Method for preparing embryos 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

Technical Field

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

Background Art

[0002] The available amount of natural fishery resources has been at a low level in recent years. In order to artificially secure fishery resources, various aquaculture methods have been developed. It is not reasonable to breed broodstock for all fish species used as food and establish seed production technologies including reproductive control. If gametes of a fish species (donor fish species) aiming at seed production can be produced using a fish species with established aquaculture methods as broodstock (host fish species), the labor required for the development of production technology can be reduced. Such a production method is also called surrogacy. A surrogacy technology has been developed to produce gametes of donor fish species with high industrial value such as tuna in closely related species (host fish species, for example, mackerel) that are easy to breed and inexpensive.

[0003] The surrogacy technology includes isolating primordial germ cells (PGCs) or germ stem cells that are the source of gametes from embryos or the gonads of adult bodies of donor species, transplanting the cells into surrogate parents (host fish species), and producing functional gametes of donor fish species. PGCs formed in the early stage of embryo development are useful as donor cells because it is easy to endow useful genetic characteristics by chromosome manipulation technology (technology for controlling the number and combination of chromosomes). In addition, if one PGC is transplanted into a host fish species, functional gametes of the donor fish species are continuously produced in the host fish species, which is useful (Non-Patent Document 1).

[0004] As a technique to increase the number of PGCs in embryos, a method has been developed in medaka fish in which mRNA encoding dead end1 (Dnd1), one of the germ plasm components (germ granule factors), is introduced into one-cell stage fertilized eggs (Non-Patent Literature 2). Non-Patent Literature 2 reports that when Dnd1 is expressed throughout the cells in the early stages of development, the number of PGCs increases by approximately 1.5 to 2 times.

[0005] Even when the method described in Non-Patent Document 2 was applied to medaka, which belongs to the Beloniformes order, and zebrafish, which belongs to the Cypriniformes order and is a distantly related species, the number of PGCs did not increase (Non-Patent Document 3). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Saito et al. Xenogenesis in teleost fish through generation of germ-line chimeras by single primordial germ cell transplantation.Biology of Reproduction 78,159-166(2008) [Non-Patent Document 2] Hong et al. Dnd is a critical specifier of primordial germ cells in the medaka fish.Stem Cell Reports 6,411-421(2016) [Non-Patent Document 3] Weidinger et al.dead end,a novel vertebrate germ plasm component,is required for zebrafish primordial germ cell migration and survival.Current Biology 13,1429-1434(2003) [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] Only about 10 to 40 primordial germ cells are formed per embryo. For example, in the field of surrogacy, there is a need for methods to significantly increase the number of primordial germ cells per embryo. [Means for solving the problem]

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

[0009] This disclosure provides the following inventions. [Item 1] A method for preparing an embryo enriched with primordial germ cells, comprising introducing Dnd1 and Nanos into an early embryo of non-human vertebrate origin; and culturing the early embryo into which Dnd1 and Nanos have been introduced. [Clause 2] The method according to Clause 1, wherein Dnd1 and Nanos are proteins or coding nucleotides. [Clause 3] The method according to Claim 1, wherein if Dnd1 and Nanos are encoding RNA, they include a polyA sequence at their 3' end, and if Dnd1 and Nanos are encoding DNA, they include an exogenous polyadenylated sequence. [Clause 4] The method according to any one of Claims 1 to 3, further comprising introducing a gamete formation factor into the early embryo. [Clause 5] The method according to Claim 4, wherein the gamete formation factor is at least one selected from the group consisting of Daz, Dazl, Boule, meioC, Ythdc2, and Rbm46. [Item 6] The method according to any one of items 1 to 5, wherein the early embryo is an embryo from the 1-cell stage to the 64-cell stage. [Clause 7] The method according to any one of Clauses 1 to 6, wherein the non-human vertebrate is an animal belonging to the fish class. [Clause 8] The method according to any one of Clauses 1 to 7, further comprising modifying the genes of the early embryo of non-human vertebrate origin. [Item 9] A method for producing a transgenic non-human vertebrate, comprising introducing Dnd1 and Nanos into an early embryo of non-human vertebrate and modifying the genes of the early embryo; and culturing the early embryo into which Dnd1 and Nanos have been introduced and the genes have been modified to produce a transgenic non-human vertebrate, wherein the early embryo is an embryo between the 4-cell stage and the 64-cell stage, and the transgenic non-human vertebrate includes genetically modified primordial germ cells in its gonadal region. [Item 10] A method for producing a germline chimeric nonhuman vertebrate, comprising: collecting donor primordial germ cells from an embryo enriched with primordial germ cells prepared by any of the methods in Items 1 to 8; injecting the collected donor primordial germ cells into a host embryo of nonhuman vertebrate origin; and culturing the host embryo into which the donor primordial germ cells have been injected, wherein the germline chimeric nonhuman vertebrate contains the donor primordial germ cells in its gonadal region, and the donor nonhuman vertebrate that provided the donor primordial germ cells is closely related to the host nonhuman vertebrate that provided the host embryo. [Item 11] A method for producing a donor non-human vertebrate, comprising mating a germline chimeric non-human vertebrate produced by the method described in Item 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. [Clause 12] The method according to Clause 10 or 11, wherein the donor nonhuman vertebrate and the host nonhuman vertebrate belong to the fish, and the host nonhuman vertebrate and the donor nonhuman vertebrate are a combination of mackerel and tuna, salmon and rainbow trout, tiger pufferfish and grass pufferfish, or goldfish and koi. [Item 13] An embryo of non-human vertebrate origin enriched with primordial germ cells, wherein the embryo is in 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 of the same species and at the same developmental stage from a non-human vertebrate. [Item 14] A primordial germ cell-enriched embryo prepared by any of the methods described in items 1 to 8, or a non-human vertebrate produced by any of the methods described in items 7 to 12. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1 is a flowchart showing the synthesis procedure for Nanos3-SV40pA mRNA and Dnd1-SV40pA mRNA. [Figure 2] Figure 2A is a schematic diagram showing the procedure for creating germline chimeric medaka. Figure 2B is a microscopic image of a donor embryo co-introduced with Dnd1 / Nanos3. Figure 2C is a microscopic image of a host embryo into which donor primordial germ cells collected from the 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 gonadal region. [Figure 3]Figure 3A is a schematic diagram showing the introduction of genes into a one-cell stage embryo to knock down endogenous Dnd1 and / or Nanos3. Figure 3B left 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. Figure 3B right is a microscopic image showing a 16-cell stage embryo expressing BuckyBall:EGFP. Figure 3C is a fluorescence image showing the gonadal region (arrowhead) of a control medaka. Figure 3D is a fluorescence image showing the gonadal region (arrowhead) of a medaka developed from an embryo co-introduced with Dnd1 / Nanos3. Figure 3E is a fluorescence image showing the gonadal region (arrowhead) of a medaka developed from an embryo introduced with Dnd1. Figure 3F is a fluorescence image showing the gonadal region (arrowhead) of a medaka developed from an embryo introduced with Nanos3. [Figure 4] Figure 4 shows microscopic images of the expression of a series of germ granule factors. [Figure 5] 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 developed from a DN-chimeric embryo. Figure 5F is a microscopic image showing donor-derived primordial germ cells in the gonadal region of a chimeric medaka developed from a DN-chimeric embryo. [Figure 6] Figure 6 shows a fluorescence microscope image of a medaka embryo. The medaka embryo is a next-generation medaka embryo that 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 a magnified view of the area enclosed by the white frame in the center of the fluorescence image. The arrow in the lower left image indicates germ cells (green fluorescence) formed in the reproductive region. [Figure 7]Figure 7A shows fluorescence microscopy images of medaka embryos co-introduced with medaka-derived Dnd1 / Nanos3. Figure 7B shows fluorescence microscopy images of medaka embryos co-introduced with medaka-derived Dnd1 / Nanos2. Figure 7C shows fluorescence microscopy images of medaka embryos co-introduced with zebrafish-derived Dnd1 / Nanos3 (Zdnd1+Znanos3). The area enclosed by the dotted line indicates the gonad region enriched with primordial germ cells. [Modes for carrying out the invention]

[0011] (Method for preparing embryos enriched with primordial germ cells) A first aspect of this disclosure provides a method for preparing a primordial germ cell-enriched embryo, comprising introducing Dnd1 and Nanos into an early embryo of non-human vertebrate origin; and culturing the early embryo into which Dnd1 and Nanos have been introduced. A primordial germ cell-enriched embryo is prepared by the method according to the first aspect. Accordingly, another aspect of this disclosure provides a primordial germ cell-enriched embryo prepared by the method according to the first aspect. Another aspect of this disclosure provides a primordial germ cell-enriched embryo of non-human vertebrate origin, wherein the embryo is in 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 of the same developmental stage from a non-human vertebrate of the same species as the embryo.

[0012] The term "primordial germ cell" means a cell that differentiates into a germ cell. Primordial germ cells can ultimately differentiate into eggs or sperm through meiosis. Primordial germ cells can be identified, for example, based on known primordial germ cell markers. Known germ cell markers can be, for example, the 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 can be, for example, vasa, piwil1, piwil2, dazl, tdrd1, tdrd9, or tdrd12. Primordial germ cells express at least one selected from the group consisting of, for example, 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 a nucleotide in which a promoter of the above-described germ cell marker or germ granule factor is 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 derived from the fluorescent protein. Known methods include introducing an mRNA containing the 3' untranslated region (UTR) of the above-described 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 of the fluorescent protein. The fluorescent protein can be, for example, green fluorescent protein, red fluorescent protein, yellow fluorescent protein, or blue fluorescent protein. The term "untranslated region" means a region in mRNA that is not translated into a protein adjacent to the coding region.

[0014] The primordial germ cells according to this aspect are induced to differentiate by introducing Dnd1 and Nanos into an early embryo of a non-human vertebrate. The term "early embryo" includes embryos from the 1-cell stage to the 64-cell stage. The early embryo is, for example, an embryo from the 1-cell stage to the 32-cell stage. The early embryo is obtained, for example, from sperm and an egg by natural mating or artificial insemination. An early embryo after the 2-cell stage can be obtained, for example, by culturing a fertilized egg.

[0015] The term "embryo" encompasses cells or cell masses in the developmental stage after the fertilized egg of vertebrates. The embryo may be, for example, an embryo at the 1-cell stage (also referred to as a "fertilized egg"). The embryo may be, for example, a developed individual (such as a larva, fry, chick or fetus). In one example, the embryo does not include a developed individual. The term "morula" includes embryos from the 64-cell stage to the blastocyst stage. The morula is, for example, an embryo containing 65 to 500 cells. The term "blastula" means an embryo containing a cell layer surrounding a cavity filled with a liquid called a blastocoel. The term "gastrula" is also referred to as a gastrula embryo and means an embryo in which the gastrula has formed. In a gastrula, for example, the differentiation of the ectoderm, mesoderm and endoderm is observed. The differentiation of the ectoderm, mesoderm and endoderm can be examined, for example, using their layer structure or known markers. The term "neurula" means an embryo from the time when the neural plate is formed until 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" means an embryo in the embryonic development stage where pharyngeal arches are seen. The pharyngeal arches have a pillar-like protruding form and refer to structures that differentiate into the head and neck.

[0016] A morula, blastula, gastrula, neurula or Pharyngula can be obtained, for example, by culturing a fertilized egg. An early embryo, morula, blastula, gastrula, neurula or Pharyngula can be identified, respectively, for example, by observing or detecting the structure of the embryo (such as the number of cells; alveolar cavity; structure of the primitive gut; layer structure such as ectoderm, mesoderm, endoderm; neural plate; pharyngeal arch) or the expression of specific markers under a microscope.

[0017] Dnd1 and Nanos, introduced into early embryos of non-human vertebrates, may be in the form of proteins, nucleotides, or a combination thereof. Both Dnd1 and Nanos may be proteins, or nucleotides that code for both, or a combination thereof. One of Dnd1 and Nanos may be a protein and the other a nucleotide that codes for it. Dnd1 and Nanos are preferably both proteins, or mRNAs that code for both, or a combination thereof, so that the effects of Dnd1 and Nanos in the developing embryo can be reduced or ignored. Dnd1 and Nanos are preferably coding DNA so that a sustained effect of Dnd1 and Nanos in the developing embryo can be expected.

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

[0020] The nucleotide 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. If the nucleotide encoding Dnd1 is DNA, it can be synthesized using known genetic engineering techniques. If the nucleotide encoding Dnd1 is RNA, it can be prepared by in vitro transcription of the DNA encoding Dnd1. The Dnd1 protein of a given species is commercially available or can be prepared according to known methods. The known methods include isolating and purifying (e.g., purification using HPLC) the Dnd1 protein from a natural source containing the Dnd1 protein. The Dnd1 protein can be prepared according to genetic engineering techniques. The genetic engineering techniques include, for example, creating a plasmid in which the nucleotide encoding Dnd1 is incorporated 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 isolating and purifying it from the host cells or culture supernatant.

[0021] Dnd1 may have an amino acid sequence different from that of wild-type Dnd1 from a non-human vertebrate that provided an early embryo into which Dnd1 was introduced, as long as it has the function described in this embodiment. Dnd1 that has the function described in this embodiment and has an amino acid sequence different from that of wild-type Dnd1 is also referred to as a "Dnd1 variant". A Dnd1 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 wild-type Dnd1. A Dnd1 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) RNA-binding affinity of wild-type Dnd1. A variant of Dnd1 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 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) RNA-binding affinity of the wild-type Dnd1. The Dnd1 variant includes wild-type Dnd1 from a non-human vertebrate and a different animal species that provided the early embryo into which it is introduced. The Dnd1 variant includes, for example, wild-type Dnd1 from a non-human vertebrate and a different animal species that provided the early embryo into which Dnd1 and Nanos are introduced.

[0022] The RNA-binding domain in Dnd1 contains an amino acid sequence that binds to RNA and is conserved among vertebrates. The RNA-binding domain in Dnd1 is publicly 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 contains the amino acid sequence represented by EVFISQIPRDVYEDLLIPLFSSVGALWEFRLMMNFSGQNRGFAYAKYGTAAIANDAIHLLHGYPLGPGARLSV (SEQ ID NO: 7).

[0023] The binding affinity of Dnd1 to RNA can be investigated by RNA immunoprecipitation. The RNA immunoprecipitation method includes, for example, crosslinking a host cell (test sample) into which a nucleotide encoding a fusion protein of Dnd1 and a labeled protein (e.g., a fluorescent protein or FLAG) has been introduced with formaldehyde; extracting the nucleus from the host cell to obtain a nuclear suspension; homogenizing the nuclear suspension to shear the chromatin and obtain an RNA solution; adding an antibody against the labeled protein to the RNA solution to immunoprecipitate the complex of the crosslinked RNA and the fusion protein; recovering the RNA from the precipitate and reverse transcribing it into cDNA; and quantifying the immunoprecipitated RNA by quantitative PCR of the cDNA. Host cells without the introduced nucleotide (negative control) can be subjected to the RNA immunoprecipitation method to quantify the immunoprecipitated RNA of the negative control. The binding affinity of Dnd1 to RNA 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 binding affinity of Dnd1 to RNA can be determined by RNA immunoprecipitation using cells derived from a non-human vertebrate that provided the early embryo into which Dnd1 was introduced as host cells. The host cells are preferably derived from the same animal species as the non-human vertebrate that provided the early embryo into which Dnd1 was introduced. The labeling protein is, for example, green fluorescent protein.

[0024] The term "Nanos" refers to an RNA-binding protein having a zinc finger motif, which is conserved among vertebrates and invertebrates. In the method according to this disclosure, Nanos 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 is, for example, an animal belonging to the same phylum or subphylum as the non-human vertebrate that provided the early embryo into which Nanos was introduced, preferably an animal belonging to the same class or subclass, an animal belonging to the same order or suborder, an animal belonging to the same family or subfamily, or an animal belonging to the same genus or subgenus. Nanos preferably includes Nanos derived from an animal of the same species as the non-human vertebrate into which it is introduced. Nanos may be, for example, Nanos1, Nanos2, or Nanos3, or a mixture thereof. Nanos is, for example, Nanos2 or Nanos3 or a mixture thereof, preferably Nanos3.

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

[0026] The nucleotide encoding Nanos of a given species can be prepared, for example, from cells of that species (e.g., ovarian cells) by RT-PCR using reverse transcriptase. If the nucleotide encoding Nanos is DNA, it can be synthesized using known genetic engineering techniques. If the nucleotide encoding Nanos is RNA, it can be prepared by in vitro transcription of the Nanos-encoding DNA. The Nanos protein of a given species is commercially available or can be prepared according to known methods. The known methods include isolating and purifying (e.g., purification by HPLC) the Nanos from a natural source containing the Nanos protein. The Nanos protein can be prepared according to genetic engineering techniques. The genetic engineering techniques include, for example, creating a plasmid in which the nucleotide encoding Nanos is incorporated 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 isolating and purifying it from the host cells or culture supernatant.

[0027] Nanos may have an amino acid sequence different from that of wild-type Nanos from a non-human vertebrate that provided an early embryo into which Nanos was introduced, as long as it has the function described in this embodiment. Nanos that has the function described in this embodiment and has an amino acid sequence different from that of wild-type Nanos are also referred to as "Nanos variants". Nanos variants have, 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 wild-type Nanos. Nanos variants have, 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) 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 the 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) RNA-binding affinity of the wild-type Nanos. The Nanos variant includes wild-type Nanos from a non-human vertebrate and a different animal species that provided the early embryo into which it is introduced. The Nanos variant includes, for example, wild-type Nanos from a non-human vertebrate and a different animal species that provided the early embryo into which Dnd1 and Nanos are introduced.

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

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

[0030] A “variant” to a wild-type protein may include deletions, substitutions, additions, or combinations thereof to the amino acid sequence of the wild-type protein. “Deletion” of an amino acid means the absence of an amino acid residue at any position in a given amino acid sequence. For example, a Dnd1 variant or Nanos variant may have an amino acid missing at the N-terminus, C-terminus, and / or the amino acid sequence between the N-terminus and C-terminus of the corresponding wild-type Dnd1 or Nanos amino acid sequence. “Addition” of an amino acid means the addition or insertion of an amino acid residue at any position in a given amino acid sequence. For example, a Dnd1 variant or Nanos variant may have an amino acid added or inserted at the N-terminus, C-terminus, and / or the 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 also be fused with, for example, a functional polypeptide (e.g., a labeled protein such as green fluorescent protein). In this example, the Dnd1 variant or Nanos variant refers to the amino acid sequence of the portion of the functional polypeptide excluding the amino acid sequence. An amino acid "substitution" means that an amino acid residue at any position in a given amino acid sequence is replaced by another amino acid residue. An amino acid substitution is, for example, a conservative substitution. A conservative amino acid substitution means that an amino acid residue in the polypeptide is replaced by another amino acid residue having similar characteristics in its side chain. 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, or sulfur-containing side chains. Side chain characteristics include, for example, charge, side chain size, and hydrophobic / hydrophilicity. The wild-type protein corresponding to the variant has the amino acid sequence with the highest sequence identity to the amino acid sequence of the variant.

[0031] The term "sequence identity" refers to the percentage of matching amino acids or nucleotides 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 exhibits 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. A protein consisting of / only a given amino acid sequence does not exclude such protein that has undergone post-translational modifications such as glycosylation.

[0032] Dnd1 and Nanos may be derived from animals of the same species, or Dnd1 may be derived from animals of different species (for example, animals belonging to the order Cypriniformes). These may be Dnd1) and Nanos (for example, Nanos from an animal belonging to the order Beloniformes). Dnd1 and Nanos may be introduced into an early embryo of non-human vertebrate origin in the form of a coding nucleotide. The coding sequences of the nucleotides coding Dnd1 and Nanos may be present in the same nucleotide molecule or in two different nucleotide molecules. The term "coding sequence" means a nucleotide sequence that codes for a specific protein. If the nucleotides coding Dnd1 and Nanos are present in two different nucleotide molecules, the nucleotides coding Dnd1 and Nanos include a first nucleotide molecule coding Dnd1 and a second nucleotide molecule coding Nanos. The ordinal numbers "first" and "second," etc., are used to distinguish each component included in a group of similar components and do not limit the order or importance of any component.

[0033] The nucleotides encoding Dnd1 and Nanos may include additional gene sequences. These additional gene sequences may be, for example, nucleotide sequences 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 translation-promoting cap structure at its 5' end. The cap structure may, for example, be 7-methylguanylate. The RNA encoding Dnd1 and Nanos may, for example, include a polyadenylate sequence at its 3' end that stabilizes the RNA or promotes translation.

[0035] The DNA encoding Dnd1 and Nanos may, for example, include a promoter at its 5' end that functions as a transcription start site. The promoter may be any known promoter. For example, the promoter may be 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), Fugu oct3 (TG913), CMV (TG912, 849), humanEF1α (TG871), or 8xHSE (artificial heat shock promoter) (TG921). The DNA encoding Dnd1 and Nanos may, for example, include a polyadenylated sequence. The polyadenylated sequence may be located, for example, at the 3' end of the DNA. The DNA encoding Dnd1 and Nanos may be linear DNA or plasmid DNA.

[0036] In one example, if the nucleotides encoding Dnd1 and Nanos are present in the same nucleotide molecule, the DNA encoding Dnd1 and Nanos includes a polyadenylated sequence, for example, located at the 3' end of the DNA. If the nucleotides encoding Dnd1 and Nanos are present in two different nucleotide molecules, both the DNA encoding Dnd1 and Nanos, and the DNA encoding Nanos, each include a polyadenylated sequence, for example, located at the 3' end of the DNA.

[0037] The term "polyadenylated sequence" refers to a DNA sequence that, when transcribed into RNA, allows for the addition of a polyadenylation sequence (also called a "poly-A sequence") to the 3' end of the transcript. Many germ granule factor mRNAs are stabilized only in primordial germ cells (PGCs) due to the regulation of their 3'UTR, and are rapidly degraded in somatic cells that make up the body after development during the early stages of 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 polyadenylated sequence may be a known polyadenylated sequence. The polyadenylated sequence may be an endogenous sequence or an exogenous sequence. The term "endogenous" means a gene, nucleic acid, or protein that is originally present in a living organism or cell, or a gene, nucleic acid, or protein that has the same structure as them. The term "exogenous" means a gene, nucleic acid, or protein that is different from the gene, nucleic acid, or protein that is originally present in a living organism or cell. The polyadenylated sequence is preferably an exogenous sequence. The polyadenylated sequence may be, for example, SV40pA, BGHpA, hGHpA, or rbGlobpA. The polyA sequence may be, for example, tens to hundreds of adenine nucleotides. The polyA sequence may include, for example, a repeating sequence of the AAUAAA sequence.

[0039] Dnd1 and Nanos may be introduced into the early embryo simultaneously or individually at any time. Preferably, Dnd1 and Nanos are introduced into the early embryo simultaneously. If the 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 coding sequence of Dnd1 and the coding sequence of Nanos. Any known self-cleaving peptide sequence can be used. For example, a 2A self-cleaving peptide sequence. Any known protease cleavage site can be used. For example, enterokinase (DDDDK↓), factor Xa (IEGR↓ / IDGR↓), tobacco etch virus (ENLYFQ↓G), or thrombin (LVPR↓GS), or pre-scission (LEVLFQ↓GP).

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

[0041] In one example, if the nucleotides encoding Dnd1 and Nanos are present in the same nucleotide molecule, the RNA encoding Dnd1 and Nanos contains a poly(A) sequence at its 3' end. In another example, if the nucleotides encoding Dnd1 and Nanos are present in two different nucleotide molecules, both the RNA encoding Dnd1 and Nanos, and the RNA encoding Nanos, each contain a poly(A) sequence at their 3' ends.

[0042] The nucleotides encoding Dnd1 and Nanos can be introduced into early embryos of non-human vertebrate origin by, for example, microinjection, electroporation, or lipofection. Dnd1 and Nanos may also be introduced into early embryos of non-human vertebrate origin in the form of proteins. Dnd1 and Nanos as proteins can be introduced into early embryos of non-human vertebrate origin by, for example, microinjection or lipofection. If Dnd1 and Nanos are a combination of proteins and nucleotides, then Dnd1 and Nanos can be introduced into early embryos of non-human vertebrate origin by microinjection or lipofection.

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

[0044] Examples of this disclosure demonstrate that introducing the nucleotides encoding Dnd1 and Nanos into a single cell of a 16-cell stage embryo can produce an individual with a significantly increased number of primordial germ cells in its gonadal region compared to an identical embryo, except that Dnd1 and Nanos have not been introduced. Examples of this disclosure also demonstrate that the cells into which Dnd1 and Nanos are introduced may be cells in which germ plasmon-constituting factors (germ granule factors) are not localized. In the method of the first embodiment, 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 gonadal region compared to an identical embryo, except that Dnd1 and Nanos have not been introduced. In one example, if the early embryo is a 32-cell stage embryo, introducing Dnd1 and Nanos into one or more cells (e.g., two, three, or four) 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 have not been introduced. The predetermined percentage may 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 be cells in which germ granule factors are localized, or they may not be localized. In one example, if the early embryo is a 16-cell stage embryo, the number of cells into which Dnd1 and Nanos are introduced may be, for example, one (about 6% = 1 / 16) or two (about 13% = 2 / 16).

[0045] In the method according to this embodiment, the number or proportion of cells to which Dnd1 and Nanos are introduced into the early embryo can be appropriately selected depending on the purpose. For example, if the purpose is to obtain a blastula in which almost or all cells are primordial germ cells, Dnd1 and Nanos are introduced into all or most cells of the early embryo. For example, if the purpose is to obtain an individual in which the number of primordial germ cells in its gonadal 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 amount of Dnd1 and Nanos introduced into the early embryo may be the known amount used when introducing proteins or genes 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 the early embryo by microinjecting a Dnd1 and Nanos-containing solution in an amount of 30 to 60% of the total embryo volume into the early embryo. When microinjecting into fish embryos (e.g., zebrafish or medaka), a 30-60% volume is, for example, 100 pl-500 pl. In one example, nucleotides encoding Dnd1 and Nanos (e.g., mRNA) are introduced by microinjecting 100 pl-500 pl of the nucleotide-containing solution at a concentration of 10-500 ng / ml into early embryos derived from medaka.

[0046] Early embryos into which Dnd1 and Nanos according to this disclosure have been introduced can undergo embryonic development by being cultured under known culture conditions. The known culture conditions are appropriately set according to the animal species of the embryo. If the animal species of the embryo is fish or amphibian, the culture of the early embryo into which Dnd1 and Nanos have been introduced includes, for example, leaving it standing in saline or freshwater at room temperature. If the animal species of the embryo is reptile or bird, the culture of the early embryo into which Dnd1 and Nanos have been introduced includes, for example, leaving it standing at a temperature appropriate to that animal species. If the animal species of the embryo is mammal, the culture of the early embryo into which Dnd1 and Nanos have been introduced includes, for example, maintaining it in a known culture medium at 37°C in a 5% CO2 environment. For the purpose of obtaining a non-human mammal in which the number of primordial germ cells in the gonadal region is significantly increased compared to wild-type animals, the culture of the early embryo into which Dnd1 and Nanos have been introduced includes, for example, maintaining it in a known medium at 37°C under a 5% CO2 environment until the blastula stage, and then transplanting it into the uterus of the non-human mammal. Therefore, the culture of 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 to the primordial germ cells in the same embryo except that Dnd1 and Nanos have not been introduced. The same embryo except that Dnd1 and Nanos have not been introduced is an embryo of the same developmental stage from the same animal species as the animal that provided the early embryo into which Dnd1 and Nanos have been introduced. The developmental stages of the embryo include, for example, the 1-cell stage to the 64-cell stage, morula stage, blastula stage, cystula stage, neurula stage, pharyngeal stage, or developed individual.

[0047] The embodiments of this disclosure demonstrate that co-introducing Dnd1 and Nanos into early embryos of medaka and zebrafish species results in individuals with a significantly increased number of primordial germ cells compared to wild-type individuals. Dnd1 and Nanos in this embodiment are RNA-binding proteins conserved in vertebrates in general, and both play important roles in germ cell formation.The aforementioned technical matters are described, for example, in relation to mice 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), and in relation to African clawed frogs in Taguchi A, Watanabe K, Orii H. Intracellular localizations of the dead end protein in Xenopus primordial germ cells. 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 vegetal cortical microtubule assembly during Xenopus axis specification. Development 140:2334-2344 (2013), and Lai et al. Xenopus Nanos1 is required to prevent endoderm gene expression and apoptosis in It is described in primordial germ cells. Development 139(8):1476-1486 (2021), and for chickens, it is described in 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). In addition, Tsuda et al. Conserved Role of nanos Proteins in Germ Cell Development. Science 301(5637):1239-1241 (2003) describes that nanos are conserved from flies to mammals and play 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, any animal including amphibians, reptiles, birds, and non-human mammals).

[0048] One embodiment provides a method for preparing embryos enriched with primordial germ cells, comprising introducing Dnd1 and Nanos into early embryos of a non-human vertebrate; introducing gamete formation factors; and culturing the early embryos into which Dnd1 and Nanos have been introduced. The gamete formation factors may be at least one selected from the group consisting of, for example, Daz, Dazl, Boule, meioC, Ythdc2, and Rbm46. Non-human vertebrates having primordial germ cells formed according to this embodiment in their gonads tend to be more fertile.

[0049] Dnd1, Nanos, and the gamete-forming factor may all be introduced into the early embryo simultaneously, or into any combination at any time, or into each individually at any time. In one example, Dnd1, Nanos, and the gamete-forming factor are introduced into the early embryo simultaneously. In another example, the combination of Dnd1 and Nanos, and the gamete-forming factor, are introduced at any time. In the above example, for example, the combination of Dnd1 and Nanos is introduced first, followed by the introduction of the gamete-forming factor. In another example, Dnd1, Nanos, and the gamete-forming factor are introduced into the early embryo individually at any time.

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

[0051] The term "DAZ family" refers to a group of three RNA-binding proteins. DAZ family members may play a crucial role in gamete formation or meiosis. DAZ family members have, for example, a sequence of 24 amino acids called 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" is an RNA-binding protein. Boule has been found in many vertebrates, including humans, and invertebrates, including sea anemones. Dazl has been found in almost all vertebrates, including mice and humans. Daz may be Daz1, Daz2, Daz3, or Daz4.

[0052] Substances involved in the transition of meiosis may be, for example, meioC, Ythdc2, or Rbm46. The term "meioC" is synonymous with "Meiosis-Specific with Coiled-Coil Domain." meioC is important for the transition of meiosis 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 gamete formation factors and the nucleotide sequences encoding them can be obtained from database sites provided by public institutions. For example, the mRNA sequence encoding Dazl in medaka can be obtained 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. [Table 2]

[0055] A gamete-forming 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. The gamete-forming factor may have a different amino acid sequence from the wild-type gamete-forming factor of the non-human vertebrate that provided the early embryo into which the gamete-forming factor was introduced, as long as it has the function described in this embodiment. A gamete-forming factor that has the function described in this embodiment and contains an amino acid sequence different from the wild-type gamete-forming factor is also referred to as a "gamete-forming factor variant." The gamete-forming 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 gamete-forming factor. For example, a gamete-forming factor variant 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 affinity of the wild-type gamete-forming factor. For example, a gamete-forming factor variant 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 the wild-type gamete-forming 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 affinity of the wild-type gamete-forming factor. Gamete-forming factor variants include wild-type gamete-forming factors derived from non-human vertebrates and other animal species that provided the early embryos into which they are introduced. The binding affinity of gamete-forming factors to RNA can be investigated by RNA immunoprecipitation.

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

[0057] Gamete formation factors may be introduced into early embryos of non-human vertebrate origin in the form of proteins or the nucleotides that encode them. The nucleotides encoding the gamete formation factors may be DNA or RNA. The nucleotides encoding the gamete formation factors may include other nucleotide sequences described herein. Gamete formation factors can be introduced into the early embryos according to the methods described herein.

[0058] In the context of Dnd1, Nanos, or gamete-forming factors, "introduction" to an early embryo refers to the operation of making Dnd1, Nanos, or gamete-forming factors present in one or more cells constituting the early embryo. The form of Dnd1, Nanos, or gamete-forming factors when introduced to an early embryo is not particularly limited. Dnd1, Nanos, or gamete-forming factors are introduced to the early embryo, for example, in the form of nucleotides, proteins, or a mixture thereof. Dnd1, Nanos, or gamete-forming factors are preferably introduced to the early embryo in the form of nucleotides (more preferably in the form of RNA).

[0059] Embryo enriched with primordial germ cells An embryo enriched with primordial germ cells is provided by a method according to the first embodiment. Accordingly, one embodiment of the present disclosure provides an embryo of non-human vertebrate origin enriched with primordial germ cells, wherein the embryo is in 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 of the same developmental stage from a non-human vertebrate of the same species as the embryo.

[0060] "Embryos enriched with primordial germ cells" have a significantly increased number of primordial germ cells compared to embryos of wild-type animals. Wild-type animal embryos are, for example, embryos obtained from the same species of animal as the animal that provided the early embryos into which Dnd1 and Nanos were introduced, and are at the same developmental stage except that Dnd1 and Nanos were not introduced. The number of primordial germ cells in the embryos is compared, for example, to the number of primordial germ cells in embryos of the same species of animal as the animal that provided the embryos, at the same developmental stage. Primordial germ cells in embryos can be separated or concentrated, for example, by subjecting the embryos to cell detachment treatment using trypsin or collagenase and subjecting the suspension containing the separated cells to density gradient centrifugation. The number of primordial germ cells in embryos is 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 immunochemical staining using antibodies against known primordial germ cell markers or germ granule factors, or by in situ hybridization using probes having sequences complementary to the mRNA of the markers or factors. The number of primordial germ cells in an embryo is compared based on the expression levels of known primordial germ cell markers or germ granule factors in the embryo. Preferably, the number of primordial germ cells in an embryo is compared based on the expression level of vasa.

[0061] Embryos derived from non-human vertebrates enriched with primordial germ cells are embryos from the blastula stage onward, such as morula, blastula, neurula, pharyngeal embryo, or embryos at any later developmental stage. The number of primordial germ cells in such embryos is at least three times greater (for example, 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) than the number of primordial germ cells in embryos of the same developmental stage derived from non-human vertebrates of the same species.

[0062] Genetically modified primordial germ cells enrich the embryo. An embryo enriched with primordial germ cells prepared by the method according to the first embodiment may have genetically modified primordial germ cells. Accordingly, one embodiment of the present disclosure provides an embryo of non-human vertebrate origin enriched with genetically modified primordial germ cells, wherein the embryo is in 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 of the same developmental stage from a non-human vertebrate of the same species.

[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," "modification of genes," or "to modify genes" refer to the intentional alteration of genes through chromosome manipulation or DNA recombination techniques. Chromosome manipulation techniques include, for example, tetraploidization of chromosomes. When the non-human vertebrate is a fish, chromosomes can be tetraploidized by treating a fertilized egg 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 chromosomes 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 of non-human vertebrate origin and modifying its genes; 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 of non-human vertebrate origin; modifying the genes of the early embryo (or cells) into which Dnd1 and Nanos have been introduced; and culturing the early embryo into which Dnd1 and Nanos have been introduced and the genes have been modified. In another example, genetically modified primordial germ cells are prepared by a method comprising modifying the genes of an early embryo (or cells) of non-human vertebrate origin; introducing Dnd1 and Nanos into the early embryo (or cells) into which the genes have been modified; and culturing the early embryo into which Dnd1 and Nanos have been introduced and the genes have been modified. In other examples, genetically modified primordial germ cells are prepared by a method comprising simultaneously introducing Dnd1 and Nanos into an early embryo (or cells) of non-human vertebrate origin and modifying its genes; and culturing the early embryo into which Dnd1 and Nanos have been introduced and which have been genetically 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 can be obtained in which most or all of the cells are genetically modified primordial germ cells. Embryos enriched with genetically modified primordial germ cells can be used to create germline chimeric non-human vertebrates, as described later.

[0066] In the above method, Dnd1 and Nanos are introduced into a predetermined proportion of cells in early embryos derived from non-human vertebrates (for example, embryos at the 4-cell, 8-cell, 16-cell, 32-cell, or 64-cell stage), and their genes are modified, thereby obtaining embryos enriched with genetically modified primordial germ cells (transgenic non-human vertebrates). Therefore, one embodiment of the first aspect provides a method for producing transgenic non-human vertebrates that contain genetically modified primordial germ cells in their gonadal region.

[0067] Method for creating 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 of non-human vertebrate origin (at least one cell from an embryo between the 4-cell and 64-cell stages) and modifying its genes; and culturing the early embryo into which Dnd1 and Nanos have been introduced and its genes have been modified to generate a transgenic non-human vertebrate, wherein the transgenic non-human vertebrate includes genetically modified primordial germ cells in its gonadal region.

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

[0069] The transgenic non-human vertebrates according to this embodiment preferably contain the genetic modification essentially only in the primordial germ cells of the gonad region (gonads). The transgenic non-human vertebrates do not contain the genetic modification in regions other than the gonad region (e.g., organs such as the brain, heart, and internal organs; somatic cells such as nerve cells, cardiomyocytes, and endothelial cells). According to the method of this embodiment, for example, even if the genetic modification is one that may cause lethality or developmental failure, it is possible to produce a transgenic non-human vertebrate that contains germ cells containing the genetic modification in its gonad region. In one example, the next generation of non-human vertebrates homozygous for the genetic modification can be produced from the male and female gametes of the transgenic non-human vertebrate. The next generation of non-human vertebrates heterozygous for the genetic modification 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 vertebrates according to this embodiment can be used, for example, for genetic analysis of diseases.

[0070] The term "vertebrate" means any animal belonging to fish, amphibians, reptiles, birds, and mammals. In this embodiment, vertebrates are non-human vertebrates, excluding humans. Non-human vertebrates are, for example, any animal belonging to fish, amphibians, reptiles, birds, or mammals, preferably any animal belonging to fish, birds, or mammals, more preferably any animal belonging to fish or mammals, and even more preferably any animal belonging to fish.

[0071] The term "fish" refers to the group of animals that exclude tetrapods from the subphylum Vertebrata. Fish may include, 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 include, for example, the genera Thunnus such as yellowfin tuna, blackfin tuna, bigeye tuna, and Pacific bluefin tuna; the genera Skipjack tuna; the genera Scomberes such as Kawakawa and Black skipjack tuna; the genera Mackerel such as King mackerel; the genera Acanthocybium; and the genera Scomberes such as Atlantic chub mackerel, chub mackerel, and Norwegian mackerel.

[0073] Fish of the Carangidae family may include, for example, animals of the genus Seriola, such as Amberjack, Yellowtail (Seriola lalandi), and Greater Amberjack (Seriola dumerili); animals of the genus Pseudocaranx, such as Striped Jack (Pseudocaranx dentex); and animals of the genus Carangidae, such as Japanese Horse Mackerel. Fish of the Salmonidae family may include, for example, animals of the genus Hucho, such as Sakhalin Hucho (Parahucho perryi); animals of the genus Oncorhynchus, such as Salmon (Oncorhynchus keta), Rainbow Trout (Oncorhynchus mykiss), Cherry Salmon (Oncorhynchus masou), and Sockeye Salmon (Oncorhynchus nerka); animals of the genus Salmon (Oncorhynchus char, such as White-spotted char); and animals of the genus Atlas.

[0074] Fish of the Gadidae family may include, for example, the Pacific cod (Gadus macrocephalus), Atlantic cod (Gadus morhua), Greenland cod (Gadus ogac), and Alaska pollock (Gadus chalcogrammus), as well as animals of the Micromesistius genus. Fish of the Anguillidae family may include, for example, the American eel (Anguilla rostrata), Japanese eel (Anguilla japonica), giant eel (Anguilla marmorata), and European eel (Anguilla anguilla), as well as animals of the Neoanguilla genus.

[0075] Fish of the family Tetraodontidae may include, for example, animals of the genus Takifugu, such as the tiger pufferfish (Takifugu rubripes) and the common pufferfish (Takifugu porphyreus); and animals of the genus Lagocephalus. Fish of the family Sparidae may include, for example, animals of the genus Pagrus, such as the red sea bream (Pagrus major); animals of the genus Acanthopagrus, such as the black sea bream (Acanthopagrus schlegelii); and animals of the genus Dentex. Fish of the family Serranidae may include, for example, animals of the genus Epinephelus, such as the common grouper (Epinephelus septemfasciatus), the longtooth grouper (Epinephelus bruneus), and the yellowback grouper (Epinephelus akaara); and animals of the genus Plectropomus. Fish of the family Paralichthyidae may include, for example, the Japanese flounder (Paralichthys olivaceus).

[0076] Fish of the Sturgeonidae family may include, for example, the Japanese sturgeon (Acipenser medirostris), the Daurian sturgeon (Huso dauricus), and the bigeye sturgeon (Huso huso). Fish of the genus Oryzias may include, for example, the Japanese rice fish (Oryzias latipes, Oryzias sakaizumii), and the Javan rice fish (Oryzias javanicus), and other animals of the genus Oryzias. Fish of the Cyprinidae family may include, for example, animals of the subfamily Cyprininae such as zebrafish, carp, crucian carp, and goldfish; the subfamily Danioninae such as rasbora; the subfamily Gobioninae such as dace; and the subfamily Leuciscinae such as chub.

[0077] The non-human vertebrate according to this embodiment is, for example, any animal belonging to the fish class. Animals belonging to the fish class may be, for example, tuna, sturgeon, skipjack tuna, yellowtail, greater amberjack, kingfish, sea bream, salmon, cod, trout, rainbow trout, flounder, pufferfish, horse mackerel, grouper, eel, or carp.

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

[0079] Amphibians of the Salamander family may include, for example, animals of the genera Hynobius and Onychodactylus. Amphibians of the Giant Salamander family may include, for example, animals of the genus Andrias. Amphibians of the Newt family may include, for example, animals of the genera Echinotriton and Cynops. Amphibians of the Toad family may include, for example, animals of the genera Rhinella and Bufo. Amphibians of the Tree Frog family may include, for example, animals of the genera Rana, Lithobates, Glandirana, Pelophylax, and Odorrana. Amphibians of the family Rhacophoridae may include, 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 class Amphibians. An example of an animal belonging to the class Amphibians may be the Japanese giant salamander.

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

[0082] Reptiles of the Chelydidae family may include, for example, animals of the genus Chelonia (green sea turtle, such as Chelonia mydas); the genus Caretta (loggerhead sea turtle, such as Caretta caretta); and the genus Eretmochelys (hawksbill sea turtle, such as Eretmochelys imbricata). Reptiles of the Emydidae family may include, for example, animals of the genus Dermochelys. Reptiles of the Geoemydidae family may include, for example, animals of the genus Mauremys (Japanese pond turtle, such as Mauremys reevesii); the genus Cuora (Rose-breasted box turtle, such as Cuora flavomarginata); and the genus Geoemyda.

[0083] Reptiles of the family Chelymidae may include, for example, animals of the genus Chelydra, such as the snapping turtle (Chelydra serpentina); and animals of the genus Macrochelys, such as the alligator snapping turtle (Macrochelys temminckii). Reptiles of the family Trionychidae may include, for example, animals of the genus Pelodiscus, such as the softshell turtle (Pelodiscus sinensis). Reptiles of the family Eublepharidae may include, for example, animals of the genus Goniurosaurus, such as the banded gecko (Goniurosaurus splendens). Reptiles of the Gekkonidae family may include, for example, animals of the genus Gekko, such as Lepidodactylus, Hemiphyllodactylus, Perochirus, Gekko japonicus, and Gekko vertebralis.

[0084] Iguanidae reptiles may include, for example, animals of the genus Iguana, such as the anolis lizard (Anolis) and the green iguana (Iguana iguana). Scincidae reptiles may include, for example, animals of the genus Plestiodon, such as the blue-striped lizard (Plestiodon elegans) and the stone lizard (Plestiodon stimpsonii). Reptiles of the Colubridae family may include, for example, animals 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 striped snake (Elaphe quadrivirgata); the genus Calamaria, such as the long-nosed snake (Calamaria pavimentata); and the genus Dinodon, such as the red-legged snake (Dinodon semicarinatum).

[0085] Reptiles of the Elapinae subfamily may include, for example, animals of the genus Sinomicrurus. Reptiles of the Ophiinae subfamily may include, for example, animals of the genera Hydrophis; Laticauda; and Emydocephalus. Reptiles of the Viperinae subfamily may include, for example, animals of the genus Protobothrops, such as the Habu (Protobothrops flavoviridis) and Sakishima Habu (Protobothrops elegans); animals of the genus Gloydius, such as the Japanese pit viper (Gloydius blomhoffii); and animals of the genus Ovophis.

[0086] The non-human vertebrate according to this embodiment is, for example, any animal belonging to the reptile class. An example of an animal belonging to the reptile class is a softshell turtle.

[0087] The term "birds" refers to the group of animals belonging to the class Aves (Aves) of the subphylum Vertebrata. Examples of birds include the Megapodiidae family, the Numididae family, and the chicken (Gallus gallus). Phasianidae (including domesticus); Struthionidae; Anatidae; Anhimidae; Columbidae; Gruidae; Rallidae; Phoeniconaias; Steatornithidae; Nyctibiidae; Caprimulgidae; Gaviidae; Aptenodytes; Eudyptes; Diomedeidae; Hydrobatidae; Ciconiidae; Ardeidae ( It may be an animal belonging to the following families: 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 class Aves. Animals belonging to the class Aves may be, for example, chickens, ostriches, parakeets, owls, or hawks.

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

[0090] Monotremate mammals may include, for example, animals of the Ornithorhynchidae or Tachyglossidae families. Marsupial mammals may include, for example, animals of the Didelphidae family; Dasyuridae family such as the Tasmanian devil (Sarcophilus harrisii); Myrmecobiidae family; Phascolarctidae family; Petauridae family; and Macropodidae family such as the red kangaroo (Macropus giganteus) and wallaby.

[0091] African insectivorous mammals may include, for example, animals of the families Chrysochloridae, Tenrecidae, Elephantulus, and Rhynchocyon. Proctoglau mammals may include, for example, animals of the families Procaviidae, Elephantidae, Dugongidae, and Trichechidae. Xenarthra mammals may include, for example, animals of the families Bradypodidae, Megalonychidae, Myrmecophagidae, Cyclopedidae, Chlamyphoridae, and Dasypodidae.

[0092] Mammals of the Euarchognathidae family may include, for example, animals of the Tarsiidae, Cercopithecidae, Hominidae, Hylobatidae, Atelidae, and Lemuridae families. Mammals of the Euarchognathidae family may include, for example, animals of the Muridae, Nesomyidae, Cricetidae, Spalacidae, Calomyscidae, Dipodidae, and Gliridae families. Mammals of the Euarchognathidae family may include, for example, animals of the Leporidae and Ochotonidae families. Mammals of the Euarchognathidae family may include, for example, animals of the Cynocephalidae family. Mammals of the Euarchognathidae family may include, for example, animals of the Tupaiidae family or Ptilocercidae family.

[0093] Laurasia mammals may include, for example, animals of the families Erinaceidae, Solenodontidae, and Talpidae. Laurasia mammals may include, for example, animals of the families Pteropodidae and Vespertilionidae. Laurasia mammals may include, for example, animals of the families Equidae, Tapiridae, and Rhinocerotidae. Laurasia mammals may include, for example, animals of the Camelidae family (camels and llamas); the Suidae family (wild boars and pigs); the Tragulidae family; the Moschidae family (musk deer); the Cervidae family (deer, reindeer, and Père David's deer); the Bovidae family (cattle, antelopes, goats, and sheep); the Giraffidae family (giraffes and okapis); the Antilocapridae family; the Hippopotamidae family (hippopotamus and pygmy hippos); the Mysticeti suborder (baleen whales), such as the Balaenidae family; the Physeteridae family; and the Delphinidae family.

[0094] The non-human vertebrates in this embodiment are non-human mammals. Non-human mammals may include, for example, any animal belonging to the rodents such as mice, rats, guinea pigs, and hamsters; any animal belonging to the non-human primates such as chimpanzees; any animal belonging to the artiodactyla order such as cattle, goats, and sheep; any animal belonging to the odd-toed ungulate order such as horses; and pet animals such as rabbits, dogs, and cats.

[0095] The terms “derived” or “of which” mean that a particular object is obtained directly or indirectly from a particular source. In one example, early embryos of non-human vertebrate origin include early embryos obtained in vitro by oviposition or artificial insemination of non-human vertebrates, or early embryos (e.g., 32-cell stage embryos) obtained by culturing fertilized eggs obtained in vitro. In another example, isolated cells of embryo origin include cells obtained by separating the cell population constituting an embryo into individual cells, or cells obtained by culturing said cells.

[0096] (Method for creating germline chimeric non-human vertebrates) A second aspect of the present disclosure provides a method for producing a germline chimeric nonhuman vertebrate, comprising: collecting primordial germ cells from an embryo enriched with primordial germ cells relating to the present disclosure; injecting the collected primordial germ cells (donor primordial germ cells) into an embryo of nonhuman vertebrate origin (host embryo); and culturing the host embryo into which the donor primordial germ cells have been injected, wherein the germline chimeric nonhuman vertebrate contains the donor primordial germ cells in its gonadal region, and the nonhuman vertebrate that provided the donor primordial germ cells (donor nonhuman vertebrate) and the nonhuman vertebrate that provided the host embryo (host nonhuman vertebrate) are closely related.

[0097] Embryos enriched with primordial germ cells can be prepared by a method relating to a first aspect of this disclosure. Such primordial germ cell-enriched embryos are, for example, blastulas, cystulas, neurulas, pharyngeal embryos, or embryos at subsequent developmental stages (including developed individuals such as juveniles, juvenile fish, chicks, or fetuses). From the viewpoint of ease of collecting primordial germ cells, such primordial germ cell-enriched embryos are blastulas, cystulas, neurulas, pharyngeal embryos, or embryos at subsequent developmental stages, and are embryos before individual development, preferably blastulas or cystulas. Primordial germ cells can be collected from embryos according to known methods depending on the developmental stage of the embryo. For example, primordial germ cells can be collected from blastulas using a suction device equipped with a glass capillary. Primordial germ cells can also be collected, for example, by subjecting embryos from the cystula stage onward to cell detachment using a protease such as trypsin or collagenase, and then separating and concentrating the detached cells by density gradient centrifugation.

[0098] To facilitate the isolation of primordial germ cells, in addition to introducing Dnd1 and Nanos, a labeling gene (e.g., a gene encoding a fluorescent protein) may be introduced into early embryos derived from non-human vertebrates. When early embryos into which Dnd1 and Nanos have been introduced are prepared by introducing Dnd1 and Nanos into all or most of the cells of an early embryo from a non-human vertebrate, an embryo obtained by culturing the early embryo and whose development has stopped at the blastula stage will consist almost entirely of primordial germ cells into which 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 derived from a non-human vertebrate (host embryo). This 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, cystula, neurula, pharyngeal embryo, or an embryo at a later developmental stage (including developed individuals such as juveniles, juvenile fish, chicks, or fetuses). Any number of donor primordial germ cells can be injected into the host embryo. For example, 10 to 1000, 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 belonging to the same family. Closely related non-human vertebrates may, for example, be non-human vertebrates belonging to the same family, and preferably, be non-human vertebrates belonging to the same genus. In this disclosure, closely related does not exclude the same species. Closely related may, for example, be non-human vertebrates belonging to the same species. Closely related non-human vertebrates may, for example, be different species but belonging to the same family. Closely related non-human vertebrates may, for example, be different species but belonging 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 the same species as the animal that provided the donor primordial germ cells. The host non-human vertebrate may be the same species as the animal that provided the host embryo. In one example, the host non-human vertebrate that provided the host embryo was 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 the fish family, 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 pufferfish and grass pufferfish, or a combination of koi and goldfish.

[0102] According to the method of the second embodiment, a chimeric non-human vertebrate is obtained that contains donor primordial germ cells in its gonadal region. 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 have their genes modified. Gene introduction into the donor primordial germ cells can be carried out according to the method of 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 its genes; culturing the early embryo into which Dnd1 and Nanos have been introduced and the genes have been modified to prepare an embryo enriched with the modified primordial germ cells; collecting the modified primordial germ cells (donor primordial germ cells) from the embryo enriched with the 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 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 above embodiment contains the genetic modification essentially only in the primordial germ cells of the gonadal region (gonads). For example, even if the genetic modification is lethal or causes developmental failure (e.g., tetraploidization of chromosomes), the method of this embodiment yields the germline chimeric non-human vertebrate (host non-human vertebrate) that contains germ cells containing the genetic modification in its gonadal region.

[0104] In fish, triploid female individuals do not produce eggs, so the nutrients that would be used for egg production are used for the growth of the individual. As a result, triploid female individuals are larger in size and have better flesh quality than normal diploid female individuals. Triploid individuals are produced from tetraploid eggs and sperm. Individuals (tetraploids) used to prepare tetraploid eggs or sperm may suffer from developmental problems or stunted growth. Therefore, there is an advantage to producing diploid individuals that contain tetraploid primordial germ cells in their gonadal region. According to the method of the second embodiment, the above-mentioned individuals can be produced. Such methods include, for example, introducing Dnd1 and Nanos into an early embryo of non-human vertebrate origin (e.g., at least one cell from an embryo between the 4-cell and 64-cell stages) and performing genetic modification to make the early embryo tetraploid; culturing the early embryo into which Dnd1 and Nanos have been introduced and which has undergone genetic modification to prepare an embryo enriched with donor primordial germ cells (tetraploid); collecting donor primordial germ cells (tetraploid) from the embryo enriched with primordial germ cells and injecting them into a host embryo (diploid) of non-human vertebrate origin; and culturing the host embryo (diploid) into which the donor primordial germ cells (tetraploid) have been injected 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 aforementioned germline chimeric vertebrate and a 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 embodiment can produce a donor non-human vertebrate that contains donor primordial germ cells, as described later, in its gonadal region.

[0106] (Method for producing non-human vertebrate donors) One aspect of the present disclosure provides a method for producing a donor nonhuman vertebrate, comprising mating a germline chimeric nonhuman vertebrate (host nonhuman vertebrate), wherein the germline chimeric nonhuman vertebrate (host nonhuman vertebrate) contains donor primordial germ cells in its gonadal region, and the donor nonhuman vertebrate contains 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 gonadal region can, for example, produce the next generation of germline chimeric non-human vertebrates (donor non-human vertebrates) through 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 gonadal region can, for example, produce the next generation of germline chimeric non-human vertebrates (donor non-human vertebrates) through natural mating or artificial insemination with the donor non-human vertebrate.

[0108] The method according to this embodiment can be used, for example, in surrogate production technology. By using an animal for which rearing methods have been established as a host non-human vertebrate (e.g., mackerel) as the host non-human vertebrate, 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 donor non-human vertebrates (e.g., tuna) from a host non-human vertebrate (e.g., mackerel) for which rearing methods have been established. More specifically, a method is provided for producing a donor non-human vertebrate (tuna), comprising: introducing Dnd1 and Nanos into an early embryo of a non-human vertebrate (e.g., an early embryo of tuna), and optionally modifying the genes; culturing the early embryo into which Dnd1 and Nanos have been introduced (and optionally modified the genes) to prepare an embryo enriched with primordial germ cells (of tuna); collecting donor primordial germ cells (of tuna) from the embryo and injecting them into a host embryo of a non-human vertebrate (e.g., a mackerel); culturing the host embryo to produce a germline chimeric non-human vertebrate (mackerel) containing the donor primordial germ cells (of tuna) 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 stage to the 8-cell stage (preferably the 1-cell or 2-cell stage, more preferably the 1-cell stage).

[0109] In this specification, the term "contains" means that the enumerated elements and / or steps exist, and other elements and / or steps may be added. In this specification, the term "consists of" means that the enumerated elements and / or steps exist, and other elements and / or steps are excluded. In this specification, the term "essentially consists of" means that the enumerated elements and / or steps exist, and other elements and / or steps may be added to the extent that they do not affect novel technical features such as methods for preparing embryos enriched with primordial germ cells, methods for producing non-human vertebrates, germline chimeric non-human vertebrates, and transgenic non-human vertebrates. In this specification, the term "substantially does not contain" does not exclude "completely does not contain".

[0110] The descriptions of specific aspects or embodiments relating to this disclosure, and the definitions of terms provided for such aspects or embodiments, shall apply to other aspects and embodiments in this disclosure as appropriate, unless otherwise specified. All publications and patents referenced in this disclosure are incorporated into this disclosure in the same way that those publications and patents individually state that their entire disclosure is incorporated by reference.

[0111] The following describes specific embodiments, which are merely preferred embodiments of the present invention and do not in any way limit the invention described in the attached claims.

[0112] [Examples] Nanos3 -SV40pA and Dnd1 - SV40pA mRNA synthesis Medaka (Japanese rice fish) Nanos3 and Dnd1 The protein-coding regions of SV40pA were amplified from cDNA of medaka ovary and from the pcs2DndChDD3' vector (Non-Patent Literature 2, provided by Dr. YunHan Hong), respectively, and cloned into the BamHI / KpnI site of the pGGEV-1_XcmI-LacZ vector (addgeneID:49296). SV40pA was amplified from the cyto-YFP-FKBPx5 vector (addgeneID:103777) and cloned into the BamHI / KpnI site of the pGGEV_2'_XcmI-LacZ (addgeneID:49303). SV40pA was then cloned 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)). Nanos3 and Dnd1 They were connected to the 3' ends of each.

[0113] Zebrafish Nanos3 and Dnd1cDNAs containing the respective protein-coding regions were prepared from mRNA expressed in zebrafish ovary cells using reverse transcriptase. Nanos3 and Dnd1 The protein-coding regions were amplified and cloned into the pGGEV-1_XcmI-LacZ vector. SV40pA was ligated using the GGW cloning method, as described above. mRNA was synthesized using the prepared DNA as a template via an in vitro transcription reaction with the T7 promoter (Figure 1).

[0114] Endogenous medaka Nanos3 and Dnd1 Mutations were introduced 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), and the endogenous Nanos3 and Dnd1 This knocked down the target cell. This allowed us to evaluate only the effects of microinjected Nanos3-SV40pA and Dnd1-SV40pA (Experiment 1-2).

[0115] Experiment (1): Enrichment of primordial germ cells in medaka embryos Test 1-1: Dnd1 and Nanos3 Co-injection induced differentiation of almost all cells in the blastula stage into primordial germ cells. In a medaka one-cell stage embryo (fertilized egg), 40 ng / μl Nanos3 -SV40pA and Dnd1 -SV40pA mRNA was microinjected (embryos overexpressing medaka-derived Dnd1 / Nanos3: Dnd1 / Nanos3-OE embryos). mCherry- Nanos3The 3'UTR was microinjected for primordial germ cell labeling, and the embryos were cultured. The embryos developed up to the blastula stage, but development did not progress thereafter. In the embryos whose development stopped at the blastula stage, almost all cells were mCherry positive, and almost all cells in the blastula became primordial germ cells (PGCs) (Figures 2A and 2B).

[0116] Approximately 50 primordial germ cells from these embryos (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 late-stage embryos, at least 200 or more PGCs were observed in the gonadal region (Figure 2D). This result indicates that a significantly larger number of PGCs were formed compared to wild-type embryos, which typically form around 10-40 PGCs.

[0117] By forcing the expression of Dnd1 alone, the number of primordial germ cells increases by approximately 1.5 to 2 times compared to the wild type (Non-Patent Literature 2). Regarding the number of primordial germ cells formed, the method according to this disclosure increases the number of primordial germ cells by more than 30 times compared to the wild type (Figure 2D). Therefore, the method according to this disclosure can significantly increase the number of primordial germ cells compared to forcing the expression of Dnd1 alone.

[0118] Experiment 1-2: In primordial germ cell formation, Dnd1 / Nanos3 The introduction of both, Dnd1 Alone or Nanos3 Comparison with standalone implementation As shown in Experiment 1-1, in a one-cell stage embryo (fertilized egg), Dnd1 / Nanos3 When both were introduced and overexpressed (OE), development did not progress beyond the blastula stage, and the individual did not develop (hatch). Therefore, only one cell of the 16-cell stage embryo was used. Dnd1 / Nanos3 We tested whether introducing this method would allow embryos with an increased number of primordial germ cells to develop (hatch) normally.

[0119] To eliminate the influence of endogenous Dnd1 and Nanos3, endogenous Dnd1, Nanos3, or both were knocked down (KD) using the CRISPR-Cas13d system (Figure 3A). Specifically, Cas13d mRNA was added to one-cell stage embryos of transgenic medaka (olvas-EGFP) whose germ cells are labeled with EGFP. Dnd and Nanos3 gRNA (Dnd1 / Nanos3 gRNA), and mCherry- Nanos3 3'UTR (for PGC labeling) is microinjected to obtain endogenous Dnd or Nanos3 He knocked out either one or both.

[0120] In a 16-cell stage embryo, 16 cells form a single layer of 4x4 cells (schematic diagram in Figure 3B). The localization of germ plasm components (germ granule factors) in a 16-cell stage embryo was investigated using BuckyBall:EGFP medaka. Germ granule factors were hardly localized in cells located outside the aforementioned single layer of cells (fluorescence image in Figure 3B). Cells destined to become cells other than primordial germ cells, where germ granule factors are not localized, Dnd1 / Nanos3 To investigate whether introducing it induces differentiation into primordial germ cells, a single cell located outside the aforementioned single cell layer (the cell marked with an asterisk in the fluorescence image of Figure 3B) was injected with 10 ng / μl of [the substance]. Dnd1 -SV40pA or Nanos3 -SV40pA alone or both ( Dnd1 / Nanos3 ) was microinjected.

[0121] Dnd1 / Nanos3 Embryos injected with microinjection developed normally (107 individuals). In the gonadal region of 99 of these 107 individuals (93%), a significantly increased number of olvas-EGFP-positive primordial germ cells was observed compared to the control individuals (Figure 3C), similar to the germline chimeras in Experiment 1-1 (Figure 3D). Dnd1 and Nanos3In embryos microinjected with each drug individually, olvas-EGFP-positive primordial germ cell formation was confirmed in 18% (19 out of 104) and 23% (23 out of 102) individuals, respectively. Furthermore, the number of primordial germ cells formed was very small, averaging 3.4 ± 2.4 cells per individual (Figures 3E and F).

[0122] Tests 1-2, importantly, involve at least one cell in a 16-cell stage embryo. Dnd1 and Nanos3 This demonstrates that injecting both allows for the development of individuals containing significantly increased primordial germ cells in their gonadal region. This is achieved in a single cell of a 4-cell cycle embryo, an 8-cell cycle embryo, and a 32-cell stage embryo. Dnd1 and Nanos3 When both were injected, individuals were produced that contained a significant increase in primordial germ cells in their gonadal region. Therefore, the embodiments of this disclosure involve injecting a predetermined percentage of cells in early embryos (4-cell to 32-cell stage embryos) Dnd1 and Nanos3 This study demonstrates that injecting both substances makes it possible to develop individuals that contain a significantly increased number of primordial germ cells in their gonadal region.

[0123] Tests 1-2 are, Dnd1 and Nanos3 This shows that the cells co-injected do not necessarily have to be cells destined to become primordial germ cells (i.e., cells in which germ granule factor is localized). Furthermore, Experiment 1-2 shows that Dnd1 and Nanos3 The injection of both genes results in the formation of a significantly larger number of primordial germ cells compared to the injection of those genes alone.

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

[0125] Tests 1-2 and 1-3 demonstrate that primordial germ cells formed by Dnd1 / Nanos3-OE possess properties similar to endogenous primordial germ cells, not only in terms of their ability to migrate to the gonads but also in terms of gene expression.

[0126] Test 1-4: Dnd1 and Nanos3 mRNA that codes for both In Test 1-1, Dnd1 mRNA that codes for and Nanos3 Two types of mRNA encoding the character were simultaneously microinjected. In Experiment 1-4, Dnd1 and Nanos3 A test similar to that in Test 1-1 was performed using single-stranded mRNA encoding both of the two genes. 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 In experiment (1), we tested whether the technique for increasing primordial germ cells, demonstrated in medaka, could be applied to other fish species. In experiment (2), we used zebrafish, which are a distant strain from medaka.

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

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

[0130] DN-OE embryos showed stronger EGFP fluorescence compared to control embryos, and Nanos3 expression was observed throughout the embryonic cells (Figure 5A). Similar to the results for medaka in Experiment 1-1, DN-OE embryos did not progress normally to the saccharid stage, and no individuals developed (hatched) (Figure 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 gonadal region were obtained. In all 16 individuals (100%), numerous EGFP-positive primordial germ cell-like cells were observed (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 gonadal region were obtained. In all 18 individuals, no EGFP-positive primordial germ cell-like cells were observed (0%) (Figure 5C).

[0132] EGFP-positive cells were observed in the gonadal regions of all four hatched DN-OE chimeras (Figure 5E). VASA protein was expressed in these EGFP-positive cells (Figure 5F).

[0133] Test (2) is, Dnd1 and Nanos3 This study demonstrates that co-introduction of [substance name] can induce the formation of embryos containing an increased number of primordial germ cells in zebrafish, and that the formed primordial germ cells possess properties similar to endogenous primordial germ cells.

[0134] Test (3): PGCs formed with Dnd1 / Nanos3-OE produce functional gametes. Olvas-EGFP / sox9b-DsRed transgenic medaka were prepared as donors. These transgenic medaka had germ cells labeled with EGFP and notochord and chondrocytes labeled with DsRed. Wild-type medaka (Cab strain) were prepared as hosts. Host embryos were prepared using the CRISPR / Cas13d system. dnd / nanos3 They knocked him out and made him infertile.

[0135] Donor cells were collected from blastula-stage embryos derived from the transgenic medaka. Donor cells were transplanted into blastula-stage host embryos by microinjection using a glass capillary, resulting in no more than 20 donor cells per embryo. Transplantation was performed on 4 to 5 host embryos. This transplantation condition is referred to as "Test N20". In the same manner, donor cells were transplanted into blastula-stage host embryos, resulting in more than 50 donor cells per embryo. Transplantation was performed on 2 to 3 host embryos. This transplantation condition is referred to as "Test N50". The transplanted chimeric embryos were reared in saline solution containing antibiotics and allowed to hatch. The hatched medaka were reared in fresh water for 3 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 aforementioned germline chimeric individuals was confirmed by pairing them with wild-type medaka. Furthermore, after mating, the chimeric individuals were dissected, and the presence or absence of donor-derived germ cells was examined based on EGFP fluorescence in the gonads.

[0137] In test N20, untreated ( Dnd1 / Nanos3 When donor cells derived from donor embryos (without injection) were used, the rate of germline chimera formation was 0% (Test Result 3-1). Dnd1 / Nanos3 When donor cells derived from donor embryos (Dnd1 / Nanos3-OE) injected via microinjection were used, the aforementioned production rate was 89%. Of the germline chimeras, 74% (14 out of 19 individuals) were fertile (Test Result 3-2).

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

[0139] Next-generation embryos were obtained from individuals whose fertility was confirmed. All next-generation embryos were positive for olvas-EGFP / sox9b-DsRed. This indicates that the next-generation embryos originated from the donor gametes. Furthermore, olvas-EGFP-positive germ cells were detected in all next-generation embryos. This indicates that normal germ cell formation progressed in the next-generation embryos (Figure 6).

[0140] Gametogenesis initiation factor dazl Co-infusion improves fertility. 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 deficient in 4 out of 7 infertile individuals. The remaining 3 individuals had underdeveloped gonads.

[0141] Furthermore, the gonads were examined 10 days after hatching. As a result, in XX individuals obtained by transplanting donor cells derived from untreated embryos into host embryos, all individuals had begun oogenesis. On the other hand, in chimeric XX individuals obtained by transplanting donor cells derived from dnd1 / nanos3-OE embryos into host embryos, oogenesis was confirmed in 5 out of 14 individuals. The remaining 8 individuals had only undifferentiated germ cells (n=8), and 1 individual lacked germ cells (n=1). These results indicate that all cells differentiated into germ cells by dnd1 / nanos3-OE lack the ability to initiate gamete formation.

[0142] We attempted to improve the fertility of germline chimeras obtained using donor cells derived from dnd1 / nanos3-OE donor embryos. dazl The mRNA dnd / nanos3mRNA was microinjected into donor embryos. More than 50 cells derived from the donor embryos were transplanted into sterilized host embryos (Study N50). As a result, the percentage of germline chimeras was 100%, and of these, 96.5% (26 out of 27 individuals) were fertile. One individual in which fertility could not be confirmed was dissected. As a result, normal testicular formation was confirmed. This indicates that gamete formation in this individual was normal. These results indicate that using dazl in conjunction with dnd / nanos3 can improve fertility in germline chimeras derived from Dnd1 / Nanos3-OE.

[0143] Test (4): Medaka (Japanese rice fish) dnd1 / nanos2 Enrichment of primordial germ cells in medaka embryos through introduction In Experiment 4, the same procedure as in Experiments 1-2 was used for 16-cell stage medaka embryos, except that nanos2 was used instead of nanos3. Specifically, 20 medaka embryos were used. In one of the outer cells of the medaka embryos, nanos2 mRNA that codes for and dnd1 mRNA encoding [the specified gene] was co-injected. As a result, 20 individuals were developed. Primordial germ cells were enriched in all 20 individuals (Figure 7B). Figure 7A shows the results of performing the same method as in Experiments 1-4 on 16-cell stage embryos.

[0144] Generally nanos Genes contain, nanos1 , nanos2 , and nanos3 There is. Nanos2 It is known to function in germ stem cells in adult fish, not in primordial germ cells in embryos. Experiment 4 was, nanos2 This demonstrated that it has the ability to form primordial germ cells in the embryo.

[0145] Test (5): Zebrafish dnd1 / nanos3 Enrichment of primordial germ cells in medaka embryos through introduction In Test 5, the medaka-derived dnd1 / nanos3 Instead of introducing zebrafish-derived dnd1 / nanos3 ( Zdnd1 , Znanos3 Except for introducing ), the procedure was essentially the same as in Experiment 1-2, but applied to 16-cell stage medaka embryos. Specifically, 13 medaka embryos were used. In one of the outer cells of the medaka embryos, Znanos3 mRNA that codes for and Zdnd1 A single-stranded mRNA encoding the gene was co-injected. As a result, 13 individuals were generated. Primordial germ cells were enriched in all 13 individuals (Figure 7C).

[0146] Experiment 5 demonstrated that even nanos and dnd from zebrafish, which belong to the order Cypriniformes, can enrich primordial germ cells in medaka embryos, which belong to the order Beloniformes. The results of Experiment 5 suggest that the invention disclosed herein can be carried out 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 phylum (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), it is possible to enrich the primordial germ cells of that animal species by co-introducing them into the embryos of the animal species from which primordial germ cells are to be obtained.

Claims

1. Introducing Dnd1 and Nanos into early embryos of fish-derived animals; and A method for preparing an embryo enriched with primordial germ cells, comprising culturing an early embryo into which the aforementioned Dnd1 and Nanos have been introduced, A method wherein Dnd1 and Nanos are each derived from animals belonging to the same phylum as the fish animal that provided the early embryo.

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

3. If Dnd1 and Nanos are encoding RNAs, they contain a polyA sequence at their 3' end, and The method according to claim 1, wherein Dnd1 and Nanos are encoding DNA, and include an exogenous polyadenylated sequence.

4. The method according to claim 1, further comprising introducing a gamete formation factor into the early embryo.

5. The method according to claim 4, wherein the gamete formation 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 early 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 genes of an early embryo of an animal belonging to the fish species.

8. A method for creating transgenic non-human vertebrates, Introducing Dnd1 and Nanos into early embryos of animals belonging to the fish class, and modifying the genes of the early embryos; and This includes culturing early embryos into which Dnd1 and Nanos have been introduced and the genes have been modified to generate transgenic non-human vertebrates, The aforementioned Dnd1 and Nanos are derived from animals belonging to the same phylum as the fish animal that provided the initial embryo. The aforementioned early embryos are embryos in the 4-cell stage to the 64-cell stage. The method wherein the transgenic non-human vertebrate contains the gene-modified primordial germ cells in its gonadal region.

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

10. To create germline chimeric non-human vertebrates, and A method for producing a non-human vertebrate, comprising crossbreeding the aforementioned germline chimeric non-human vertebrate, To create the aforementioned germline chimeric non-human vertebrates, To collect donor primordial germ cells from an embryo enriched with primordial germ cells prepared by any one of claims 1 to 7; Injecting collected donor primordial germ cells into host embryos derived from non-human vertebrates; and This includes culturing the host embryo into which the donor primordial germ cells have been injected, 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 the fish family and are closely related to each other, and A method wherein the non-human vertebrate produced 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, salmon and rainbow trout, tiger pufferfish and grass pufferfish, or 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 pufferfish and grass pufferfish, or a combination of goldfish and koi.

13. An embryo of an animal belonging to the fish species, enriched with primordial germ cells, The aforementioned embryo is an embryo from the blastula stage onward. At least some of the primordial germ cells contained in the embryo contain exogenous Dnd1 and exogenous Nanos, or a nucleotide encoding exogenous Dnd1 and a nucleotide encoding exogenous Nanos, The aforementioned exogenous Dnd1 and exogenous Nanos are derived from animals belonging to the same phylum as the fish that provided the embryo.

14. The embryo according to claim 13, wherein the number of primordial germ cells in the embryo is five times greater than the number of primordial germ cells in an embryo of the same developmental stage derived from an animal of the same species of fish as the embryo.

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