 (the disclosure of which is incorporated herein by reference). The RNA-binding domain in medaka Dnd1 comprises the amino acid sequence shown by EVFISQIPRDVYEDLLIPLFSSVGALWEFRLMMNFSGQNRGFAYAKYGTAAIANDAIHLLHGYPLGPGARLSV (SEQ ID NO: 7).

[0023] The binding of Dnd1 to RNA can be examined by RNA immunoprecipitation. The RNA immunoprecipitation method includes, for example, subjecting host cells (test samples) into which nucleotides encoding a fusion protein of Dnd1 and a labeled protein (e.g., a fluorescent protein or FLAG) have been introduced to a crosslinking treatment with formaldehyde; extracting nuclei from the host cells to obtain a nuclear suspension; subjecting the nuclear suspension to a homogenizer to shear the chromatin and obtain an RNA solution; adding an antibody against the labeled protein to the RNA solution to immunoprecipitate a complex between the crosslinked RNA and the fusion protein; recovering RNA from the precipitate and reverse transcribing it into cDNA; and quantifying the immunoprecipitated RNA from the cDNA by quantitative PCR. Host cells (negative control) into which the nucleotides have not been introduced can be subjected to the RNA immunoprecipitation method, and the immunoprecipitated RNA for the negative control can be quantified. The RNA binding ability of Dnd1 can be calculated by subtracting the amount of RNA of the negative control obtained by the immunoprecipitation method from the amount of RNA of the test sample obtained by the immunoprecipitation method. The RNA binding ability of Dnd1 can be determined by RNA immunoprecipitation using cells derived from a non-human vertebrate that provided the early embryo into which Dnd1 is introduced as host cells. The host cells are preferably cells derived from an animal of the same species as the non-human vertebrate that provided the early embryo into which Dnd1 is introduced. The labeling protein is, for example, green fluorescent protein.

[0024] The term "Nanos" refers to an RNA-binding protein with a zinc finger motif that is conserved among vertebrates and invertebrates. Nanos in the methods of the present disclosure includes wild-type Nanos derived from an animal of the same or different species as the non-human vertebrate into which it is introduced. The different species of animal is, for example, an animal of the same phylum or subphylum as the non-human vertebrate that provided the early embryo into which Nanos is introduced, preferably an animal of the same class or subclass, the same order or suborder, the same family or subfamily, or the same genus or subgenus. The Nanos preferably includes Nanos derived from an animal of the same species as the non-human vertebrate into which it is introduced. The Nanos may be, for example, Nanos 1, Nanos 2, or Nanos 3, or a mixture thereof. The Nanos may be, for example, Nanos 2 or Nanos 3, or a mixture thereof, and is preferably Nanos 3.

[0025] Sequence information for the amino acid sequence of Nanos and the nucleotide sequence encoding Nanos can be obtained from data bank sites provided by public institutions. For example, the nucleotide sequence encoding medaka Nanos1 can be obtained from NCBI under Gene ID: 100144352. For example, the nucleotide sequence encoding medaka Nanos2 can be obtained from NCBI under Gene ID: 100301601. For example, the nucleotide sequence encoding medaka Nanos3 can be obtained from NCBI under Gene ID: 100144282. For example, the nucleotide sequence encoding bluefin tuna Nanos3 can be obtained from NCBI under Gene ID: 121909102.

[0026] Nucleotides encoding a specific species of Nanos can be prepared, for example, from cells of that species (e.g., ovarian cells) by RT-PCR using reverse transcriptase. When the nucleotides encoding Nanos are DNA, they can be synthesized using known genetic engineering techniques. When the nucleotides encoding Nanos are RNA, they can be prepared by in vitro transcription of the DNA encoding the Nanos. Nanos proteins of a specific species are commercially available or can be prepared according to known methods. These known methods include isolating and purifying the Nanos from a natural source containing the Nanos protein (e.g., purification using HPLC). Nanos proteins can be prepared according to genetic engineering techniques. Genetic engineering techniques include, for example, preparing a plasmid incorporating nucleotides encoding Nanos into an expression vector, introducing the plasmid into host cells, and culturing the host cells to express the Nanos. The expressed Nanos can be obtained by isolation and purification from the host cells or culture supernatant.

[0027] As long as Nanos retains the function of this embodiment, it may have an amino acid sequence that differs from that of wild-type Nanos from the non-human vertebrate that provided the early embryo into which Nanos is introduced. Nanos that retains the function of this embodiment and contains an amino acid sequence that differs from that of wild-type Nanos is also referred to as a "Nanos variant." Nanos variants, for example, have at least 70% (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) sequence identity with the amino acid sequence of the RNA-binding domain in wild-type Nanos. Nanos variants, for example, have at least 50% (e.g., 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 150% or more, or 200% or more) of the RNA-binding ability of wild-type Nanos. A Nanos variant, for example, has at least 70% (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) sequence identity with the amino acid sequence of the RNA-binding domain in wild-type Nanos, and has at least 50% (e.g., 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 150% or more, or 200% or more) of the RNA-binding ability of wild-type Nanos. Nanos variants include wild-type Nanos derived from animals of a different species than the non-human vertebrate that provided the early embryo into which it is introduced. Nanos variants include, for example, wild-type Nanos derived from animals of a different species than the non-human vertebrate that provided the early embryo into which Dnd1 and Nanos are introduced.

[0028] The RNA-binding domain in Nanos binds to RNA and contains an amino acid sequence conserved among vertebrates. The RNA-binding domain in Nanos is known and is disclosed, for example, in Comparative Biochemistry and Physiology, Part B 218 (2018) 13-22 (the disclosure of which is incorporated herein by reference). The RNA-binding domain in medaka Nanos3 contains an N-terminal domain (FHLWKDYMGLSDTVK (SEQ ID NO: 8)) and a zinc finger domain (CSFCRHNGESEMVYRSHWLKNQKGDVLCPYLRQYVCPLCGATGAKAHTKRFCPK (SEQ ID NO: 9)).

[0029] The binding ability of Nanos to RNA can be examined according to the RNA immunoprecipitation method described above as a method for examining the binding ability of Dnd1 to RNA.

[0030] A "variant" of a wild-type protein may include deletions, substitutions, or additions, or a combination thereof, in the amino acid sequence of the wild-type protein. An amino acid "deletion" means that an amino acid residue is missing at any position in a specified amino acid sequence. A Dnd1 variant or Nanos variant, for example, has an amino acid deleted at the N-terminus, C-terminus, and / or amino acid sequence between the N-terminus and C-terminus of the corresponding wild-type Dnd1 or Nanos amino acid sequence. An amino acid "addition" means that an amino acid residue is added or inserted at any position in a specified amino acid sequence. A Dnd1 variant or Nanos variant, for example, has an amino acid added or inserted at the N-terminus, C-terminus, and / or amino acid sequence between the N-terminus and C-terminus of the corresponding wild-type Dnd1 or Nanos amino acid sequence. A Dnd1 variant or Nanos variant may, for example, be fused to a functional polypeptide (e.g., a labeling protein such as green fluorescent protein). In this example, a Dnd1 variant or Nanos variant refers to the amino acid sequence of a portion excluding the amino acid sequence of the functional polypeptide. An amino acid "substitution" means that an amino acid residue at any position in a given amino acid sequence is replaced with another amino acid residue. An amino acid substitution is, for example, a conservative substitution. An amino acid "conservative substitution" means that an amino acid residue in a polypeptide is replaced with another amino acid residue having similar side chain characteristics. Examples of side chains include aliphatic side chains, aliphatic hydroxyl side chains, amide-containing side chains, aromatic side chains, basic side chains, acidic side chains, and sulfur-containing side chains. Side chain characteristics include, for example, charge, side chain size, and hydrophobicity / hydrophilicity. A wild-type protein corresponding to a variant has an amino acid sequence that has the highest sequence identity to the amino acid sequence of the variant.

[0031] The term "sequence identity" refers to the percentage of amino acids or nucleotides that match between two optimally aligned polynucleotide sequences or two amino acid sequences. Sequence identity can be calculated using commercially or publicly available software, such as BLAST+. A Dnd1 variant or Nanos variant consists of an amino acid sequence that shows at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the amino acid sequence of the RNA-binding domain in Dnd1 or Nanos, for example. A protein consisting of / consisting solely of a given amino acid sequence does not exclude said protein including post-translational modifications such as glycosylation.

[0032] Dnd1 and Nanos may be Dnd1 and Nanos derived from the same species of animal, or Dnd1 and Nanos derived from different species of animals (e.g., Dnd1 derived from an animal belonging to the order Cypriniformes) (e.g., Nanos derived from an animal belonging to the order Garfishiformes). Dnd1 and Nanos may be introduced into an early embryo derived from a non-human vertebrate in the form of nucleotides encoding them. The nucleotides encoding Dnd1 and Nanos may be present in the same coding sequence on the same nucleotide molecule, or in two different nucleotide molecules. The term "coding sequence" refers to a nucleotide sequence encoding a specific protein. When the nucleotides encoding Dnd1 and Nanos are present in two different nucleotide molecules, the nucleotides encoding Dnd1 and Nanos include a first nucleotide molecule encoding Dnd1 and a second nucleotide molecule encoding Nanos. Ordinal numbers such as "first" and "second" are used to distinguish between elements within a group of similar elements and do not limit the order or importance of a given element.

[0033] The nucleotides encoding Dnd1 and Nanos may include an additional gene sequence, which may be, for example, a nucleotide sequence encoding a fluorescent protein (e.g., green fluorescent protein).

[0034] The nucleotides encoding Dnd1 and Nanos may be DNA or RNA. The RNA encoding Dnd1 and Nanos may, for example, include a cap structure at its 5' end that promotes translation. The cap structure may be, for example, 7-methylguanylic acid. The RNA encoding Dnd1 and Nanos may, for example, include a polyadenylation sequence at its 3' end that stabilizes the RNA or promotes translation.

[0035] The DNA encoding Dnd1 and Nanos may contain, for example, a promoter at its 5' end that functions as a transcription initiation site. The promoter may be any known promoter. Examples of the promoter include zebrafish heat shock protein (hsp) 70 (TG1096), zebrafish hspa9 (TG1182), zebrafish ubiquitin (TG1057), medaka βacitn (TG1147), medaka hsp70 (TG908), medaka EF-1α (TG844), medaka gapdh (TG923), medaka vasa (olvas) (TG870), and Fugu. The promoter may be oct3 (TG913), CMV (TG912, 849), human EF1α (TG871), or 8xHSE (artificial heat shock promoter) (TG921). The DNA encoding Dnd1 and Nanos may, for example, contain a polyadenylation sequence. The polyadenylation sequence may, for example, be present on the 3' end of the DNA. The DNA encoding Dnd1 and Nanos may be linear DNA or a plasmid.

[0036] In one example, when nucleotides encoding Dnd1 and Nanos are present in the same nucleotide molecule, the DNA encoding Dnd1 and Nanos contains a polyadenylation sequence, for example, the polyadenylation sequence is present on the 3'-end side of the DNA. When nucleotides encoding Dnd1 and Nanos are present in two different nucleotide molecules, the DNA encoding Dnd1 and Nanos and the DNA encoding Nanos each contain a polyadenylation sequence, for example, the polyadenylation sequence is present on the 3'-end side of the DNA.

[0037] The term "polyadenylation sequence" refers to a DNA sequence that, when transcribed into RNA, enables the addition of a polyadenylation sequence (also referred to as a "polyA sequence") to the 3' end of the transcript. The mRNAs of many germ granule factors are stabilized only in primordial germ cells (PGCs) under the control of their 3'UTR, and are rapidly degraded in the early stages of development in somatic cells that constitute the body after development (Mishima et al. Differential regulation of germline mRNAs in soma and germ cells by zebrafish miR-430. Current Biology 16, 2135-2142 (2006)).

[0038] The polyadenylation sequence may be a known polyadenylation sequence. The polyadenylation sequence may be an endogenous sequence or an exogenous sequence. The term "endogenous" refers to a gene, nucleic acid, or protein that is native to an organism or cell, or a gene, nucleic acid, or protein with the same structure as the gene, nucleic acid, or protein. The term "exogenous" refers to a gene, nucleic acid, or protein that is different from the gene, nucleic acid, or protein that is native to an organism or cell. The polyadenylation sequence is preferably an exogenous sequence. The polyadenylation sequence may be, for example, SV40pA, BGHpA, hGHpA, or rbGlobpA. The polyA sequence may be, for example, several tens to several hundreds of adenine nucleotides. The polyA sequence may include, for example, a repeat sequence of the AAUAAA sequence.

[0039] Dnd1 and Nanos may be introduced into the early embryo simultaneously, or may be introduced separately at any time. Preferably, Dnd1 and Nanos are introduced into the early embryo simultaneously. When nucleotides encoding Dnd1 and Nanos are present in the same nucleotide molecule, Dnd1 and Nanos are introduced into the early embryo simultaneously. The nucleotide molecule may contain a self-cleaving peptide sequence or a protease cleavage site between the Dnd1 coding sequence and the Nanos coding sequence. The self-cleaving peptide sequence may be any known self-cleaving peptide sequence. The self-cleaving peptide sequence is, for example, a 2A self-cleaving peptide sequence. The protease cleavage site may be any known protease cleavage site. Protease cleavage sites are, for example, enterokinase (DDDDK↓), factor Xa (IEGR↓ / IDGR↓), tobacco etch virus (ENLYFQ↓G) or thrombin (LVPR↓GS), PreScission (LEVLFQ↓GP).

[0040] When nucleotides encoding Dnd1 and Nanos are present in the same nucleotide molecule, the order and number of the Dnd1 coding sequence and the Nanos coding sequence within the molecule are not particularly limited. In one example, the nucleotides include a Dnd1 coding sequence on the 5'-end side and a Nanos coding sequence on the 3'-end side. In another example, the nucleotides include a Nanos coding sequence on the 5'-end side and a Dnd1 coding sequence on the 3'-end side. In one example, the nucleotides include two Dnd1 coding sequences and at least one Nanos coding sequence. In one example, the nucleotides include at least one Dnd1 coding sequence and two Nanos coding sequences.

[0041] In one example, when the nucleotides encoding Dnd1 and Nanos are present in the same nucleotide molecule, the RNA encoding Dnd1 and Nanos contains a polyA sequence at its 3' end. In one example, when the nucleotides encoding Dnd1 and Nanos are present in two different nucleotide molecules, the RNA encoding Dnd1 and Nanos and the RNA encoding Nanos each contain a polyA sequence at their 3' end.

[0042] Nucleotides encoding Dnd1 and Nanos can be introduced into early embryos derived from non-human vertebrates, for example, by microinjection, electroporation, or lipofection. Dnd1 and Nanos may be introduced into early embryos derived from non-human vertebrates in the form of proteins. The proteins Dnd1 and Nanos can be introduced into early embryos derived from non-human vertebrates, for example, by microinjection or lipofection. When Dnd1 and Nanos are a combination of protein and nucleotide, the Dnd1 and Nanos can be introduced into early embryos derived from non-human vertebrates by microinjection or lipofection.

[0043] Examples of the present disclosure demonstrate that introducing the nucleotides into a one-cell stage embryo can produce a blastula in which most or all of the cells are primordial germ cells (PGCs). In the method of this embodiment, introducing Dnd1 and Nanos into all or almost all of the cells of an early embryo can produce a blastula in which most or all of the cells are PGCs. In one example, when an early embryo is a four-cell stage embryo, introducing Dnd1 and Nanos into all four cells or three cells can produce a blastula in which most or all of the cells are PGCs. The ratio of the number of cells into which the nucleotides are introduced to the number of cells in the early embryo is, for example, at least 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100%).

[0044] Examples of the present disclosure demonstrate that introducing nucleotides encoding Dnd1 and Nanos into a single cell of a 16-cell stage embryo can generate an individual with a significantly increased number of primordial germ cells in its gonad region compared to an identical embryo except that Dnd1 and Nanos are not introduced. Examples of the present disclosure also demonstrate that the cells into which Dnd1 and Nanos are introduced may be cells in which factors that constitute germ plasm (germ granule factors) are not localized. In the method of the first aspect, introducing Dnd1 and Nanos into a predetermined proportion of cells in an early embryo can produce an individual with a significantly increased number of primordial germ cells in its gonad region compared to an identical embryo except that Dnd1 and Nanos are not introduced. In one example, when the early embryo is at the 32-cell stage, introducing Dnd1 and Nanos into one or more (e.g., two, three, or four) cells can produce an individual with a significantly increased number of primordial germ cells in its gonadal region compared to an identical embryo (individual) except that Dnd1 and Nanos are not introduced. The predetermined percentage can be, for example, 2% to 30%, 2% to 25%, 2% to 20%, 2% to 15%, or 2% to 10%. The cells in the early embryo into which Dnd1 and Nanos are introduced may or may not be cells in which germ granule factors are localized. In one example, when the early embryo is at the 16-cell stage, the number of cells into which Dnd1 and Nanos are introduced can be, for example, one (approximately 6% = 1 / 16) or two (approximately 13% = 2 / 16).

[0045] In the method according to this embodiment, the number or proportion of cells into which Dnd1 and Nanos are introduced into an early embryo can be appropriately selected depending on the purpose. For example, when the aim is to obtain a blastula in which most or all cells are primordial germ cells, Dnd1 and Nanos are introduced into all or almost all cells of the early embryo. For example, when the aim is to obtain an individual in which the number of primordial germ cells in its gonad region is significantly increased compared to a wild-type animal, Dnd1 and Nanos are introduced into a predetermined proportion of cells in the early embryo. The amounts of Dnd1 and Nanos introduced into the early embryo may be known amounts used when introducing a protein or gene into an embryo. The ratio of Dnd1 to Nanos introduced into the early embryo is, for example, 10:1 to 1:10, 5:1 to 1:5, 3:1 to 1:3, 2:1 to 1:2, or 1:1. In one example, Dnd1 and Nanos are introduced into early embryos by microinjecting a solution containing Dnd1 and Nanos at a volume that is 30 to 60% of the total embryo volume. When microinjecting into fish (e.g., zebrafish or medaka) embryos, the 30 to 60% volume is, for example, 100 pl to 500 pl. In one example, nucleotides (e.g., mRNA) encoding Dnd1 and Nanos are introduced into early embryos derived from medaka by microinjecting 100 pl to 500 pl of the nucleotide-containing solution at 10 to 500 ng / ml.

[0046] The early embryo into which Dnd1 and Nanos according to the present disclosure have been introduced can be cultured under known culture conditions to allow embryonic development to proceed. Known culture conditions are appropriately set depending on the animal species of the embryo. When the animal species of the embryo is a fish or amphibian, culturing the early embryo into which Dnd1 and Nanos have been introduced includes, for example, leaving it standing in saline or freshwater at room temperature. When the animal species of the embryo is a reptile or a bird, culturing the early embryo into which Dnd1 and Nanos have been introduced includes, for example, leaving it standing at a temperature appropriate for the animal species. When the animal species of the embryo is a mammal, culturing the early embryo into which Dnd1 and Nanos have been introduced can be, for example, left in a known medium at 37°C under 5% CO ２For the purpose of obtaining a non-human mammal in which the number of primordial germ cells in the gonad region is significantly increased compared to a wild-type animal, the early embryo into which Dnd1 and Nanos have been introduced can be cultured, for example, in a known medium at 37°C under 5% CO ２ The method includes maintaining the embryo in an environment until the blastula stage, and then implanting it into the uterus of the non-human mammal. Thus, culturing the early embryo into which Dnd1 and Nanos have been introduced includes in vitro culture, and optionally in vivo culture. The number of primordial germ cells in the embryo is compared with that of an identical embryo except that Dnd1 and Nanos have not been introduced. The identical embryo except that Dnd1 and Nanos have not been introduced is an embryo of the same developmental stage derived from an animal of the same species as the animal that provided the early embryo into which Dnd1 and Nanos have been introduced. Developmental stages of the embryo include, for example, the 1-cell stage to the 64-cell stage, the morula stage, the blastula stage, the neurula stage, the pharyngeal stage, or a developed individual.

[0047] The examples of the present disclosure demonstrate that co-introduction of Dnd1 and Nanos into early embryos of medaka and zebrafish results in individuals with a significantly increased number of primordial germ cells compared to wild-type individuals. Dnd1 and Nanos according to this embodiment are RNA-binding proteins conserved across vertebrates, and both play important roles in the formation of germ cells.The technical matters are described, for example, in Youngren et al., "The Ter mutation in the dead end gene causes germ cell loss and testicular germ cell tumors" (Nature 435, 360-364 (2005) for mice, and in Taguchi A, Watanabe K, Orii H. "Intracellular localizations of the dead end protein in Xenopus primordial germ cells" (Xenopus laevis), for Xenopus laevis. Int J Dev Biol 58:793-798 (2014), Mei W, Jin Z, Lai F, Schwend T, Houston DW, King ML, Yang J. Maternal Dead-End1 is required for vegetable cortical microtubule assembly during Xenopus axis specification. Development 140:2334-2344 (2013), and Lai et al. Xenopus Nanos1 is required to prevent endodermal gene expression and apoptosis in primordial germ cells. Development 139(8):1476-1486(2021), and for chickens, Aramaki et al. Chicken Dead End Homologue Protein is a Nucleoprotein of Germ Cells Including Primordial Germ Cells. J. Reprod. and Dev. 55(2):214-218 (2009). Tsuda et al., "Conserved Role of Nanos Proteins in Germ Cell Development," Science 301(5637):1239-1241 (2003), describes that Nanos is conserved from flies to mammals and plays an important role in germ cell formation.Therefore, the method according to this embodiment may be applicable not only to fish such as medaka and zebrafish, but also to other vertebrates (for example, amphibians, reptiles, birds, and any non-human mammals).

[0048] One embodiment provides a method for preparing an embryo enriched in primordial germ cells, comprising introducing Dnd1 and Nanos into an early embryo derived from a non-human vertebrate; introducing a gametogenesis factor; and culturing the early embryo into which the Dnd1 and Nanos have been introduced. The gametogenesis factor may be, for example, at least one selected from the group consisting of Daz, Dazl, Boule, meioC, Ythdc2, and Rbm46. A non-human vertebrate having primordial germ cells formed according to this embodiment in its gonads has a high tendency to be fertile.

[0049] Dnd1, Nanos, and the gametogenesis factor may be introduced into the early embryo all at the same time, or may be introduced in any combination at any time, or may be introduced all individually at any time. In one example, Dnd1, Nanos, and the gametogenesis factor are introduced into the early embryo simultaneously. In one example, the combination of Dnd1 and Nanos and the gametogenesis factor are each introduced at any time. In the example, for example, the combination of Dnd1 and Nanos is introduced first, and then the gametogenesis factor is introduced. In one example, Dnd1, Nanos, and the gametogenesis factor are introduced individually into the early embryo at any time.

[0050] The term "gametogenesis factor" refers to any substance involved in gametogenesis. The gametogenesis factor may be, for example, a DAZ family member or any substance involved in the meiotic transition.

[0051] The term "DAZ family" refers to a group of three RNA-binding proteins. DAZ family members may play important roles in gametogenesis or meiosis. DAZ family members have, for example, a sequence of 24 amino acids termed the DAZ repeat. DAZ family members may be, for example, Daz, Dazl, or Boule. Daz, Dazl, and Boule can differentiate germ cells from human and mouse embryonic stem cells (Nature volume 462, pages 222-225 (2009)). The term "Daz" is synonymous with "Deleted in Azoospermia" and is an RNA-binding protein. The term "Dazl" is synonymous with "Deleted in azoospermia-like" and is an RNA-binding protein. The term "Boule" refers to an RNA-binding protein. Boule is found in many vertebrates, including humans, and invertebrates, including sea anemones. Dazl is found in almost all vertebrates, including mice and humans. Daz can be Daz1, Daz2, Daz3, or Daz4.

[0052] The substance involved in meiotic transition may be, for example, meioC, Ythdc2, or Rbm46. The term "meioC" is synonymous with "Meiosis-Specific with Coiled-Coil Domain." meioC is important for meiotic transition in medaka. The term "Ythdc2" is synonymous with "YTH domain containing 2." The term "Rbm46" is synonymous with "RNA Binding Motif Protein 46."

[0053] Information on the amino acid sequences of gametogenesis factors and the nucleotide sequences encoding them can be obtained from data bank sites provided by public institutions. For example, the mRNA sequence encoding medaka Dazl is available from the National Center for Biotechnology Information (NCBI) under Accession: NM_001104799.1.

[0054] The amino acid sequences of Dazl derived from medaka, zebrafish, and tuna are shown below. The amino acid sequence of Dazl derived from tuna is a predicted sequence.

[0055] A gametogenesis factor of a given species can be prepared, for example, from cells of that species (e.g., ovarian cells) by RT-PCR using reverse transcriptase. As long as the gametogenesis factor has the function of this embodiment, it may have an amino acid sequence that differs from that of a wild-type gametogenesis factor of the non-human vertebrate that provided the early embryo into which the gametogenesis factor is introduced. A gametogenesis factor that has the function of this embodiment and comprises an amino acid sequence that differs from that of the wild-type gametogenesis factor is also referred to as a "gametogenesis factor variant." A gametogenesis factor variant has, for example, at least 70% (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) sequence identity with the amino acid sequence of the RNA-binding domain of a wild-type gametogenesis factor. A gametogenesis factor variant has, for example, at least 50% (e.g., 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 150% or more, or 200% or more) of the RNA binding ability of a wild-type gametogenesis factor. A gametogenesis factor variant has, for example, at least 70% (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) sequence identity with the amino acid sequence of the RNA-binding domain in the wild-type gametogenesis factor, and has at least 50% (e.g., 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 150% or more, or 200% or more) of the RNA binding ability of a wild-type gametogenesis factor. Gametogenesis factor variants include wild-type gametogenesis factors derived from animals of a different species than the non-human vertebrate that provided the early embryo into which the gametogenesis factor is introduced. Gametogenesis factor variants include, for example, wild-type gametogenesis factors derived from animals of a different species than the non-human vertebrate that provided the early embryo into which the gametogenesis factor is introduced. The binding ability of gametogenesis factors to RNA can be examined by RNA immunoprecipitation.

[0056] The gametogenesis factor is, for example, at least one selected from the group consisting of Daz, Dazl, Boule, meioC, Ythdc2, and Rbm46. The gametogenesis factor is, for example, at least one selected from the group consisting of Daz, Dazl, Boule, and meioC. The gametogenesis factor is, for example, Daz, Dazl, Boule, or meioC, or a mixture thereof. The gametogenesis factor is preferably Daz, Dazl, or meioC, or a mixture thereof. The gametogenesis factor is preferably Daz. The gametogenesis factor is preferably Dazl. The gametogenesis factor is preferably meioC. The gametogenesis factor is preferably Boule.

[0057] The gametogenesis factor may be introduced into an early embryo derived from a non-human vertebrate in the form of a protein or in the form of a nucleotide encoding the same. The nucleotide encoding the gametogenesis factor may be DNA or RNA. The nucleotide encoding the gametogenesis factor may include other nucleotide sequences described herein. The gametogenesis factor can be introduced into the early embryo according to the methods described herein.

[0058] "Introducing" Dnd1, Nanos or a gametogenesis factor into an early embryo in the context of Dnd1, Nanos or a gametogenesis factor refers to a procedure in which Dnd1, Nanos or a gametogenesis factor is present in one or more cells that constitute the early embryo. The form of Dnd1, Nanos or a gametogenesis factor when introduced into the early embryo is not particularly limited. Dnd1, Nanos or a gametogenesis factor is introduced into the early embryo, for example, in the form of a nucleotide or a protein, or a mixture thereof. Dnd1, Nanos or a gametogenesis factor is preferably introduced into the early embryo in the form of a nucleotide (more preferably in the form of RNA).

[0059] Embryos enriched in primordial germ cells The method according to the first aspect provides embryos enriched in primordial germ cells. Accordingly, one aspect of the present disclosure provides an embryo derived from a non-human vertebrate that is enriched in primordial germ cells, the embryo being at or after the blastula stage, and in which the number of primordial germ cells in the embryo is at least three times greater than the number of primordial germ cells in an embryo of the same developmental stage derived from the same non-human vertebrate species as the embryo.

[0060] An "embryo enriched in primordial germ cells" has a significantly increased number of primordial germ cells compared to an embryo from a wild-type animal. A wild-type animal embryo is, for example, an embryo obtained from an animal of the same species as the animal that provided the early embryo into which Dnd1 and Nanos according to the present disclosure have been introduced, and is at the same developmental stage except that Dnd1 and Nanos have not been introduced. The number of primordial germ cells in the embryo is compared, for example, with the number of primordial germ cells in an embryo derived from an animal of the same species as the animal that provided the embryo and at the same developmental stage. Primordial germ cells in an embryo can be separated or concentrated, for example, by subjecting the embryo to cell detachment treatment using trypsin or collagenase, and then subjecting the suspension containing the separated cells to density gradient centrifugation. The number of primordial germ cells in an embryo can be compared, for example, by counting the number of cells expressing known primordial germ cell markers or germ granule factors. The number of primordial germ cells in an embryo is compared based on the labeled cells, for example, by labeling the primordial germ cells by immunochemical staining using an antibody against a known primordial germ cell marker or germ granule factor, or by in situ hybridization using a probe having a sequence complementary to the mRNA of the marker or factor. The number of primordial germ cells in an embryo is compared based on, for example, the expression level of a known primordial germ cell marker or germ granule factor in the embryo. The number of primordial germ cells in an embryo is preferably compared based on the expression level of vasa.

[0061] The primordial germ cell-enriched embryo from a non-human vertebrate may be an embryo at or after the blastula stage, such as a morula, blastula, neurula, pharyngeal embryo, or an embryo at a later developmental stage, in which the number of primordial germ cells is at least three times (e.g., five times or more, ten times or more, fifteen times or more, twenty times or more, twenty-five times or more, or thirty times or more) greater than the number of primordial germ cells in an embryo at the same developmental stage from the same non-human vertebrate species as the embryo.

[0062] Embryo enriched in genetically modified primordial germ cells The embryo enriched in primordial germ cells prepared by the method according to the first aspect may be genetically modified in the primordial germ cells. Accordingly, one aspect of the present disclosure provides an embryo derived from a non-human vertebrate and enriched in genetically modified primordial germ cells, the embryo being at the blastula stage or later, in which the number of primordial germ cells in the embryo is at least three times greater than the number of primordial germ cells in an embryo at the same developmental stage derived from the same non-human vertebrate species as the embryo.

[0063] The genes of the early embryo can be modified before, simultaneously with, or after the introduction of Dnd1 and Nanos into the early embryo derived from a non-human vertebrate. For example, the genes may be modified simultaneously with the introduction of Dnd1 and Nanos into the early embryo. For example, the genes may be modified before or after the introduction of Dnd1 and Nanos into the early embryo.

[0064] The terms "genetic modification," "genetic modification," or "modifying a gene" mean intentionally modifying a gene by chromosome manipulation techniques or DNA recombination techniques. Chromosome manipulation techniques include, for example, tetraploidization of chromosomes. When the non-human vertebrate is a fish, the chromosomes can be made tetraploid by treating fertilized eggs with pressure, temperature, or chemicals such as cytochalasin B. DNA recombination techniques include, for example, genome editing techniques. DNA recombination techniques include introducing deletions, substitutions, additions, or combinations thereof into a chromosome using the CRISPR-CAS system.

[0065] Genetically modified primordial germ cells are prepared by a method comprising introducing Dnd1 and Nanos into an early embryo derived from a non-human vertebrate and modifying the genes thereof; and culturing the early embryo. In one example, genetically modified primordial germ cells are prepared by a method comprising introducing Dnd1 and Nanos into an early embryo derived from a non-human vertebrate; modifying the genes of the early embryo (or cell) into which Dnd1 and Nanos have been introduced; and culturing the early embryo into which Dnd1 and Nanos have been introduced and modified. In another example, genetically modified primordial germ cells are prepared by a method comprising modifying the genes of an early embryo (or cell) derived from a non-human vertebrate; introducing Dnd1 and Nanos into the genetically modified early embryo (or cell); and culturing the early embryo into which Dnd1 and Nanos have been introduced and modified. In another example, genetically modified primordial germ cells are prepared by a method comprising simultaneously introducing Dnd1 and Nanos into an early embryo (or cell) derived from a non-human vertebrate and modifying its genes; and culturing the early embryo into which Dnd1 and Nanos have been introduced and into which genes have been modified. By introducing Ddn1 and Nanos into all or most of the cells of the early embryo (e.g., a 1-cell, 2-cell, 4-cell, or 8-cell embryo) and modifying its genes, a blastula in which most or all of the primordial germ cells are genetically modified can be obtained. Embryos enriched for genetically modified primordial germ cells can be used to create germline chimeric non-human vertebrates, as described below.

[0066] In the method, Dnd1 and Nanos are introduced into a predetermined proportion of cells in an early embryo (e.g., a 4-cell, 8-cell, 16-cell, 32-cell, or 64-cell stage embryo) derived from a non-human vertebrate, and the genes are modified to obtain an embryo (transgenic non-human vertebrate) enriched in genetically modified primordial germ cells. Thus, one embodiment of the first aspect provides a method for producing a transgenic non-human vertebrate comprising genetically modified primordial germ cells in its gonadal region.

[0067] Method for Producing Transgenic Non-Human Vertebrates One aspect of the present disclosure provides a method for producing a transgenic non-human vertebrate, comprising introducing Dnd1 and Nanos into an early embryo (at least one cell of an embryo at the 4-cell to 64-cell stage) derived from a non-human vertebrate and modifying its genes; and culturing the Dnd1- and Nanos-introduced, genetically modified early embryo to generate a transgenic non-human vertebrate, wherein the transgenic non-human vertebrate comprises genetically modified primordial germ cells in its gonadal region.

[0068] The term "gonad region" refers to a region in an embryo that becomes an organ that produces germ cells in the developing individual. The gonad region may be, for example, a region in an embryo where primordial germ cells are present. When referring to an individual from which the gonad region develops, the gonad region is synonymous with gonads. The term "gonad" refers to any organ that produces germ cells in an animal. Gonads include the ovaries of females, which produce eggs, or the testes of males, which produce sperm. The term "germ cell" refers to a cell lineage that gives rise to gametes (eggs or sperm).

[0069] The transgenic non-human vertebrate of this embodiment preferably contains the genetic modification essentially only in the primordial germ cells of the gonadal region (gonads). For example, the transgenic non-human vertebrate does not contain the genetic modification in regions other than the gonadal region (e.g., organs such as the brain, heart, and internal organs; somatic cells such as neurons, cardiomyocytes, and endothelial cells). According to the method of this embodiment, for example, even if the genetic modification may cause lethality or developmental defects, it is possible to create a transgenic non-human vertebrate containing germ cells containing the genetic modification in its gonadal region. In one example, a next-generation non-human vertebrate having the genetic modification homozygously can be produced from the male and female gametes of the transgenic non-human vertebrate. A next-generation non-human vertebrate having the genetic modification heterozygously can be produced from the male or female gametes of the transgenic non-human vertebrate and the female or male gametes of an animal of the same species as the non-human vertebrate. The transgenic non-human vertebrate according to this embodiment can be used, for example, in genetic analysis of diseases.

[0070] The term "vertebrate" refers to any animal belonging to the classes fish, amphibians, reptiles, birds, and mammals. In this embodiment, the vertebrate is a non-human vertebrate, excluding humans. The non-human vertebrate is, for example, any animal belonging to the classes fish, amphibians, reptiles, birds, or mammals, preferably any animal belonging to the classes fish, birds, or mammals, more preferably any animal belonging to the classes fish or mammals, and even more preferably any animal belonging to the class fish.

[0071] The term "fish" refers to the group of animals in the subphylum Vertebrata, excluding tetrapods. Fish may be, for example, animals of the families Scombridae, Amberjack, Salmonidae, Gadidae, Anguillidae, Tetraodontidae (puffers), Sparidae (sea breams and porgies), Serranidae (sea basses), Paralichthys, Acipenseridae, Oryzias (medakas), or Cyprinidae.

[0072] Fish of the Scombridae family may be, for example, animals of the genus Thunnus, such as Yellowfin tuna, Blackfin tuna, Bigeye tuna, and Pacific bluefin tuna; the genus Bonito; the genus Scombridae, such as Kawakawa and Black skipjack tuna; the genus Scombridae, such as King mackerel; the genus Acanthocybium; and animals of the genus Scombridae, such as Atlantic chub mackerel, Chub mackerel, and Atlantic mackerel.

[0073] Fish of the Carangidae family may be, for example, animals of the Seriola genus, such as Amberjack, Seriola lalandi, and Seriola dumerili; Carangidae, such as Pseudocaranx dentex; or Carangidae, such as Japanese horse mackerel. Fish of the Salmonidae family may be, for example, animals of the Hucho genus, such as Japanese huchen (Parahucho perryi); Salmon, such as Oncorhynchus keta, rainbow trout (Oncorhynchus mykiss), cherry salmon (Oncorhynchus masou), and sockeye salmon (Oncorhynchus nerka); Salmon, such as white-spotted char; or Salmonidae.

[0074] Examples of fish of the Gadidae family include animals of the genus Cod (Gadus macrocephalus), Atlantic cod (Gadus morhua), Greenland cod (Gadus ogac), and Alaska pollock (Gadus chalcogrammus); animals of the genus Micromesistius. Examples of fish of the Anguillididae family include animals of the genus Anguilla (Anguilla), such as the American eel (Anguilla rostrata), the Japanese eel (Anguilla japonica), the giant mottled mottle mollusks (Anguilla marmorata), and the European eel (Anguilla anguilla); animals of the genus Neoanguilla.

[0075] Examples of fish of the Tetraodontidae family include animals of the genus Takifugu, such as the tiger pufferfish (Takifugu rubripes) and the red pufferfish (Takifugu porphyreus); and animals of the genus Lagocephalus. Examples of fish of the Sparidae family include animals of the genus Pagrus, such as the red sea bream (Pagrus major); animals of the genus Acanthopagrus, such as the black porgy (Acanthopagrus schlegelii); and animals of the genus Dentex. Examples of fish of the Grouper family include animals of the genus Epinephelus, such as the yellow spotted grouper (Epinephelus septemfasciatus), the grouper (Epinephelus bruneus), and the red spotted grouper (Epinephelus akaara); and animals of the genus Plectropomus. The fish of the family Paralichthyidae may be, for example, a flounder (Paralichthys olivaceus).

[0076] Examples of fish of the Acipenseridae family include sturgeon (Acipenser medirostris), Daurian sturgeon (Huso dauricus), and Beluga sturgeon (Huso huso). Examples of fish of the genus Oryzias include animals of the genus Oryzias, such as medaka (Oryzias latipes, Oryzias sakaizumii), and Javan medaka (Oryzias javanicus). Examples of fish of the family Cyprinidae include animals of the subfamily Cyprininae, such as zebrafish, carp, crucian carp, and goldfish; the subfamily Danioninae, such as rasboras; the subfamily Gobioninae, such as black carp; and the subfamily Leuciscinae, such as Japanese dace.

[0077] The non-human vertebrate according to this embodiment may be, for example, any animal belonging to the fish family, such as tuna, sturgeon, bonito, yellowtail, amberjack, yellowtail amberjack, sea bream, salmon, cod, trout, rainbow trout, flounder, tiger pufferfish, horse mackerel, grouper, eel, or carp.

[0078] The term "amphibians" refers to the group of animals belonging to the subphylum Vertebrate, class Amphibia. Amphibians may be, for example, animals of the families Hynobiidae, Cryptobranchidae, Salamandridae, Bufonidae, Hylidae, Ranidae, or Rhacophoridae.

[0079] Amphibians of the salamander family may be, for example, animals of the genus Hynobius and the genus Onychodactylus. Amphibians of the giant salamander family may be, for example, animals of the genus Andrias. Amphibians of the salamander family may be, for example, animals of the genus Echinotriton and the genus Cynops. Amphibians of the toad family may be, for example, animals of the genus Rhinella and the genus Bufo. Amphibians of the tree frog family may be, for example, animals of the genus Rana, the genus Lithobates, the genus Glandirana, the genus Pelophylax, and the genus Odorrana. Amphibians of the Rhacophoridae family may be, for example, animals of the genera Kurixalus, Rhacophorus, and Buergeria.

[0080] The non-human vertebrate according to this embodiment is, for example, any animal belonging to the amphibian class, and may be, for example, a giant salamander.

[0081] The term "reptile" refers to the group of animals belonging to the subphylum Vertebrate, class Reptilia. Reptiles may be, for example, animals of the families Cheloniidae, Dermochelyidae, Geoemydidae, Chelydridae, Trionychidae, Eublepharidae, Gekkonidae, Iguanidae, Scincidae, Colubridae, Elapinae, Hydrophiinae, or Crotalinae.

[0082] Reptiles of the Chelonidae family may be, for example, animals of the genus Cheloniae (Chelonia mydas), such as the green turtle (Chelonia mydas); the genus Caretta (Caretta caretta), such as the loggerhead turtle (Caretta caretta); or the genus Eretmochelys (Eretmochelys imbricata), such as the hawksbill turtle (Eretmochelys imbricata). Reptiles of the leatherback family may be, for example, animals of the genus Dermochelys. Reptiles of the Geomydae family may be, for example, animals of the genus Mauremys (Mauremys reevesii), such as the pond turtle (Mauremys reevesii); the genus Cuora (Cuora flavomarginata), such as the box turtle (Cuora flavomarginata); or the genus Geoemyda.

[0083] Reptiles of the family Chelydidae may be, for example, animals of the genus Chelydra, such as the common snapping turtle (Chelydra serpentina); or animals of the genus Macrochelys, such as the alligator snapping turtle (Macrochelys temminckii). Reptiles of the family Pelopidae may be, for example, animals of the genus Pelodiscus, such as the soft-shelled turtle (Pelodiscus sinensis). Reptiles of the family Geckonidae may be, for example, animals of the genus Goniurosaurus, such as the banded gecko (Goniurosaurus splendens). The reptile of the Gekkonidae family may be, for example, an animal of the genus Gekko, such as the genus Lepidodactylus; the genus Hemiphyllodactylus; the genus Perochirus; the Japanese gecko Gekko japonicus, and the Amami gecko Gekko vertebralis.

[0084] The reptile of the Iguanidae family may be, for example, an animal of the Iguana genus, such as the Anolis lizard or the Green Iguana (Iguana iguana). The reptile of the Lacertidae family may be, for example, an animal of the Plestiodon genus, such as the Blue-striped Skink (Plestiodon elegans) or the Stone-streaked Skink (Plestiodon stimpsonii). The Colubridae reptile may be, for example, an animal of the genus Cyclophiops, such as the Sakishima green snake (Cyclophiops herminae); the genus Elaphe, such as the Japanese rat snake (Elaphe climacophora) and the Japanese striped snake (Elaphe quadrivirgata); the genus Calamaria, such as the Japanese long-legged snake (Calamaria pavimentata); or the genus Dinodon, such as the Japanese spotted snake (Dinodon semicarinatum).

[0085] The reptiles of the Elaphinae subfamily may be, for example, animals of the genus Sinomicrurus. The reptiles of the Ophididae subfamily may be, for example, animals of the genus Hydrophis, the genus Laticauda, ​​or the genus Emydocephalus. The reptiles of the Viperinae subfamily may be, for example, animals of the genus Protobothrops, such as the Habu (Protobothrops flavoviridis) and the Sakishima Habu (Protobothrops elegans), the genus Gloydius, such as the Japanese Mamushi (Gloydius blomhoffii), or the genus Ovophis.

[0086] The non-human vertebrate according to this embodiment is, for example, any animal belonging to the reptile family, such as a soft-shelled turtle.

[0087] The term "birds" refers to the group of animals belonging to the subphylum Vertebrata and class Aves. Birds include, for example, the families Megapodiidae, Numididae, chickens (Gallus gallus), Phasianidae (Phasianidae), such as Phasianidae (Phasianidae), Struthionidae (Ostriches), Anatidae (Ducks), Anhimidae (Salmonbirds), Columbidae (Doves), Gruidae (Cranes), Rallidae (Rallidae), Phoeniconaias (Flamingos), Steatornithidae (Nyctibiidae), Caprimulgidae (Nightjars), Gaviidae (Loons), Aptenodytes (King Penguins), Eudyptes (Rockhopper Penguins), Diomedeidae (Albatrosses), Hydrobatidae (Storks), Ciconiidae (Storks), Herons ( The animal may be from the family Ardeidae; Threskiornithidae; Pelecanidae; Anhingidae; Accipitridae; Cathartidae; Strigidae; Colius; Harpactes; Bucerotidae; Picidae; Alcedinidae; Meropidae; Falconinae; Cacatuidae; Psittaculidae; Strigopidae; or Alaudidae.

[0088] The non-human vertebrate according to this embodiment is, for example, any animal belonging to the genus Avian, which may be, for example, a chicken, an ostrich, a parakeet, an owl, or a hawk.

[0089] The term "mammal" refers to the group of animals belonging to the class Mammalia of the subphylum Vertebrata. Mammals may be, for example, Monotremata, Marsupials, Afroinsectiphilia, Paenungulata, Xenarthra, Euarchontoglires, or Laurasiatheria.

[0090] Monotreme mammals may be, for example, animals from the families Ornithorhynchidae or Tachyglossidae. Marsupial mammals may be, for example, animals from the families Didelphidae, Dasyuridae such as the Tasmanian devil (Sarcophilus harrisii), Myrmecobiidae, Phascolarctidae, Petauridae, Macropodidae such as the eastern grey kangaroo (Macropus giganteus) and wallaby.

[0091] African insectivorous mammals may be, for example, animals from the Chrysochloridae family, the Tenrecidae family, the Elephantulus family, or the Rhynchocyon family. Protounge mammals may be, for example, animals from the Procaviidae family, the Elephantidae family, the Dugongidae family, or the Trichechidae family. Xenarthra mammals may be, for example, animals from the Bradypodidae family, the Megalonychidae family, the Myrmecophagidae family, the Cyclopedae ​​family, the Chlamyphoridae family, or the Dasypodidae family.

[0092] The mammals of the primate family may be, for example, animals of the Tarsiidae (Tarsiidae), Cercopithecidae (Cercopithecidae), Hominidae (Hominidae), Hylobatidae (Hylobatidae), Spider monkeys (Atelidae), or Lemuridae (Lemuria). The mammals of the primate family may be, for example, animals of the Muridae (Muridae), Nesomyidae (Nesomyidae), Cricetidae (Cricetidae), Mole rats (Spalacidae), Calomyscidae (Calomyscidae), Jerboas (Dipodidae), or Gliridae (Dormice). The mammals of the primate family may be, for example, animals of the Leporidae (Leporidae) or Ochotonidae (Ochotonidae). The mammal of the Eucoptera may be, for example, an animal of the Cynocephalidae family. The mammal of the Eucoptera may be, for example, an animal of the Tupaiidae family;

[0093] The Laurasian mammal may be, for example, an animal from the families Erinaceidae, Solenodontidae, or Talpidae. The Laurasian mammal may be, for example, an animal from the families Pteropodidae or Vespertilionidae. The Laurasian mammal may be, for example, an animal from the families Equidae, Tapiridae, or Rhinocerotidae. Laurasian mammals may be, for example, animals from the Camelidae family (Camelidae), such as camels and llamas; Suidae family (Suidae), such as wild boars and pigs; Tragulidae family (Moschidae); Cervidae family (Cervidae), such as deer, reindeer and elephants; Bovidae family (Bovidae), such as cattle, antelopes, goats and sheep; Giraffidae family (Giraffidae), such as giraffes and okapi; Antilocapridae family (Pronghorns); Hippopotamidae family (Hippopotamidae), such as hippos and pygmy hippos; Mysticeti family (Mysticeti), such as right whales (Balaenidae); Sperm whales (Physeteridae); Delphinidae family.

[0094] The non-human vertebrate according to this embodiment is a non-human mammal, and may be, for example, any animal belonging to rodents such as mice, rats, guinea pigs, hamsters, etc., any animal belonging to non-human primates such as chimpanzees, any animal belonging to artiodactyla such as cows, goats, sheep, etc., any animal belonging to perissodactyla such as horses, or a pet animal such as a rabbit, dog, or cat.

[0095] The terms "derived" or "derived from" mean that a particular object is obtained directly or indirectly from a particular source. In one example, an early embryo derived from a non-human vertebrate includes an early embryo obtained in vitro by oviposition or artificial insemination of a non-human vertebrate, or an early embryo (e.g., a 32-cell stage embryo) obtained by culturing a fertilized egg obtained in vitro. In another example, an isolated cell derived from an embryo includes a cell obtained by separating a group of cells constituting an embryo into individual cells, or a cell obtained by culturing such a cell.

[0096] (Method for Producing Germline Chimeric Non-Human Vertebrate) A second aspect of the present disclosure provides a method for producing a germline chimeric non-human vertebrate, comprising: collecting primordial germ cells from a primordial germ cell-enriched embryo according to the present disclosure; injecting the collected primordial germ cells (donor primordial germ cells) into an embryo (host embryo) derived from a non-human vertebrate; and culturing the host embryo into which the donor primordial germ cells have been injected, wherein the germline chimeric non-human vertebrate contains the donor primordial germ cells in its gonadal region, and the non-human vertebrate that provided the donor primordial germ cells (donor non-human vertebrate) is closely related to the non-human vertebrate that provided the host embryo (host non-human vertebrate).

[0097] Embryos enriched in primordial germ cells can be prepared by the method according to the first aspect of the present disclosure. The embryos enriched in primordial germ cells are, for example, embryos at the blastula, blastula, neurula, pharyngeal embryo, or later developmental stage (including developed individuals such as juveniles, fry, chicks, or fetuses). From the viewpoint of ease of collection of primordial germ cells, the embryos enriched in primordial germ cells are embryos at the blastula, blastula, neurula, pharyngeal embryo, or later developmental stage, which are embryos before individual development, preferably blastula or blastula. Primordial germ cells can be collected from embryos according to known methods depending on the developmental stage of the embryo. Primordial germ cells can be collected from blastulas, for example, using a suction device equipped with a glass capillary. Primordial germ cells can be collected, for example, by subjecting embryos at the blastula stage or later to cell detachment treatment using a protease such as trypsin or collagenase, and then subjecting the detached cells to density gradient centrifugation to separate and concentrate the primordial germ cells.

[0098] To facilitate the isolation of primordial germ cells, a marker gene (e.g., a gene encoding a fluorescent protein) may be introduced into an early embryo derived from a non-human vertebrate in addition to the introduction of Dnd1 and Nanos. When an early embryo into which Dnd1 and Nanos have been introduced is prepared by introducing the Dnd1 and Nanos into all or most of the cells of an early embryo of a non-human vertebrate, an embryo whose development has stopped at the blastula stage obtained by culturing the early embryo will have almost all cells that are primordial germ cells into which the Dnd1 and Nanos have been introduced and which express the target gene.

[0099] The collected primordial germ cells (donor primordial germ cells) are injected into an embryo (host embryo) derived from a non-human vertebrate. The injection can be performed, for example, by microinjection. The host embryo into which the donor primordial germ cells are injected or transplanted is, for example, a blastula, a blastula, a pharyngeal embryo, or an embryo at a later developmental stage (including, for example, developed individuals such as juveniles, fry, chicks, or fetuses). Any number of donor primordial germ cells can be injected into the host embryo. For example, 10 to 1,000, 10 to 500, 10 to 250, 10 to 150, 10 to 100, 20 to 100, 10 to 75, 20 to 75, 10 to 60, 20 to 60, or 30 to 60 donor primordial germ cells can be injected into the host embryo.

[0100] In this embodiment, the non-human vertebrate that provided the donor primordial germ cells (donor non-human vertebrate) and the non-human vertebrate that provided the host embryo (host non-human vertebrate) are closely related. The term "closely related" refers to non-human vertebrates that belong to the same family. Closely related non-human vertebrates may be, for example, non-human vertebrates that belong to the same family, and preferably non-human vertebrates that belong to the same genus. In the present disclosure, "closely related" does not exclude the same species. Closely related non-human vertebrates may be, for example, non-human vertebrates that belong to the same species. Closely related non-human vertebrates are, for example, non-human vertebrates that are different species but belong to the same family. Closely related non-human vertebrates are, for example, non-human vertebrates that are different species but belong to the same genus. In one example, the donor non-human vertebrate and the host non-human vertebrate are non-human vertebrates of the same species. The donor non-human vertebrate may be of the same species as the animal that provided the donor primordial germ cells. The host non-human vertebrate may be of the same species as the animal that provided the host embryo. In one example, the host non-human vertebrate that provided the host embryo is sterilized. Sterilization of the non-human vertebrate can be carried out using known methods (e.g., genome editing technology).

[0101] In this embodiment, when the donor non-human vertebrate and the host non-human vertebrate belong to fish, the donor non-human vertebrate and the host non-human vertebrate are, for example, a combination of tuna and mackerel, a combination of salmon and rainbow trout, a combination of tiger pufferfish and grass pufferfish, or a combination of koi carp and goldfish.

[0102] According to the method of the second aspect, a chimeric non-human vertebrate containing donor primordial germ cells in its gonadal region can be obtained. The chimeric non-human vertebrate is a non-human vertebrate of the same species as the non-human vertebrate that provided the host embryo. The donor primordial germ cells may be genetically modified. Gene transfer into the donor primordial germ cells can be carried out according to the method described in the present disclosure. One embodiment of this aspect provides a method for producing a germline chimeric non-human vertebrate, comprising: introducing Dnd1 and Nanos into an early embryo derived from a donor non-human vertebrate and modifying the genes thereof; culturing the early embryo into which Dnd1 and Nanos have been introduced and whose genes have been modified to prepare an embryo enriched in primordial germ cells whose genes have been modified; collecting the primordial germ cells whose genes have been modified (donor primordial germ cells) from the embryo enriched in primordial germ cells; injecting the collected donor primordial germ cells into a host embryo derived from a non-human vertebrate; and culturing the host embryo into which the donor primordial germ cells have been injected, wherein the germline chimeric non-human vertebrate contains the genetically modified donor primordial germ cells in its gonadal region, and the donor non-human vertebrate that provided the donor primordial germ cells and the host non-human vertebrate that provided the host embryo are closely related.

[0103] The germline chimeric non-human vertebrate (host non-human vertebrate) according to the embodiment contains a genetic modification essentially only in primordial germ cells in the gonadal region (gonads). For example, even if the genetic modification is lethal or a developmentally defective modification (e.g., chromosomal tetraploidization), the method of the present embodiment can provide a germline chimeric non-human vertebrate (host non-human vertebrate) containing germ cells containing the genetic modification in its gonadal region.

[0104] In fish, triploid females do not produce eggs, so the nutrients used for egg production are used for individual growth. As a result, triploid females are larger in size and have better meat quality than normal diploid females. Triploid individuals are produced from tetraploid eggs and sperm. Individuals (tetraploids) used to prepare tetraploid eggs or sperm may exhibit poor development or growth. Therefore, it is advantageous to produce diploid individuals that contain tetraploid primordial germ cells in their gonad regions. The method of the second aspect allows the production of the aforementioned individuals. Such methods include, for example, introducing Dnd1 and Nanos into an early embryo (e.g., at least one cell of an embryo at the 4-cell to 64-cell stage) derived from a non-human vertebrate and genetically modifying the early embryo to make it tetraploid; culturing the early embryo into which Dnd1 and Nanos have been introduced and genetically modified to prepare an embryo enriched in donor primordial germ cells (tetraploid); collecting donor primordial germ cells (tetraploid) from the embryo enriched in primordial germ cells and injecting it into a host embryo (diploid) derived from a non-human vertebrate; and culturing the host embryo (diploid) injected with the donor primordial germ cells (tetraploid) to produce a germline chimeric non-human vertebrate. The germline chimeric non-human vertebrate contains donor primordial germ cells (tetraploid) in its gonadal region and its somatic cells are diploid. A triploid individual can be obtained from an egg (tetraploid) derived from the germline chimeric vertebrate and sperm (diploid) derived from a wild-type non-human vertebrate of the same species.

[0105] The germline chimeric non-human vertebrate (host non-human vertebrate) produced by the method according to the second aspect can produce a donor non-human vertebrate that contains donor primordial germ cells, as described below, in its gonad region.

[0106] (Method of Producing a Donor Non-Human Vertebrate) One aspect of the present disclosure provides a method of producing a donor non-human vertebrate, comprising mating germline chimeric non-human vertebrates (host non-human vertebrates), wherein the germline chimeric non-human vertebrate (host non-human vertebrate) comprises donor primordial germ cells in its gonadal region, and the donor non-human vertebrate comprises the donor primordial germ cells in its gonadal region.

[0107] A germline chimeric non-human vertebrate (host non-human vertebrate) containing donor primordial germ cells in its gonad region can produce a next-generation germline chimeric non-human vertebrate (donor non-human vertebrate) by, for example, natural mating or artificial insemination with the same germline chimeric non-human vertebrate (e.g., host non-human vertebrate). A germline chimeric non-human vertebrate (host non-human vertebrate) containing donor primordial germ cells in its gonad region can produce a next-generation germline chimeric non-human vertebrate (donor non-human vertebrate) by, for example, natural mating or artificial insemination with a donor non-human vertebrate.

[0108] The method according to this embodiment can be used, for example, in borrowed-litter production technology. By using an animal for which a breeding method has been established as the host non-human vertebrate (e.g., mackerel) and using a non-human vertebrate with high economic value as the donor non-human vertebrate (e.g., tuna), it is possible to easily produce a donor non-human vertebrate (e.g., tuna) from the host non-human vertebrate (e.g., mackerel) for which a breeding method has been established. More specifically, a method for producing a donor non-human vertebrate (tuna) is provided, comprising: introducing Dnd1 and Nanos into a non-human vertebrate-derived early embryo (e.g., a tuna-derived early embryo) and optionally modifying its genes; culturing the Dnd1- and Nanos-introduced early embryo (optionally modified) to prepare an embryo (tuna-derived) enriched in primordial germ cells; collecting donor primordial germ cells (tuna-derived) from the embryo and injecting them into a non-human vertebrate-derived host embryo (e.g., mackerel-derived); culturing the host embryo to produce a germline chimeric non-human vertebrate (mackerel) containing the donor primordial germ cells (tuna-derived) in its gonadal region; and mating the germline chimeric non-human vertebrate (mackerel). The early embryo is, for example, an embryo at the 1-cell to 8-cell stage (preferably the 1-cell or 2-cell stage, more preferably the 1-cell stage).

[0109] As used herein, the term "comprising" means that the recited elements and / or steps are present, and that other elements and / or steps may be added. As used herein, the term "consisting of" means that the recited elements and / or steps are present, and that other elements and / or steps are excluded. As used herein, the term "essentially consisting of" means that the recited elements and / or steps are present, and that other elements and / or steps may be added to the extent that they do not affect the novel technical features of the method for preparing an embryo enriched in primordial germ cells, the method for producing a non-human vertebrate, the germline chimeric non-human vertebrate, and the transgenic non-human vertebrate, etc. As used herein, the term "substantially free" does not exclude "completely free."

[0110] Descriptions of particular aspects or embodiments of this disclosure, and explanations of terms provided for those aspects or embodiments, also apply to other aspects and embodiments in this disclosure, as appropriate, unless expressly stated otherwise. All publications and patents mentioned in this disclosure are incorporated herein in their entireties, to the same extent as if each individual publication or patent were specifically and individually indicated to be incorporated by reference in its entirety.

[0111] Specific examples will be described below, but they are intended to illustrate preferred embodiments of the present invention and are not intended to limit the invention described in the appended claims in any way.

[0112] [Example] Synthesis of Nanos3-SV40pA and Dnd1-SV40pA mRNA The protein coding regions of medaka Nanos3 and Dnd1 were amplified from medaka ovary cDNA and the pcs2DndChDD3' vector (Non-Patent Document 2, provided by Dr. YunHan Hong), respectively, and cloned into the BamHI / KpnI site of the pGGEV-1_XcmI-LacZ vector (addgene ID: 49296). SV40pA was amplified from the cyto-YFP-FKBPx5 vector (addgene ID: 103777) and cloned into the BamHI / KpnI site of pGGEV_2'_XcmI-LacZ (addgene ID: 49303). According to the Golden GATEway (GGW) cloning method (Kirchmaier et al. Golden GATEway Cloning - A combinatorial approach to generate fusion and recombination constructs. PLoS One 8 (10): e76117. (2013)), SV40pA was ligated to the 3' ends of Nanos3 and Dnd1, respectively.

[0113] cDNAs containing the protein-coding regions of zebrafish Nanos3 and Dnd1 were prepared from mRNA expressed in zebrafish ovarian cells using reverse transcriptase. The protein-coding regions of Nanos3 and Dnd1 were amplified from the cDNAs and cloned into the pGGEV-1_XcmI-LacZ vector. SV40pA was ligated using the GGW cloning method as described above. mRNA was synthesized by in vitro transcription using the prepared DNA as a template and the T7 promoter (Figure 1).

[0114] Endogenous medaka Nanos3 and Dnd1 were mutated using the CRISPR / Cas13d system (Kushawah et al. CRISPR-Cas13d induces efficient mRNA knockdown in animal embryos. Developmental Cell 54(6):805-817.e7(2020)) (Figure 1), knocking down endogenous Nanos3 and Dnd1. This allows us to evaluate only the effects of microinjected Nanos3-SV40pA and Dnd1-SV40pA (Test 1-2).

[0115] Experiment (1): Enrichment of primordial germ cells in medaka embryos Experiment 1-1: Co-injection of Dnd1 and Nanos3 induced differentiation of almost all cells at the blastula stage into primordial germ cells. Medaka one-cell embryos (fertilized eggs) were microinjected with 40 ng / μl of Nanos3-SV40pA and Dnd1-SV40pA mRNA (medaka-derived Dnd1 / Nanos3-overexpressing embryos: Dnd1 / Nanos3-OE embryos). The mCherry-Nanos3 3'UTR was microinjected into the one-cell embryos to label primordial germ cells, and the embryos were cultured. The embryos developed to the blastula stage, but development did not progress beyond that. In embryos whose development was arrested at the blastula stage, almost all cells became mCherry-positive, and almost all cells in the blastula became primordial germ cells (PGCs) (FIGS. 2A and 2B).

[0116] Approximately 50 primordial germ cells from these donor embryos were microinjected into wild-type host embryos, and the embryos were cultured. Surprisingly, many of the transplanted cells migrated to the gonads, similar to PGCs (Figure 2C). In embryos at later stages of development, at least 200 PGCs were observed in the gonad region (Figure 2D). This result indicates that a significantly greater number of PGCs were formed than in wild-type embryos, which typically form around 10 to 40 PGCs.

[0117] Forced expression of Dnd1 alone increases the number of primordial germ cells by approximately 1.5 to 2 times compared to the wild-type (Non-Patent Document 2). Regarding the number of primordial germ cells formed, the method according to the present disclosure increases the number of primordial germ cells by approximately 30 times or more compared to the wild-type ( FIG. 2D ). Therefore, the method according to the present disclosure can significantly increase the number of primordial germ cells compared to the forced expression of Dnd1 alone.

[0118] Test 1-2: Comparison of introduction of both Dnd1 / Nanos3 with introduction of Dnd1 alone or Nanos3 alone in primordial germ cell formation As shown in Test 1-1, when both Dnd1 / Nanos3 were introduced into one-cell stage embryos (fertilized eggs) and overexpressed (OE), development did not progress beyond the blastula stage, and the individual did not develop (hatch). Therefore, we tested whether embryos with an increased number of primordial germ cells would develop (hatch) normally by introducing Dnd1 / Nanos3 into only one cell of a 16-cell stage embryo.

[0119] To eliminate the effects of endogenous Dnd1 and Nanos3, we knocked down (KD) endogenous Dnd1, Nanos3, or both using the CRISPR-Cas13d system (Figure 3A). Specifically, we microinjected Cas13d mRNA, gRNAs for Dnd and Nanos3 (Dnd1 / Nanos3 gRNA), and mCherry-Nanos3 3'UTR (for PGC labeling) into one-cell embryos of transgenic medaka fish (olvas-EGFP), whose germ cells are labeled with EGFP, to knock down endogenous Dnd, Nanos3, or both.

[0120] In 16-cell embryos, 16 cells form a single cell layer of 4 cells x 4 cells (schematic diagram in Figure 3B). The localization of germ plasm factors (germ granule factors) in 16-cell embryos was examined using BuckyBall:EGFP medaka fish. Germ granule factors were barely localized in cells outside the single cell layer (fluorescence image in Figure 3B). To investigate whether cells destined to become cells other than primordial germ cells (PGCs) without germ granule factors could be induced to differentiate into PGCs by introducing Dnd1 / Nanos3, 10 ng / μl of Dnd1-SV40pA or Nanos3-SV40pA alone, or both (Dnd1 / Nanos3) were microinjected into a single cell outside the single cell layer (the cell marked with an * in the fluorescence image in Figure 3B).

[0121] Embryos microinjected with Dnd1 / Nanos3 developed normally (107 individuals). Similar to the germline chimeras in Experiment 1-1, 99 of the 107 individuals (93%) had significantly increased numbers of olvas-EGFP-positive primordial germ cells in the gonadal regions compared to control individuals (Figure 3C) (Figure 3D). In embryos microinjected with Dnd1 or Nanos3 alone, olvas-EGFP-positive primordial germ cells were confirmed in 18% (19 of 104 individuals) and 23% (23 of 102 individuals), respectively. Furthermore, the number of primordial germ cells formed per individual was very low, averaging 3.4 ± 2.4 cells (Figures 3E and F).

[0122] Importantly, Experiments 1-2 demonstrate that injection of both Dnd1 and Nanos3 into at least one cell of a 16-cell stage embryo can result in the development of an individual with a significantly increased number of primordial germ cells in its gonadal region. Injection of both Dnd1 and Nanos3 into a single cell of a 4-cell stage embryo, an 8-cell stage embryo, and a 32-cell stage embryo also resulted in the development of an individual with a significantly increased number of primordial germ cells in its gonadal region. Thus, the examples of the present disclosure demonstrate that injection of both Dnd1 and Nanos3 into a predetermined percentage of cells in an early embryo (4-cell to 32-cell stage embryos) can result in the development of an individual with a significantly increased number of primordial germ cells in its gonadal region.

[0123] Experiments 1-2 show that cells co-injected with Dnd1 and Nanos3 do not necessarily have to be cells destined to become primordial germ cells (i.e., cells in which germ granule factors are localized). Furthermore, experiments 1-2 show that injection of both Dnd1 and Nanos3 results in the formation of significantly greater numbers of primordial germ cells than injection of either gene alone.

[0124] Test 1-3: Primordial germ cells formed with Dnd1 / Nanos3-OE express representative germ granule factors. In Test 1-2, expression of olvas-EGFP was observed in most of the numerous primordial germ cells formed by Dnd1 / Nanos3-OE. olvas-EGFP is a marker for monitoring the expression of the germ granule factor vasa with EGFP. The medaka embryos used in Test 1-2 had wild-type mothers and olvas-EGFP transgenic fathers. Therefore, the expression of the vasa gene observed in the developed embryos was not of maternal origin accumulated in the eggs, but rather of genes derived from the formed primordial germ cells themselves (embryonic origin). We examined the expression of representative germ granule factors (piwil1, piwil2, dazl, tdrd1, tdrd9, and tdrd12) other than vasa by in situ hybridization. As a result, in addition to the vasa gene, all of the germ granule factors examined were expressed in primordial germ cells formed in Dnd1 / Nanos3-OE embryos (Fig. 4).

[0125] Experiments 1-2 and 1-3 show that the primordial germ cells formed by Dnd1 / Nanos3-OE have similar properties to endogenous primordial germ cells in terms of gene expression, in addition to their ability to migrate to the gonads.

[0126] Test 1-4: mRNA encoding both Dnd1 and Nanos3 In Test 1-1, two types of mRNA, mRNA encoding Dnd1 and mRNA encoding Nanos3, were simultaneously microinjected. In Test 1-4, a test similar to Test 1-1 was performed using single-stranded mRNA encoding both Dnd1 and Nanos3. As a result, similar to Test 1-1, embryos microinjected with the single-stranded mRNA stopped developing at the blastula stage, and almost all cells in the blastula became primordial germ cells.

[0127] Experiment (2): Enrichment of primordial germ cells in zebrafish embryos We tested whether the technique for increasing primordial germ cells demonstrated in medaka in Experiment (1) can be applied to other fish species. In Experiment (2), we used zebrafish, which are lineage-distinct from medaka.

[0128] Following the method described in Experiment 1-1 (Figure 2A), 75 ng / μl of Nanos3-SV40pA, 90 ng / μl of Dnd1-SV40pA, and 40 ng / μl of EGFP-Nanos3 3'UTR (PGC marker) mRNA were microinjected into one-cell stage zebrafish embryos (fertilized eggs). Donor embryos (embryos overexpressing zebrafish-derived Nanos3 and Dnd1: DN-OE embryos) transfected with the mRNA were cultured to the blastula stage (Figure 5A, right). Primordial germ cells (donor primordial germ cells) were collected from the cultured blastula and microinjected into untreated zebrafish embryos (host embryos) (Figure 5B, right).

[0129] Control donor embryos were prepared by microinjecting 40 ng / μl EGFP-Nanos3 3'UTR mRNA alone into one-cell stage zebrafish embryos (donor embryos) (Fig. 5A, left). The control donor embryos were cultured to the blastula stage, and primordial germ cells (PGCs) were collected and injected into untreated host embryos (Fig. 5B, left).

[0130] Compared to control embryos, DN-OE embryos had stronger EGFP fluorescence and Nanos3 expression was observed throughout the cells of the embryo (Fig. 5A). Similar to the results of medaka in Test 1-1, DN-OE embryos did not progress normally to the sacculture stage, and individuals did not develop (hatch) (Fig. 5A).

[0131] By culturing host embryos injected with donor primordial germ cells derived from DN-OE embryos, 16 germline chimeras (DN-OE chimeras) containing donor primordial germ cells in their gonad regions were obtained. Numerous EGFP-positive primordial germ cell-like cells were observed in 16 of the 16 individuals (100%) (Figure 5D). By culturing host embryos injected with donor primordial germ cells derived from control embryos, 18 germline chimeras containing control donor primordial germ cells in their gonad regions were obtained. None of the 18 individuals (0%) showed EGFP-positive primordial germ cell-like cells (Figure 5C).

[0132] EGFP-positive cells were observed in the gonad region of all four hatched DN-OE chimeras (Fig. 5E), and VASA protein was expressed in these EGFP-positive cells (Fig. 5F).

[0133] Study (2) shows that co-introduction of Dnd1 and Nanos3 can induce the formation of embryos containing an increased number of primordial germ cells in zebrafish, and that the formed primordial germ cells have properties similar to those of endogenous primordial germ cells.

[0134] Test (3): PGCs formed by Dnd1 / Nanos3-OE produce functional gametes. solvas-EGFP / sox9b-DsRed transgenic medaka fish were prepared as donors. In these transgenic medaka fish, germ cells are labeled with EGFP, and notochord and chondrocytes are labeled with DsRed. Wild-type medaka fish (Cab strain) were prepared as hosts. Host embryos were sterilized by knocking out dnd / nanos3 using the CRISPR / Cas13d system.

[0135] Donor cells were collected from blastula-stage embryos derived from the transgenic medaka fish. They were transplanted into blastula-stage host embryos by microinjection using a glass capillary, resulting in 20 or fewer donor cells per embryo. Transplantation was performed on four to five host embryos. These transplantation conditions are referred to as "Test N20." Using a similar method, donor cells were transplanted into blastula-stage host embryos, resulting in 50 or more donor cells per embryo. Transplantation was performed on two to three host embryos. These transplantation conditions are referred to as "Test N50." The transplanted chimeric embryos were reared in saline containing antibiotics and then hatched. The hatched medaka fish were reared in fresh water for three months to reach sexual maturity. The creation of germline chimeras was determined by whether or not olvas-EGFP-positive PGCs were incorporated into the gonads.

[0136] The fertility of the germline chimeric individuals was confirmed by pair-mating them with wild-type medaka fish. After mating, the chimeric individuals were dissected and examined for the presence or absence of donor-derived germ cells based on EGFP fluorescence in the gonads.

[0137] In Test N20, when donor cells derived from untreated donor embryos (not injected with Dnd1 / Nanos3) were used, the rate of germline chimeras was 0% (Test Result 3-1). When donor cells derived from donor embryos microinjected with Dnd1 / Nanos3 (Dnd1 / Nanos3-OE) were used, the rate of germline chimeras was 89%. Of the germline chimeras, 74% (14 of 19) were fertile (Test Result 3-2).

[0138] In Test N50, when donor cells derived from untreated donor embryos were used, the rate of germline chimerism was 48%, of which 100% (7 out of 7 individuals) were fertile (Test Result 3-3).When donor cells derived from Dnd1 / Nanos3-OE donor embryos were used, the rate of chimerism was 100%, of which 87% (16 out of 18 individuals) were fertile (Test Result 3-4).

[0139] Next-generation embryos were obtained from individuals in which fertility was confirmed. All of the next-generation embryos were olvas-EGFP / sox9b-DsRed positive, indicating that the next-generation embryos were derived from donor gametes. Furthermore, olvas-EGFP-positive germ cells were detected in all of the next-generation embryos, indicating that germ cell formation had progressed normally in the next-generation embryos (Figure 6).

[0140] Fertility improved by co-injection of the gametogenesis initiation factor dazl. Test results 3-2 and 3-4 showed that some germline chimeras obtained using donor cells derived from Dnd1 / Nanos3-OE donor embryos were infertile. The cause of infertility in germline chimeras was investigated. As a result, it was found that germ cells were missing in four of the seven infertile individuals. The remaining three individuals had underdeveloped gonads.

[0141] Furthermore, the gonads were examined 10 days after hatching. As a result, all XX individuals obtained by transplanting donor cells derived from untreated embryos into host embryos had begun oogenesis. On the other hand, oogenesis was confirmed in 5 out of 14 chimeric XX individuals obtained by transplanting donor cells derived from dnd1 / nanos3-OE embryos into host embryos. The remaining 8 individuals had only undifferentiated germ cells (n = 8), and 1 individual lacked germ cells (n = 1). These results indicate that none of the cells differentiated into germ cells by dnd1 / nanos3-OE had the ability to initiate gametogenesis.

[0142] We attempted to improve the fertility of germline chimeras obtained using donor cells derived from dnd1 / nanos3-OE donor embryos. Dazl mRNA, required for the initiation of gametogenesis, was microinjected into donor embryos together with dnd / nanos3 mRNA. More than 50 cells from these donor embryos were transplanted into sterilized host embryos (Test N50). The resulting germline chimeras were 100%, of which 96.5% (26 of 27) were fertile. One individual in which fertility could not be confirmed was dissected. Normal testis formation was confirmed, indicating that gametogenesis in this individual was normal. These results demonstrate that the use of dazl together with dnd / nanos3 can improve fertility in germline chimeras derived from Dnd1 / Nanos3-OE.

[0143] Test (4): Enrichment of primordial germ cells in medaka embryos by introducing medaka dnd1 / nanos2 In Test 4, a method essentially similar to Test 1-2 was performed on 16-cell stage medaka embryos, except that nanos2 was used instead of nanos3. Specifically, 20 medaka embryos were used. mRNA encoding nanos2 and mRNA encoding dnd1 were co-injected into one outer cell of the medaka embryos. As a result, 20 individuals were generated. Primordial germ cells were enriched in all 20 individuals (Figure 7B). Figure 7A shows the results of a method similar to Test 1-4 performed on 16-cell stage embryos.

[0144] Generally, nanos genes include nanos1, nanos2, and nanos3. Nanos2 is known to function in germline stem cells in adult fish, but not in primordial germ cells in embryos. Test 4 demonstrated that nanos2 has the ability to form primordial germ cells in embryos.

[0145] Experiment (5): Enrichment of primordial germ cells in medaka embryos by introduction of zebrafish dnd1 / nanos3 In Experiment 5, a method essentially similar to Experiments 1-2 was performed on 16-cell stage medaka embryos, except that zebrafish-derived dnd1 / nanos3 (Zdnd1, Znanos3) was introduced instead of medaka-derived dnd1 / nanos3. Specifically, 13 medaka embryos were used. One outer cell of the medaka embryo was co-injected with mRNA encoding Znanos3 and single-stranded mRNA encoding Zdnd1. As a result, 13 individuals were generated. Primordial germ cells were enriched in all 13 individuals (Figure 7C).

[0146] Experiment 5 demonstrated that nanos and dnd from zebrafish, a member of the Cypriniformes, can enrich for primordial germ cells in embryos of medaka, a member of the Garfishes. The results of Experiment 5 suggest that the present invention can be practiced without obtaining (cloning) Nanos and Dnd1 from the animal species from which primordial germ cells are to be obtained. For example, if Nanos and Dnd1 from another animal species belonging to the same division (preferably the same class) as the animal species from which primordial germ cells are to be obtained (e.g., a fish species with high economic value) are available, they can be co-introduced into embryos of the animal species from which primordial germ cells are to be obtained, thereby enriching for primordial germ cells of that animal species.

Claims

Introducing Dnd1 and Nanog into an initial embryo derived from an animal belonging to fish; and A method for preparing an embryo enriched with primordial germ cells, comprising culturing the initial embryo into which Dnd1 and Nanog have been introduced, wherein Dnd1 and Nanog are each derived from an animal belonging to the same phylum as the animal belonging to fish that provided the initial embryo.

2. The method according to claim 1, wherein Dnd1 and Nanog are proteins or nucleotides encoding them.

3. When Dnd1 and Nanog are RNAs encoding them, they contain a poly-A sequence at their 3'-end side, and when Dnd1 and Nanog are DNAs encoding them, they contain an exogenous polyadenylation sequence, according to the method of claim 1.

4. The method according to claim 1, further comprising introducing a gametogenesis factor into the initial embryo.

5. The method according to claim 4, wherein the gametogenesis factor is at least one selected from the group consisting of Daz, Dazl, Boule, meioC, Ythdc2, and Rbm46.

6. The method according to claim 1, wherein the initial embryo is an embryo from the 1-cell stage to the 64-cell stage.

7. The method according to claim 1, further comprising modifying the gene of the initial embryo derived from a non-human vertebrate.

8. A method for producing a transgenic non-human vertebrate, comprising introducing Dnd1 and Nanog into an initial embryo derived from an animal belonging to fish and modifying the gene of the initial embryo; and culturing the initial embryo into which Dnd1 and Nanog have been introduced and the gene of which has been modified to generate a transgenic non-human vertebrate, wherein Dnd1 and Nanog are each derived from an animal belonging to the same phylum as the animal belonging to fish that provided the initial embryo, the initial embryo is an embryo from the 4-cell stage to the 64-cell stage, and the transgenic non-human vertebrate contains primordial germ cells with modified genes in its gonadal region.

9. Collecting donor primordial germ cells from an embryo enriched with primordial germ cells prepared by the method according to any one of claims 1 to 7; injecting the collected donor primordial germ cells into a host embryo derived from a non-human vertebrate; and culturing the host embryo into which the donor primordial germ cells have been injected, a method for producing a germline chimeric non-human vertebrate, comprising The germline chimeric non-human vertebrate contains the donor primordial germ cells in its gonadal region, and a method in which the donor non-human vertebrate that provided the donor primordial germ cells and the host non-human vertebrate that provided the host embryo each belong to fish and are closely related to each other.

10. Producing a germline chimeric non-human vertebrate, and A method for producing a non-human vertebrate, comprising mating the germline chimeric non-human vertebrate, wherein producing the germline chimeric non-human vertebrate comprises collecting donor primordial germ cells from an embryo enriched with primordial germ cells prepared by the method according to any one of Claims 1 to 7; injecting the collected donor primordial germ cells into a host embryo derived from a non-human vertebrate; and culturing the host embryo into which the donor primordial germ cells have been injected, wherein the germline chimeric non-human vertebrate contains the donor primordial germ cells in its gonadal region, the donor non-human vertebrate that provided the donor primordial germ cells and the host non-human vertebrate that provided the host embryo each belong to fish and are closely related to each other, and the produced non-human vertebrate contains donor primordial germ cells derived from the donor primordial germ cells in its gonadal region.

11. The method according to Claim 9, wherein the host non-human vertebrate and the donor non-human vertebrate are a combination of mackerel and tuna, a combination of salmon and rainbow trout, a combination of tiger pufferfish and grass pufferfish, or a combination of goldfish and koi.

12. The method according to Claim 10, wherein the host non-human vertebrate and the donor non-human vertebrate are a combination of mackerel and tuna, a combination of salmon and rainbow trout, a combination of tiger pufferfish and grass pufferfish, or a combination of goldfish and koi.

13. An embryo derived from an animal belonging to fish, enriched with primordial germ cells, wherein the embryo is an embryo after the blastula stage, and the number of primordial germ cells in the embryo is at least 5 times greater than the number of primordial germ cells in an embryo of the same developmental stage derived from a non-human vertebrate of the same species as the embryo.

14. An embryo enriched with primordial germ cells, prepared by the method according to any one of Claims 1 to 7.