WO2021132205A1 - Method for producing fish having ability to self-fertilize and self-fertilizing eggs - Google Patents

Method for producing fish having ability to self-fertilize and self-fertilizing eggs Download PDF

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WO2021132205A1
WO2021132205A1 PCT/JP2020/047811 JP2020047811W WO2021132205A1 WO 2021132205 A1 WO2021132205 A1 WO 2021132205A1 JP 2020047811 W JP2020047811 W JP 2020047811W WO 2021132205 A1 WO2021132205 A1 WO 2021132205A1
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fish
gene
self
foxl3
agent
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PCT/JP2020/047811
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French (fr)
Japanese (ja)
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田中　実
俊哉 西村
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国立大学法人東海国立大学機構
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

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  • the present invention relates to a method for producing fish having self-fertilizing ability, a method for producing self-fertilized eggs, and the like.
  • Patent Document 1 it is reported that both fertilizable sperm and fertilizable egg are produced in the ovary of a fish in which the expression of the foxl3 gene in germ cells is suppressed. However, they cannot be self-fertilized because they are made in separate compartments within the ovary (Fig. 1).
  • An object of the present invention is to provide a self-fertilization technique for fish.
  • the present inventor (1) self-fertilization ability was improved by administering a maleizing agent to fish in which the function and / or expression of the foxl3 gene in germ cells was deficient or decreased. It has been found that it is possible to produce fish having a fertilized egg and also to produce self-fertilized eggs.
  • the present inventor has completed the present invention as a result of further research based on this finding. That is, the present invention includes the following aspects.
  • Item 1 A method for producing a fish having self-fertilizing ability, which comprises a step of administering a virilizing agent to a fish in which the function and / or expression of the foxl3 gene in germ cells is deficient or decreased.
  • Item 2. The production method according to Item 1, wherein the fish is a female.
  • Item 3 The production method according to Item 1 or 2, wherein the fish is in the oogenesis stage.
  • Item 4. The production method according to any one of Items 1 to 3, wherein the administration of the virilizing agent is continuous administration.
  • Item 5 The production method according to any one of Items 1 to 4, wherein the period of administration of the virilizing agent is 5 to 100 days.
  • Item 6 Fish having self-fertilizing ability obtained by the production method according to any one of Items 1 to 5.
  • Item 7. A method for producing a self-fertilized egg, which comprises a step of inducing ovulation in the fish according to item 6.
  • Item 8 The production method according to Item 7, wherein the ovulation induction treatment is mating with a male fish or administration of an ovulation inducer.
  • Item 9 The production method according to Item 8, wherein the ovulation induction treatment is mating with a sterile male fish.
  • Item 10 A method for producing a self-fertilized egg, which comprises a step of administering a virilizing agent to a fish in which the function and / or expression of the foxl3 gene in germ cells is deficient or decreased.
  • Item 11 The production method according to Item 10, further comprising (3) a step of inducing ovulation in a fish in which the function and / or expression of the foxl3 gene in germ cells is deficient or decreased.
  • Item 12. The manufacturing method according to Item 11, wherein the step 2 is performed after the step 1 is completed.
  • Item 13 The production method according to Item 11 or 12, wherein the ovulation induction treatment is mating with a male fish or administration of an ovulation inducer.
  • Item 14 A self-fertilized egg obtained by the production method according to any one of Items 7 to 13.
  • Item 15 Fish from which the self-fertilized egg according to item 14 is generated.
  • a self-fertilization technique for fish it is possible to provide a self-fertilization technique for fish. Specifically, it is possible to provide a method for producing a fish having a self-fertilizing ability, a method for producing a self-fertilized egg, a fish having a self-fertilizing ability, a self-fertilized egg, a fish in which a self-fertilized egg is generated, and the like.
  • FIG. 1 A schematic cross-sectional view of a normal fish ovary and a fish ovary in which the expression of the foxl3 gene in germ cells is suppressed, and an enlarged schematic view of a site where germ stem cells are present are shown.
  • the figure above shows the ovaries of foxl3-/-female medaka before the start of mating with the sterile male medaka.
  • the lower figure shows the section image of the dotted line part in the upper figure.
  • Identity of amino acid sequences refers to the degree of agreement between two or more contrastable amino acid sequences with respect to each other. Therefore, the higher the match between two amino acid sequences, the higher the identity or similarity of those sequences.
  • the level of amino acid sequence identity is determined, for example, using FASTA, a sequence analysis tool, using default parameters.
  • FASTA a sequence analysis tool
  • the algorithm BLAST by Karlin and Altschul Karlin and Altschul (KarlinS, Altschul SF. “Methods for assessment the statistical signature of molecular sequence features by using general scoring schemes” Proc Natl Acad Sci USA. 87: 2264-2268 (1990), K. It can be determined using “Applications and statistics for multiple high-scoring segments in molecular sequences.” Proc Natl Acad Sci USA.
  • BLASTX based on such a BLAST algorithm has been developed. Specific methods for these analysis methods are known, and the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/) can be referred to. In addition, the "identity" of the base sequence is also defined according to the above.
  • conservative substitution means that an amino acid residue is replaced with an amino acid residue having a similar side chain.
  • substitution between amino acid residues having a basic side chain such as lysine, arginine, and histidine is a conservative substitution.
  • amino acid residues with acidic side chains such as aspartic acid and glutamic acid
  • amino acid residues with non-charged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine and cysteine
  • Amino acid residues with non-polar side chains such as proline, phenylalanine, methionine and tryptophan
  • amino acid residues with ⁇ -branched side chains such as threonine, valine and isoleucine
  • aromatic side chains such as tyrosine, phenylalanine, tryptophan and histidine Substitution between amino acid residues is also a conservative substitution.
  • the present invention comprises (1) administration of a virilizing agent to fish in which the function and / or expression of the foxl3 gene in germ cells is deficient or reduced.
  • the present invention relates to a method for producing a fish having fertilizing ability (in the present specification, it may be referred to as “the production method 1 of the present invention”). This will be described below.
  • Fish are not particularly limited as long as they have the foxl3 gene endogenously.
  • fish for various purposes such as experimental or research, edible, ornamental, and aquaculture can be applied. Specific examples include medaka, zebrafish, goldfish, dojo, tilapia, eel, anago, carp, crucian carp, red sea bream, red sea bream, salmon, rainbow trout, amago, bristle, tuna, pufferfish, flatfish, and yamame trout.
  • those belonging to any classification such as teleost fish, cartilaginous fish, and jaw mouth fish can be applied. It is preferably applicable to teleost fish, and examples thereof include fish belonging to the genus Oryzias and its close relatives.
  • the sex of the fish is preferably a female chromosome type fish in the case of a fish having a sex chromosome, and is not particularly limited in the case of a fish having no sex chromosome (for example, Thailand).
  • the female chromosome type fish is not particularly limited as long as the combination of sex chromosomes is a female type fish.
  • sex determination based on sex chromosomes there are various types of sex determination based on sex chromosomes, and for example, XY type, XO type, modified type, ZW type, ZO type and the like are known.
  • XY type fish the case where the sex chromosome is XX corresponds to the female chromosome type
  • ZW type fish the case where the sex chromosome is ZW corresponds to the female chromosome type.
  • the foxl3 gene is a gene belonging to a protein family with a forkhead-type DNA-binding domain.
  • the foxl3 gene belongs to the same protein family as the foxl2 gene, which is conserved across species.
  • the foxl2 gene often means a female-specific gene expressed in somatic cells of an egg follicle, and is widely found in mammals, birds, and fish. In fish, foxl2 is also referred to as foxl2a.
  • the foxl3 gene is a gene different from foxl2a, and in Patent Document 1, fertilizable sperm and fertilizable sperm can be fertilized in the ovary of a fish in which the function and / or expression of the foxl3 gene in germ cells is deficient or reduced. It has been reported that both ovarian eggs are produced.
  • the nucleotide sequence and amino acid sequence of the foxl3 gene are known, or can be easily determined based on the known nucleotide sequence and amino acid sequence of the foxl3 gene (for example, by identity analysis with these). ..
  • the foxl3 gene of medaka is a protein consisting of the amino acid sequence specified by NCBI RefSeq accession number: NP_058673 (from the amino acid sequence encoded by the nucleotide sequence specified by NCBI RefSeq accession number: NM_016977). Protein).
  • SEQ ID NO: 1 is the nucleotide sequence of the foxl3 gene of medaka
  • SEQ ID NO: 2 is the amino acid sequence of the protein encoded by the foxl3 gene.
  • the foxl3 gene may be called the "foxl2b gene" depending on the classification and naming method.
  • the foxl3 gene targeted by the present invention also includes mutants that can occur in nature.
  • the foxl3 gene targeted by the present invention may have a base mutation such as substitution, deletion, addition, or insertion as long as the properties of the encoding protein (DNA binding ability, etc.) are not significantly impaired.
  • a mutation that does not cause an amino acid substitution in the protein translated from the mRNA or a mutation that causes a conservative substitution of an amino acid is preferable.
  • the amino acid sequence of the protein encoded by the gene is 95% or more, preferably 98% or more, the amino acid sequence of the protein encoded by the wild-type foxl3 gene of the same fish. , More preferably a gene having 99% or more identity.
  • the amino acid sequence of the protein encoded by the gene is the same as the amino acid sequence of the protein encoded by the wild-type foxl3 gene of the same fish, or the amino acid sequence thereof.
  • a gene in which one or more eg, 2-10, preferably 2-5, more preferably 2-3, even more preferably 2 are substituted, deleted, added, or inserted amino acid sequences. Is.
  • the function and / or expression of the foxl3 gene targeted by the present invention described above is deficient or decreased.
  • a mutation is introduced into the foxl3 gene so that the function and / or expression of the foxl3 gene targeted by the present invention is deleted or reduced.
  • the "function" of the foxl3 gene indicates the properties of the foxl3 protein (DNA binding ability, etc.).
  • the "expression" of the foxl3 gene includes both the expression of foxl3 mRNA and the expression of foxl3 protein, but is preferably the expression of foxl3 protein.
  • “Deficiency” indicates that the activity and / or expression level of the foxl3 gene is below the detection limit for a sample obtained from a fish of the present invention.
  • “decrease” means that the activity and / or expression level of the foxl3 gene is the foxl3 gene (the foxl3 gene targeted by the present invention) before the introduction of the mutation in the sample obtained from the fish targeted by the present invention. Less than activity and / or expression level (eg 1/2, 1/5, 1/10, 1/20, 1/50, 1/100, 1/200, 1/500, 1/1000, 1/2000) , 1/5000, 1/10000 or less).
  • the activity and / or expression level of the foxl3 gene can be measured according to a known method.
  • Suppression of gene expression can be evaluated by detecting at least one of the mRNA transcribed from the target gene and the protein encoded by the target gene. Specifically, when evaluating based on mRNA, mRNA transcribed from the target gene by Northern hybridization using a primer or probe specific to the target gene, RT-PCR, quantitative PCR, microarray, etc. It can be done by measuring the amount. When evaluating based on a protein, the expression level of the protein can be measured by an immunological method such as Western blotting or ELISA using an antibody specific to the protein encoded by the target gene. it can.
  • the preferred time to control the foxl3 gene in germ cells can be appropriately selected according to the development of the gonads of the target fish. Specifically, in order to differentiate germ cells into sperms, it is preferable to suppress the expression of the foxl3 gene in germ cells at the same time. For example, in medaka, the differentiation time of the gonad is before and after hatching, and it is possible to suppress the expression of the foxl3 gene in germ cells at this time. In addition, since stem cell type and cyst type germ cells are considered to maintain sexual undifferentiation or sexual plasticity, it is possible to control the stage of juveniles after sexual differentiation and in the mature gonads (ovaries). In addition, for individuals such as hagfish, which are thought to undergo sexual differentiation several years after hatching, the expression of the foxl3 gene in germ cells can be suppressed at the time of sexual differentiation according to the individual.
  • the mutation is not particularly limited as long as it is a mutation in which the function and / or expression of the foxl3 gene is deleted or decreased.
  • the mutation include a gene defect, a mutation in a protein coding region, a mutation in a splicing regulatory region, a mutation in an expression control region (for example, a promoter, an activator, an enhancer, etc.) and the like.
  • gene deletion is preferably mentioned.
  • Mutation introduction into the foxl3 gene includes, for example, gene knockout using an artificial nuclease (TALEN, Cas protein), gene knockout using an ES cell into which a targeting vector containing at least a part of the foxl3 gene has been introduced by homologous recombination, and foxl3 gene. It can be achieved by gene knockdown using shRNA, antisense, ribozyme, etc., irradiation, chemical mutant administration, transposon introduction, and the like. Construction of targeting vectors used for gene knockout, gene knockdown, gene mutation introduction, design and synthesis of shRNA, antisense, ribozyme, etc., DNA to ES cells, unfertilized eggs, fertilized eggs, embryos, primordial germ cells, etc. The method of introducing the gene, irradiation, administration of a chemical mutation source, introduction of a transposon, etc. can be performed by applying a conventionally known method to those skilled in the art.
  • TALEN artificial nuclease
  • the "fishes in which the function and / or expression of the foxl3 gene is deficient or decreased" used in the production method of the present invention include foxl3 gene knockout fish (foxl3 gene deficient fish), foxl3 gene knockdown fish, and foxl3 gene mutation as described above. Introduced fish and the like can be mentioned, but foxl3 gene knockout fish are preferable.
  • sperm In the ovary of fish in which the function and / or expression of the foxl3 gene in germ cells is deficient or reduced, only sperm are initially produced, but then eggs are also produced. This can be determined by a method known to those skilled in the art, for example, by observing a stained image of a section of an ovary, whether or not not only a sperm but also an egg is produced.
  • the target fish in step 1 is preferably at the time when an egg is formed (oogenesis stage: a time when the presence of an egg can be confirmed in the ovary). In the case of medaka, the oogenesis period is, for example, 90 to 120 days after hatching. By adopting fish at such a time, it is possible to improve the production efficiency of fish having self-fertilizing ability.
  • the maleizing agent is not particularly limited as long as it is a substance that causes virilization, and examples thereof include substances that promote virilization such as androgens and substances that suppress femaleization such as antiestrogen agents.
  • androgens include 11-ketotestosterone, adrenosterone, testosterone, dihydrotestosterone, dehydroepiandrosterone and the like.
  • anti-estrogen agent include an estrogen receptor inhibitor, an aromatase inhibitor and the like. Androgen is preferable as the virilizing agent.
  • the virilizing agent may be used alone or in combination of two or more.
  • the administration method of the virilizing agent is not particularly limited, and an appropriate method can be adopted according to the type of fish and the breeding form.
  • Examples of the administration method include oral administration, transdermal administration, intraperitoneal administration, intravenous administration, subcutaneous administration and the like.
  • the present invention for example, by dissolving the maleizing agent in the breeding water, it is possible to administer it easily and reliably.
  • a feed containing a virilizing agent as a feed, it is possible to administer the feed easily and reliably.
  • the form of the feed is not particularly limited as long as it is ingestible by the target fish. Examples of the form of the feed include powder, granule, crumble, pellet, cube, paste, liquid and the like.
  • the maleizing agent-containing feed includes other substances / components constituting the feed, such as fish meal; soybean meal, concentrated soybean protein, corn gluten meal, corn distilled meal, rapeseed oil meal, and other vegetable feed raw materials; Animal feed raw materials such as meat-and-bone meal, cod liver oil, feather meal, blood powder; microalgae raw materials; single-cell biological raw materials such as yeast and bacteria; binders (for example, sodium carboxymethyl cellulose (CMC), guar gum, sodium alginate, polyacrylic Soy acid, sodium caseinate, propylene glycol, etc.), grain flour (eg wheat flour, starch, etc.), minerals, vitamins (eg, vitamin C, vitamin B1, vitamin A, vitamin D, vitamin E, etc.), amino acids (eg, lysine) , Methionin, histidine, etc.), pigments (eg, ⁇ -carotene, astaxanthin, cantaxanthin, etc.), liver oil (eg, cod liver oil, etc.
  • CMC carboxymethyl
  • a virilizing agent causes a part of sperm to move into the ovarian cavity where mature eggs are ovulated (see Test Example 2).
  • the dose of the virilizing agent is not particularly limited as long as the amount of sperm transfer to the ovarian cavity occurs, and the presence or absence of the phenomenon can be appropriately set as an index.
  • the concentration of the maleizing agent in the water is, for example, 1 to 1000 ng / ml, preferably 5 to 500 ng / ml, more preferably 10 to 200 ng / ml, and further preferably. It is 20 to 100 ng / ml.
  • the content of the virilizing agent in the feed is, for example, 0.00001 to 50% by mass.
  • the upper and lower limits of the content are, for example, 0.0001% by mass, 0.001% by mass, 0.01% by mass, 0.1% by mass, 0.2% by mass, 0.5% by mass, 1% by mass, 2% by mass, 5% by mass, 10% by mass, It can be 20% by mass, 30% by mass, 40% by mass, and the like.
  • the number of administrations of the virilizing agent is not particularly limited, but the administration of the virilizing agent is preferably continuous administration. That is, it is preferable to administer the virilizing agent continuously or continuously at regular intervals (for example, 1 to 3 times a day, once every 2 to 3 days, etc.).
  • the administration period of the virilizing agent is not particularly limited as long as the period of sperm transfer to the ovarian cavity occurs, and the presence or absence of the phenomenon can be appropriately set as an index.
  • the period is, for example, 1 to 200 days, preferably 5 to 100 days, more preferably 10 to 50 days, and even more preferably 20 to 40 days. This period can vary depending on the time to maturity of the fish.
  • This breeding period is not particularly limited, and is, for example, 0.1 to 10, preferably 0.3 to 5, and more preferably 0.7 to 3 with respect to the maleizing agent administration period 1. More specifically, the breeding period is, for example, 1 to 400 days, preferably 5 to 200 days, more preferably 10 to 100 days, and even more preferably 20 to 80 days.
  • the fish obtained by the production method 1 of the present invention (sometimes referred to as "the fish of the present invention” in the present specification) has a self-fertilizing ability. That is, since a part of sperm is transferred from the parenchyma of the ovary to the outside (for example, to the ovarian cavity or body cavity), when a mature egg is transferred from the parenchyma of the ovary to the outside, it is possible to fertilize with its own sperm. Is.
  • a method for producing a self-fertilized egg including (2) a step of inducing ovulation in the fish of the present invention may be referred to as "the production method 2A of the present invention" in the present specification. .). This will be described below.
  • the ovulation induction treatment is not particularly limited, and examples thereof include mating with male fish and administration of an ovulation inducer.
  • Male fish are preferably sterile male fish in order to obtain only self-fertilized eggs.
  • Sterile male fish can be produced, for example, by mutation of a gene involved in sterility (for example, nanos gene).
  • the ovulation inducer include clomiphene, gonadotropin, chorionic gonadotropin, GnRH agonist, GnRH antagonist and the like.
  • fertilization with self-fertilized sperm occurs when ovulation occurs, and a self-fertilized egg can be obtained.
  • the obtained self-fertilized egg can be cultivated and generated in an appropriate environment to obtain an individual fish.
  • a method for producing a self-fertilized egg which comprises (1) administering a virilizing agent to fish in which the function and / or expression of the foxl3 gene in germ cells is deficient or decreased (the present specification). In the above, it may be referred to as “the production method 2B of the present invention”). This will be described below.
  • the production method 2B of the present invention preferably further includes (3) a step of inducing ovulation in fish in which the function and / or expression of the foxl3 gene in germ cells is deficient or reduced. By this step, self-fertilization is more likely to occur, and the production efficiency of self-fertilized eggs can be improved.
  • the ovulation induction treatment is the same as the production method 2A of the present invention.
  • the obtained self-fertilized egg can be cultivated and generated in an appropriate environment to obtain an individual fish.
  • Reference example 1 Preparation of sterile male medaka A sterile male medaka of a nanos homozygous mutant (nanos-/-) in which both alleles of the nanos gene were disrupted was prepared as follows using TALEN technology. The TALEN target site for the nanos3 gene was searched using the TALEN Targeter program (https://tale-nt.cac.cornell.edu/node/add/talen). The TALEN plasmid was linearized by NotI digestion and used as a template for in vitro RNA synthesis using the mMESSAGE mMACHINE® T7 Transcription Kit (Thermo Fisher).
  • TALEN mRNA 250 ng / ⁇ l left and right was injected into 1-cell or 2-cell stage embryos.
  • the F0 founder was crossed with a wild-type medaka.
  • Males and females of the obtained nanos heterozygous mutant (nanos +/-) were crossed to obtain a sterile male medaka of the nanos homozygous mutant (nanos-/-).
  • Test example 1 Production of self-fertilizing fish and self-fertilized eggs Foxl3 homozygous mutation by mating medaka (Patent Document 1) of foxl3 heterozygous mutant (foxl3 +/-) in which one allele of the foxl3 gene is disrupted A female chromosome type (XX type) medaka of the body (foxl3-/-) was obtained.
  • foxl3-/-Medaka was bred in an aquarium according to a conventional method, and from 90 to 120 days after hatching, it was bred in a virilizing agent aqueous solution containing a virilizing agent (11-ketotestosterone) at a concentration of 50 ng / ml for 30 days.
  • the virilizing agent aqueous solution was changed once every 24 hours. After breeding in an aqueous virilizing agent, the animals were bred in normal water containing no maleizing agent for at least 30 days (30 to 60 days). A sterile male medaka fish (Reference Example 1) was placed in an aquarium and bred for 30 days for mating. During this period, the laid eggs were collected and the presence or absence of fertilization was judged using the activation of the egg membrane and the normal development up to the blastogenic stage as indicators.
  • Test example 2 Histological analysis of ovaries
  • the gonads of foxl3-/-female medaka before the start of mating with sterile male medaka in Test Example 1 were dissected and fixed overnight at 4 ° C with Buin solution.
  • a 4 ⁇ m thick plastic section was prepared using Technovit® 8100 (Heraeus Kulzer). Sections were stained with periodic acid Schiff and hematoxyli according to a conventional method.

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Abstract

The present invention addresses the problem of providing a means of self-fertilizing in fish, and solves said problem by providing a method for producing a fish having the ability to self-fertilize or self-fertilizing eggs, the method including (1) a step for administering a masculinizing agent to a fish exhibiting deficient or reduced function and/or expression of the foxl3 gene in the reproductive cells thereof.

Description

自家受精能を有する魚類及び自家受精卵の製造方法Method for producing self-fertilizing fish and self-fertilized eggs
　本発明は、自家受精能を有する魚類の製造方法、自家受精卵の製造方法等に関する。 The present invention relates to a method for producing fish having self-fertilizing ability, a method for producing self-fertilized eggs, and the like.
　各種動物が、有用形質を指標として品種改良されている。例えば、メダカ等の観賞用魚類は、審美的観点等に基づいて、体色、模様、目の形状、ヒレの形状等を指標として品種改良されており、メダカについては近年では数百種類もの品種が報告されている。また、タイ、ブリ、マグロ等の食用養殖魚においても、成長速度、病気抵抗性、美味しさ等を指標として、各種品種改良されている。しかし、通常、動物の受精は他家受精であるので、有用形質が見出されたとしても、それが固定された品種の作出には手間や時間を要する。 Various animals have been bred using useful traits as indicators. For example, ornamental fish such as medaka have been bred based on aesthetic viewpoints, using body color, pattern, eye shape, fin shape, etc. as indicators. In recent years, there are hundreds of varieties of medaka. Has been reported. In addition, various varieties of edible farmed fish such as Thailand, yellowtail, and tuna have been bred using the growth rate, disease resistance, and deliciousness as indicators. However, since animal fertilization is usually allogeneic fertilization, even if a useful trait is found, it takes time and effort to produce a variety in which it is fixed.
　自家受精が可能であれば、有用形質が固定された品種の作出を、より効率的に行うことができる。しかしながら、自家受精は、一部の非脊椎動物において起こるものの、大多数の動物、特に脊椎動物においては、通常は起こり得ない。 If self-fertilization is possible, it is possible to more efficiently produce varieties with fixed useful traits. However, although self-fertilization occurs in some non-vertebrates, it usually does not occur in the majority of animals, especially vertebrates.
特開2015-126729号公報JP-A-2015-126729
　特許文献１においては、生殖細胞中のfoxl3遺伝子の発現が抑制された魚類の卵巣には、受精可能な精子と受精可能な卵子の両方が作られることが報告されている。しかし、これらは卵巣内の別の区画で作られるので、自家受精することはできない（図１）。 In Patent Document 1, it is reported that both fertilizable sperm and fertilizable egg are produced in the ovary of a fish in which the expression of the foxl3 gene in germ cells is suppressed. However, they cannot be self-fertilized because they are made in separate compartments within the ovary (Fig. 1).
　本発明は、魚類における自家受精技術を提供することを課題とする。 An object of the present invention is to provide a self-fertilization technique for fish.
　本発明者は上記課題に鑑みて鋭意研究を進めた結果、（1）生殖細胞中のfoxl3遺伝子の機能及び／又は発現が欠損又は低下した魚類に雄性化剤投与することにより、自家受精能を有する魚類を製造でき、さらに自家受精卵を製造できることを見出した。本発明者はこの知見に基づいてさらに研究を進めた結果、本発明を完成させた。即ち、本発明は、下記の態様を包含する。 As a result of diligent research in view of the above problems, the present inventor (1) self-fertilization ability was improved by administering a maleizing agent to fish in which the function and / or expression of the foxl3 gene in germ cells was deficient or decreased. It has been found that it is possible to produce fish having a fertilized egg and also to produce self-fertilized eggs. The present inventor has completed the present invention as a result of further research based on this finding. That is, the present invention includes the following aspects.
　項１．　（1）生殖細胞中のfoxl3遺伝子の機能及び／又は発現が欠損又は低下した魚類に雄性化剤投与する工程を含む、自家受精能を有する魚類の製造方法。 Item 1. (1) A method for producing a fish having self-fertilizing ability, which comprises a step of administering a virilizing agent to a fish in which the function and / or expression of the foxl3 gene in germ cells is deficient or decreased.
　項２．　前記魚類が雌である、項１に記載の製造方法。 Item 2. Item 2. The production method according to Item 1, wherein the fish is a female.
　項３．　前記魚類が卵形成期である、項１又は２に記載の製造方法。 Item 3. Item 2. The production method according to Item 1 or 2, wherein the fish is in the oogenesis stage.
　項４．　前記雄性化剤投与が持続投与である、項１～３のいずれかに記載の製造方法。 Item 4. Item 8. The production method according to any one of Items 1 to 3, wherein the administration of the virilizing agent is continuous administration.
　項５．　前記雄性化剤投与の期間が5～100日間である、項１～４のいずれかに記載の製造方法。 Item 5. Item 8. The production method according to any one of Items 1 to 4, wherein the period of administration of the virilizing agent is 5 to 100 days.
　項６．　項１～５のいずれかに記載の製造方法で得られる、自家受精能を有する魚類。 Item 6. Fish having self-fertilizing ability obtained by the production method according to any one of Items 1 to 5.
　項７．　（2）項６に記載の魚類に排卵誘発処理する工程を含む、自家受精卵の製造方法。 Item 7. (2) A method for producing a self-fertilized egg, which comprises a step of inducing ovulation in the fish according to item 6.
　項８．　前記排卵誘発処理が雄魚類との交配又は排卵誘発剤の投与である、項７に記載の製造方法。 Item 8. Item 8. The production method according to Item 7, wherein the ovulation induction treatment is mating with a male fish or administration of an ovulation inducer.
　項９．　前記排卵誘発処理が不稔の雄魚類との交配である、項８に記載の製造方法。 Item 9. Item 8. The production method according to Item 8, wherein the ovulation induction treatment is mating with a sterile male fish.
　項１０．　（1）生殖細胞中のfoxl3遺伝子の機能及び／又は発現が欠損又は低下した魚類に雄性化剤投与する工程を含む、自家受精卵の製造方法。 Item 10. (1) A method for producing a self-fertilized egg, which comprises a step of administering a virilizing agent to a fish in which the function and / or expression of the foxl3 gene in germ cells is deficient or decreased.
　項１１．　さらに、（3）生殖細胞中のfoxl3遺伝子の機能及び／又は発現が欠損又は低下した魚類を排卵誘発処理する工程を含む、項１０に記載の製造方法。 Item 11. Item 10. The production method according to Item 10, further comprising (3) a step of inducing ovulation in a fish in which the function and / or expression of the foxl3 gene in germ cells is deficient or decreased.
　項１２．　前記工程1終了後に前記工程2を行う、項１１に記載の製造方法。 Item 12. Item 2. The manufacturing method according to Item 11, wherein the step 2 is performed after the step 1 is completed.
　項１３．　前記排卵誘発処理が雄魚類との交配又は排卵誘発剤の投与である、項１１又は１２に記載の製造方法。 Item 13. Item 4. The production method according to Item 11 or 12, wherein the ovulation induction treatment is mating with a male fish or administration of an ovulation inducer.
　項１４．　項７～１３のいずれかに記載の製造方法で得られる、自家受精卵。 Item 14. A self-fertilized egg obtained by the production method according to any one of Items 7 to 13.
　項１５．　項１４に記載の自家受精卵が発生してなる、魚類。 Item 15. Fish from which the self-fertilized egg according to item 14 is generated.
　本発明によれば、魚類における自家受精技術を提供することができる。具体的には、自家受精能を有する魚類の製造方法、自家受精卵の製造方法、自家受精能を有する魚類、自家受精卵、自家受精卵が発生してなる魚類等を提供することができる。 According to the present invention, it is possible to provide a self-fertilization technique for fish. Specifically, it is possible to provide a method for producing a fish having a self-fertilizing ability, a method for producing a self-fertilized egg, a fish having a self-fertilizing ability, a self-fertilized egg, a fish in which a self-fertilized egg is generated, and the like.
通常の魚類の卵巣と生殖細胞中のfoxl3遺伝子の発現が抑制された魚類の卵巣との断面模式図、及び生殖幹細胞が存在する部位の拡大模式図を示す。A schematic cross-sectional view of a normal fish ovary and a fish ovary in which the expression of the foxl3 gene in germ cells is suppressed, and an enlarged schematic view of a site where germ stem cells are present are shown. 上図は、不稔雄メダカとの交配開始前のfoxl3 -/-雌メダカの卵巣を示す。下図は、上図の点線部の切片像を示す。The figure above shows the ovaries of foxl3-/-female medaka before the start of mating with the sterile male medaka. The lower figure shows the section image of the dotted line part in the upper figure.
　本明細書中において、「含有」及び「含む」なる表現については、「含有」、「含む」、「実質的にからなる」及び「のみからなる」という概念を含む。 In the present specification, the expressions "contains" and "contains" include the concepts of "contains", "contains", "substantially consists" and "consists of only".
　1．定義
　本明細書中において、「含有」及び「含む」なる表現については、「含有」、「含む」、「実質的にからなる」及び「のみからなる」という概念を含む。 1. 1. Definitions In the present specification, the expressions "contains" and "contains" include the concepts of "contains", "contains", "substantially consists" and "consists of only".
　アミノ酸配列の「同一性」とは、2以上の対比可能なアミノ酸配列の、お互いに対するアミノ酸配列の一致の程度をいう。従って、ある2つのアミノ酸配列の一致性が高いほど、それらの配列の同一性又は類似性は高い。アミノ酸配列の同一性のレベルは、例えば、配列分析用ツールであるFASTAを用い、デフォルトパラメータを用いて決定される。若しくは、Karlin及びAltschulによるアルゴリズムBLAST（KarlinS，Altschul SF．“Methods for assessing the statistical significance of molecular sequence features by using general scoringschemes”Proc Natl Acad Sci USA．87:2264－2268（1990）、KarlinS，Altschul SF．“Applications and statistics for multiple high－scoring segments in molecular sequences．”Proc Natl Acad Sci USA．90:5873－7（1993））を用いて決定できる。このようなBLASTのアルゴリズムに基づいたBLASTXと呼ばれるプログラムが開発されている。これらの解析方法の具体的な手法は公知であり、National Center of Biotechnology Information（NCBI）のウェエブサイト（http://www.ncbi.nlm.nih.gov/）を参照すればよい。また、塩基配列の『同一性』も上記に準じて定義される。 "Identity" of amino acid sequences refers to the degree of agreement between two or more contrastable amino acid sequences with respect to each other. Therefore, the higher the match between two amino acid sequences, the higher the identity or similarity of those sequences. The level of amino acid sequence identity is determined, for example, using FASTA, a sequence analysis tool, using default parameters. Alternatively, the algorithm BLAST by Karlin and Altschul (KarlinS, Altschul SF. “Methods for assessment the statistical signature of molecular sequence features by using general scoring schemes” Proc Natl Acad Sci USA. 87: 2264-2268 (1990), K. It can be determined using “Applications and statistics for multiple high-scoring segments in molecular sequences.” Proc Natl Acad Sci USA. 90: 5873-7 (1993). A program called BLASTX based on such a BLAST algorithm has been developed. Specific methods for these analysis methods are known, and the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/) can be referred to. In addition, the "identity" of the base sequence is also defined according to the above.
　本明細書中において、「保存的置換」とは、アミノ酸残基が類似の側鎖を有するアミノ酸残基に置換されることを意味する。例えば、リジン、アルギニン、ヒスチジンといった塩基性側鎖を有するアミノ酸残基同士で置換されることが、保存的な置換にあたる。その他、アスパラギン酸、グルタミン酸といった酸性側鎖を有するアミノ酸残基；グリシン、アスパラギン、グルタミン、セリン、スレオニン、チロシン、システインといった非帯電性極性側鎖を有するアミノ酸残基；アラニン、バリン、ロイシン、イソロイシン、プロリン、フェニルアラニン、メチオニン、トリプトファンといった非極性側鎖を有するアミノ酸残基；スレオニン、バリン、イソロイシンといったβ－分枝側鎖を有するアミノ酸残基；チロシン、フェニルアラニン、トリプトファン、ヒスチジンといった芳香族側鎖を有するアミノ酸残基同士での置換も同様に、保存的な置換にあたる。 In the present specification, "conservative substitution" means that an amino acid residue is replaced with an amino acid residue having a similar side chain. For example, substitution between amino acid residues having a basic side chain such as lysine, arginine, and histidine is a conservative substitution. Other amino acid residues with acidic side chains such as aspartic acid and glutamic acid; amino acid residues with non-charged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine and cysteine; alanine, valine, leucine, isoleucine, Amino acid residues with non-polar side chains such as proline, phenylalanine, methionine and tryptophan; amino acid residues with β-branched side chains such as threonine, valine and isoleucine; with aromatic side chains such as tyrosine, phenylalanine, tryptophan and histidine Substitution between amino acid residues is also a conservative substitution.
　2．自家受精能を有する魚類の製造方法
　本発明は、その一態様において、（1）生殖細胞中のfoxl3遺伝子の機能及び／又は発現が欠損又は低下した魚類に雄性化剤投与する工程を含む、自家受精能を有する魚類の製造方法（本明細書において、「本発明の製造方法1」と示すこともある。）に関する。以下に、これについて説明する。 2. Method for producing fish having self-fertilizing ability In one aspect of the present invention, the present invention comprises (1) administration of a virilizing agent to fish in which the function and / or expression of the foxl3 gene in germ cells is deficient or reduced. The present invention relates to a method for producing a fish having fertilizing ability (in the present specification, it may be referred to as “the production method 1 of the present invention”). This will be described below.
　魚類としては、foxl3遺伝子を内在的に有する限り特に制限されない。魚類としては、例えば、実験又は研究用、食用、観賞用、養殖用など種々の用途の魚類を適用することができる。具体的には、例えばメダカ、ゼブラフィッシュ、キンギョ、ドジョウ、ティラピア、ウナギ、アナゴ、コイ、フナ、ヘダイ、マダイ、サケ、ニジマス、アマゴ、ブリ、マグロ、フグ、ヒラメ、ヤマメ等が挙げられる。当該魚類は、硬骨魚類、軟骨魚類、顎口魚類等のいずれの分類に属するものも適用することができる。硬骨魚類に好ましく適用可能であり、例えばメダカ属（Oryzias）及びその近縁に属する魚類などが挙げられる。 Fish are not particularly limited as long as they have the foxl3 gene endogenously. As the fish, for example, fish for various purposes such as experimental or research, edible, ornamental, and aquaculture can be applied. Specific examples include medaka, zebrafish, goldfish, dojo, tilapia, eel, anago, carp, crucian carp, red sea bream, red sea bream, salmon, rainbow trout, amago, bristle, tuna, pufferfish, flatfish, and yamame trout. As the fish concerned, those belonging to any classification such as teleost fish, cartilaginous fish, and jaw mouth fish can be applied. It is preferably applicable to teleost fish, and examples thereof include fish belonging to the genus Oryzias and its close relatives.
　魚類の雌雄は、性染色体を有する魚類の場合は、雌染色体型魚類であることが好ましく、性染色体を有しない魚類（例えばタイ等）の場合は、特に制限されない。 The sex of the fish is preferably a female chromosome type fish in the case of a fish having a sex chromosome, and is not particularly limited in the case of a fish having no sex chromosome (for example, Thailand).
　雌染色体型魚類は、性染色体の組合せが雌型である魚類である限り、特に制限されない。性染色体による性決定は各種存在し、例えばXY型、XO型、変形型、ZW型、ZO型等が知られている。例えばXY型の魚類であれば、性染色体がXXである場合が雌染色体型に該当し、ZW型の魚類であれば、性染色体がZWである場合が雌染色体型に該当する。 The female chromosome type fish is not particularly limited as long as the combination of sex chromosomes is a female type fish. There are various types of sex determination based on sex chromosomes, and for example, XY type, XO type, modified type, ZW type, ZO type and the like are known. For example, in the case of XY type fish, the case where the sex chromosome is XX corresponds to the female chromosome type, and in the case of ZW type fish, the case where the sex chromosome is ZW corresponds to the female chromosome type.
　foxl3遺伝子は、フォークヘッド型のDNA結合ドメインを持つ蛋白質ファミリーに属する遺伝子である。foxl3遺伝子は、種を超えて保存されているfoxl2遺伝子と同じ蛋白質ファミリーに属している。ここで、一般的にfoxl2遺伝子は、卵ろ胞の体細胞で発現する雌特異的遺伝子を意味することが多く、哺乳類や鳥類、魚類でも広く見出される。なお、魚類ではfoxl2をfoxl2aとも表記する。一方、foxl3遺伝子はfoxl2aとは別の遺伝子であり、特許文献１においては、生殖細胞中のfoxl3遺伝子の機能及び／又は発現が欠損又は低下した魚類の卵巣には、受精可能な精子と受精可能な卵子の両方が作られることが報告されている。各種魚類において、foxl3遺伝子の塩基配列及びアミノ酸配列は公知である、或いは公知のfoxl3遺伝子の塩基配列及びアミノ酸配列に基づいて（例えば、これらとの同一性解析等により）容易に決定することができる。一例として、メダカ（Oryzias latipes）のfoxl3遺伝子としては、NCBI RefSeqアクセッション番号：NP＿058673で特定されるアミノ酸配列からなるタンパク質（NCBI RefSeqアクセッション番号：NM＿016977で特定されるヌクレオチド配列がコードするアミノ酸配列からなるタンパク質）が挙げられる。配列番号1はメダカのfoxl3遺伝子の塩基配列、配列番号2はfoxl3遺伝子によってコードされるタンパク質のアミノ酸配列である。なお、foxl3遺伝子は、分類や呼称の仕方によっては「foxl2b遺伝子」と呼ばれることがある。 The foxl3 gene is a gene belonging to a protein family with a forkhead-type DNA-binding domain. The foxl3 gene belongs to the same protein family as the foxl2 gene, which is conserved across species. Here, in general, the foxl2 gene often means a female-specific gene expressed in somatic cells of an egg follicle, and is widely found in mammals, birds, and fish. In fish, foxl2 is also referred to as foxl2a. On the other hand, the foxl3 gene is a gene different from foxl2a, and in Patent Document 1, fertilizable sperm and fertilizable sperm can be fertilized in the ovary of a fish in which the function and / or expression of the foxl3 gene in germ cells is deficient or reduced. It has been reported that both ovarian eggs are produced. In various fish, the nucleotide sequence and amino acid sequence of the foxl3 gene are known, or can be easily determined based on the known nucleotide sequence and amino acid sequence of the foxl3 gene (for example, by identity analysis with these). .. As an example, the foxl3 gene of medaka (Oryzias laminates) is a protein consisting of the amino acid sequence specified by NCBI RefSeq accession number: NP_058673 (from the amino acid sequence encoded by the nucleotide sequence specified by NCBI RefSeq accession number: NM_016977). Protein). SEQ ID NO: 1 is the nucleotide sequence of the foxl3 gene of medaka, and SEQ ID NO: 2 is the amino acid sequence of the protein encoded by the foxl3 gene. The foxl3 gene may be called the "foxl2b gene" depending on the classification and naming method.
　本発明が対象とするfoxl3遺伝子には、自然界において生じ得る変異体も含まれる。本発明が対象とするfoxl3遺伝子は、コードするタンパク質の性質（DNA結合能等）が著しく損なわれない限りにおいて、置換、欠失、付加、挿入等の塩基変異を有していてもよい。変異としては、該mRNAから翻訳されるタンパク質においてアミノ酸置換が生じない変異やアミノ酸の保存的置換が生じる変異が好ましい。 The foxl3 gene targeted by the present invention also includes mutants that can occur in nature. The foxl3 gene targeted by the present invention may have a base mutation such as substitution, deletion, addition, or insertion as long as the properties of the encoding protein (DNA binding ability, etc.) are not significantly impaired. As the mutation, a mutation that does not cause an amino acid substitution in the protein translated from the mRNA or a mutation that causes a conservative substitution of an amino acid is preferable.
　本発明が対象とするfoxl3遺伝子は、例えば、それによりコードされるタンパク質のアミノ酸配列が、同種魚類の野生型foxl3遺伝子によりコードされるタンパク質のアミノ酸配列と、例えば95％以上、好ましくは98％以上、より好ましくは99％以上の同一性を有する、遺伝子である。また、本発明が対象とするfoxl3遺伝子は、例えば、それによりコードされるタンパク質のアミノ酸配列が、同種魚類の野生型foxl3遺伝子によりコードされるタンパク質のアミノ酸配列と同一であるか又は該アミノ酸配列に対して1もしくは複数個（例えば2～10、好ましくは2～5、より好ましくは2～3個、さらに好ましくは2個）が置換、欠失、付加、又は挿入されたアミノ酸配列である、遺伝子である。 In the foxl3 gene targeted by the present invention, for example, the amino acid sequence of the protein encoded by the gene is 95% or more, preferably 98% or more, the amino acid sequence of the protein encoded by the wild-type foxl3 gene of the same fish. , More preferably a gene having 99% or more identity. Further, in the foxl3 gene targeted by the present invention, for example, the amino acid sequence of the protein encoded by the gene is the same as the amino acid sequence of the protein encoded by the wild-type foxl3 gene of the same fish, or the amino acid sequence thereof. A gene in which one or more (eg, 2-10, preferably 2-5, more preferably 2-3, even more preferably 2) are substituted, deleted, added, or inserted amino acid sequences. Is.
　本発明が対象とする魚類の生殖細胞においては、上記した本発明が対象とするfoxl3遺伝子の機能及び／又は発現が欠損又は低下している。本発明の一態様において、本発明が対象とする魚類においては、上記した本発明が対象とするfoxl3遺伝子の機能及び／又は発現が欠損又は低下するように、該foxl3遺伝子に変異が導入されている。ここで、foxl3遺伝子の「機能」は、foxl3タンパク質の性質（DNA結合能等）を示す。また、foxl3遺伝子の「発現」は、foxl3 mRNAの発現、及びfoxl3タンパク質の発現の両方を包含するが、好ましくはfoxl3タンパク質の発現である。「欠損」とは、本発明が対象とする魚類から得られたサンプルについて、foxl3遺伝子の活性及び／又は発現量が検出限界以下であることを示す。また、「低下」とは、本発明が対象とする魚類から得られたサンプルについて、foxl3遺伝子の活性及び／又は発現量が、変異導入前のfoxl3遺伝子（本発明が対象とするfoxl3遺伝子）の活性及び／又は発現量よりも低い（例えば1／2、1／5、1／10、1／20、1／50、1／100、1／200、1／500、1／1000、1／2000、1／5000、1／10000以下である）ことを示す。なお、foxl3遺伝子の活性及び／又は発現量は、公知の方法に従って測定することが可能である。 In the germ cells of fish targeted by the present invention, the function and / or expression of the foxl3 gene targeted by the present invention described above is deficient or decreased. In one aspect of the present invention, in the fish targeted by the present invention, a mutation is introduced into the foxl3 gene so that the function and / or expression of the foxl3 gene targeted by the present invention is deleted or reduced. There is. Here, the "function" of the foxl3 gene indicates the properties of the foxl3 protein (DNA binding ability, etc.). The "expression" of the foxl3 gene includes both the expression of foxl3 mRNA and the expression of foxl3 protein, but is preferably the expression of foxl3 protein. “Deficiency” indicates that the activity and / or expression level of the foxl3 gene is below the detection limit for a sample obtained from a fish of the present invention. In addition, "decrease" means that the activity and / or expression level of the foxl3 gene is the foxl3 gene (the foxl3 gene targeted by the present invention) before the introduction of the mutation in the sample obtained from the fish targeted by the present invention. Less than activity and / or expression level (eg 1/2, 1/5, 1/10, 1/20, 1/50, 1/100, 1/200, 1/500, 1/1000, 1/2000) , 1/5000, 1/10000 or less). The activity and / or expression level of the foxl3 gene can be measured according to a known method.
　遺伝子の発現の抑制は、標的遺伝子から転写されたmRNA及び標的遺伝子によってコードされる蛋白質の少なくともいずれかを検出することにより評価することができる。具体的には、mRNAに基づき評価する場合には、標的遺伝子に特異的なプライマーやプローブを用いたノーザンハイブリダイゼーション、RT-PCR、定量的PCR、マイクロアレイなどにより、標的遺伝子から転写されたmRNAの量を測定することにより行うことができる。蛋白質に基づき評価する場合には、標的遺伝子によってコードされる蛋白質に特異的な抗体を用いたウエスタンブロッティング、ELISAなどの免疫学的な方法により、その蛋白質の発現量を測定することにより行うことができる。 Suppression of gene expression can be evaluated by detecting at least one of the mRNA transcribed from the target gene and the protein encoded by the target gene. Specifically, when evaluating based on mRNA, mRNA transcribed from the target gene by Northern hybridization using a primer or probe specific to the target gene, RT-PCR, quantitative PCR, microarray, etc. It can be done by measuring the amount. When evaluating based on a protein, the expression level of the protein can be measured by an immunological method such as Western blotting or ELISA using an antibody specific to the protein encoded by the target gene. it can.
　生殖細胞中のfoxl3遺伝子を制御する好ましい時期は、対象となる魚類の生殖腺の発達に応じて適宜選択することができる。具体的には、生殖細胞を精子に分化させるには、生殖細胞でfoxl3遺伝子が発現する時期にその発現を抑制することが好ましい。例えばメダカでは生殖腺の分化時期が孵化前後であり、この時期に生殖細胞中のfoxl3遺伝子の発現を抑制することが可能である。また、幹細胞型及びシスト型生殖細胞は性的未分化又は性的可塑性を保っていると考えられるので、性分化後の幼魚の時期、成熟生殖腺（卵巣）においても制御することが可能である。また、ヌタウナギのように孵化から数年後に性分化すると考えられている個体については、個体に応じた性分化の時期に合わせて生殖細胞中のfoxl3の遺伝子の発現を抑制することができる。 The preferred time to control the foxl3 gene in germ cells can be appropriately selected according to the development of the gonads of the target fish. Specifically, in order to differentiate germ cells into sperms, it is preferable to suppress the expression of the foxl3 gene in germ cells at the same time. For example, in medaka, the differentiation time of the gonad is before and after hatching, and it is possible to suppress the expression of the foxl3 gene in germ cells at this time. In addition, since stem cell type and cyst type germ cells are considered to maintain sexual undifferentiation or sexual plasticity, it is possible to control the stage of juveniles after sexual differentiation and in the mature gonads (ovaries). In addition, for individuals such as hagfish, which are thought to undergo sexual differentiation several years after hatching, the expression of the foxl3 gene in germ cells can be suppressed at the time of sexual differentiation according to the individual.
　foxl3遺伝子に変異を導入する場合、その変異としては、foxl3遺伝子の機能及び／又は発現が欠損又は低下する変異である限り特に制限されるものではない。当該変異としては、例えば遺伝子欠損、タンパク質コード領域における変異、スプライシング調節領域における変異、発現制御領域（例えば、プロモーター、アクチベーター、エンハンサー等）における変異等が挙げられる。これらの中でも、好ましくは遺伝子欠損が挙げられる。また、本発明が対象とする魚類においては、当該変異を、対の染色体の両方において有することが好ましい。 When a mutation is introduced into the foxl3 gene, the mutation is not particularly limited as long as it is a mutation in which the function and / or expression of the foxl3 gene is deleted or decreased. Examples of the mutation include a gene defect, a mutation in a protein coding region, a mutation in a splicing regulatory region, a mutation in an expression control region (for example, a promoter, an activator, an enhancer, etc.) and the like. Among these, gene deletion is preferably mentioned. Moreover, in the fish targeted by the present invention, it is preferable to have the mutation on both of the paired chromosomes.
　foxl3遺伝子への変異導入は、例えば人工ヌクレアーゼ（TALEN、Casタンパク質）を用いる遺伝子ノックアウト、foxl3遺伝子の少なくとも一部を含むターゲティングベクターが相同組み換えによって導入されたES細胞等を用いる遺伝子ノックアウト、foxl3遺伝子に対するshRNA、アンチセンス、リボザイム等を用いる遺伝子ノックダウン、放射線照射、化学変異原投与、トランスポゾン導入等によって達成され得る。遺伝子ノックアウト、遺伝子ノックダウン、遺伝子変異導入に用いられる、ターゲッティングベクターの構築、shRNA、アンチセンス、リボザイム等の設計及び合成、ES細胞、未受精卵、受精卵、胚、始原生殖細胞等へのDNAの導入方法、放射線照射、化学変異原投与、トランスポゾン導入等は、当業者であれば従来公知の方法を適用して行うことができる。 Mutation introduction into the foxl3 gene includes, for example, gene knockout using an artificial nuclease (TALEN, Cas protein), gene knockout using an ES cell into which a targeting vector containing at least a part of the foxl3 gene has been introduced by homologous recombination, and foxl3 gene. It can be achieved by gene knockdown using shRNA, antisense, ribozyme, etc., irradiation, chemical mutant administration, transposon introduction, and the like. Construction of targeting vectors used for gene knockout, gene knockdown, gene mutation introduction, design and synthesis of shRNA, antisense, ribozyme, etc., DNA to ES cells, unfertilized eggs, fertilized eggs, embryos, primordial germ cells, etc. The method of introducing the gene, irradiation, administration of a chemical mutation source, introduction of a transposon, etc. can be performed by applying a conventionally known method to those skilled in the art.
　本発明の製造方法で用いられる「foxl3遺伝子の機能及び／又は発現が欠損又は低下した魚類」としては、前述の通りfoxl3遺伝子ノックアウト魚類（foxl3遺伝子欠損魚類）、foxl3遺伝子ノックダウン魚類、foxl3遺伝子変異導入魚類等が挙げられるが、好ましくはfoxl3遺伝子ノックアウト魚類である。 The "fishes in which the function and / or expression of the foxl3 gene is deficient or decreased" used in the production method of the present invention include foxl3 gene knockout fish (foxl3 gene deficient fish), foxl3 gene knockdown fish, and foxl3 gene mutation as described above. Introduced fish and the like can be mentioned, but foxl3 gene knockout fish are preferable.
　生殖細胞中のfoxl3遺伝子の機能及び／又は発現が欠損又は低下した魚類の卵巣内では、当初は精子のみが製造されるものの、その後卵子も製造されるようになる。これは、当業者に公知の方法、例えば卵巣の切片の染色像を観察することにより、精子のみならず卵子も製造されているか否かを判別することができる。工程1の対象となる魚類は、卵子が形成される時期（卵形成期：卵巣内に卵子の存在が確認できる時期）であることが好ましい。メダカの場合、卵形成期は、例えば孵化後90～120日である。このような時期の魚類を採用することにより、自家受精能を有する魚類の製造効率を向上させることができる。 In the ovary of fish in which the function and / or expression of the foxl3 gene in germ cells is deficient or reduced, only sperm are initially produced, but then eggs are also produced. This can be determined by a method known to those skilled in the art, for example, by observing a stained image of a section of an ovary, whether or not not only a sperm but also an egg is produced. The target fish in step 1 is preferably at the time when an egg is formed (oogenesis stage: a time when the presence of an egg can be confirmed in the ovary). In the case of medaka, the oogenesis period is, for example, 90 to 120 days after hatching. By adopting fish at such a time, it is possible to improve the production efficiency of fish having self-fertilizing ability.
　雄性化剤は、雄化を引き起こす物質である限り特に制限されず、例えばアンドロゲン等の雄化を促進する物質、抗エストロゲン剤等の雌化を抑制する物質等が挙げられる。アンドロゲンとしては、11-ケトテストステロン、アドレノステロン、テストステロン、ジヒドロテストステロン、デヒドロエピアンドロステロン等が挙げられる。抗エストロゲン剤としては、例えばエストロゲン受容体阻害剤、アロマターゼ阻害剤等が挙げられる。雄性化剤としては、アンドロゲンが好ましい。雄性化剤は、1種単独であってもよいし、2種以上の組合せであってもよい。 The maleizing agent is not particularly limited as long as it is a substance that causes virilization, and examples thereof include substances that promote virilization such as androgens and substances that suppress femaleization such as antiestrogen agents. Examples of androgens include 11-ketotestosterone, adrenosterone, testosterone, dihydrotestosterone, dehydroepiandrosterone and the like. Examples of the anti-estrogen agent include an estrogen receptor inhibitor, an aromatase inhibitor and the like. Androgen is preferable as the virilizing agent. The virilizing agent may be used alone or in combination of two or more.
　雄性化剤の投与方法は、特に制限されず、魚類の種類、飼育形態に応じて、適切な方法を採用することができる。投与方法としては、例えば経口投与、経皮投与、腹腔内投与、静脈内投与、皮下投与等が挙げられる。 The administration method of the virilizing agent is not particularly limited, and an appropriate method can be adopted according to the type of fish and the breeding form. Examples of the administration method include oral administration, transdermal administration, intraperitoneal administration, intravenous administration, subcutaneous administration and the like.
　本発明の一態様においては、例えば、飼育する水中に雄性化剤を溶解させることにより、簡便且つ確実に投与することが可能である。 In one aspect of the present invention, for example, by dissolving the maleizing agent in the breeding water, it is possible to administer it easily and reliably.
　本発明の一態様においては、例えば、飼料として、雄性化剤含有飼料を使用することにより、簡便且つ確実に投与することが可能である。飼料の形態は、対象魚類が摂取可能な形態である限り、特に制限されない。飼料の形態としては、粉末状、顆粒状、クランブル状、ペレット状、キューブ状、ペースト状、液状等が挙げられる。雄性化剤含有飼料は、雄性化剤以外に、飼料を構成する他の物質・成分、例えば魚粉；　大豆粕、濃縮大豆タンパク質、コーングルテンミール、トウモロコシ蒸留粕、ナタネ油粕等の植物性飼料原料；　肉粉、肉骨粉、フェザーミール、血粉等の動物性飼料原料；　微細藻類原料；　酵母、バクテリア等の単細胞生物原料等；　粘結剤（例えば、カルボキシメチルセルロースナトリウム（CMC）、グアーガム、アルギン酸ナトリウム、ポリアクリル酸ナトリウム、カゼインナトリウム、プロピレングリコール等）、穀粉（例えば小麦粉、デンプン等）、ミネラル類、ビタミン類（例えばビタミンC、ビタミンB1、ビタミンA、ビタミンD、ビタミンE等）、アミノ酸類（例えば、リジン、メチオニン、ヒスチジン等）、色素（例えばβ－カロチン、アスタキサンチン、カンタキサンチン等）、肝油（例えばタラ肝油等）等が挙げられる。 In one aspect of the present invention, for example, by using a feed containing a virilizing agent as a feed, it is possible to administer the feed easily and reliably. The form of the feed is not particularly limited as long as it is ingestible by the target fish. Examples of the form of the feed include powder, granule, crumble, pellet, cube, paste, liquid and the like. In addition to the maleizing agent, the maleizing agent-containing feed includes other substances / components constituting the feed, such as fish meal; soybean meal, concentrated soybean protein, corn gluten meal, corn distilled meal, rapeseed oil meal, and other vegetable feed raw materials; Animal feed raw materials such as meat-and-bone meal, cod liver oil, feather meal, blood powder; microalgae raw materials; single-cell biological raw materials such as yeast and bacteria; binders (for example, sodium carboxymethyl cellulose (CMC), guar gum, sodium alginate, polyacrylic Soy acid, sodium caseinate, propylene glycol, etc.), grain flour (eg wheat flour, starch, etc.), minerals, vitamins (eg, vitamin C, vitamin B1, vitamin A, vitamin D, vitamin E, etc.), amino acids (eg, lysine) , Methionin, histidine, etc.), pigments (eg, β-carotene, astaxanthin, cantaxanthin, etc.), liver oil (eg, cod liver oil, etc.) and the like.
　雄性化剤の投与により、成熟卵が排卵される卵巣腔の中に精子の一部が移行するようになる（試験例2参照）。 Administration of a virilizing agent causes a part of sperm to move into the ovarian cavity where mature eggs are ovulated (see Test Example 2).
　雄性化剤の投与量は、卵巣腔への精子の移行が起こる程度の量である限り特に制限されず、当該現象の有無を指標として、適宜設定することができる。例えば魚類を飼育する水中に雄性化剤を溶解させる場合、水中の雄性化剤濃度は、例えば1～1000ng/ml、好ましくは5～500ng/ml、より好ましくは10～200ng/ml、さらに好ましくは20～100ng/mlである。例えば、雄性化剤含有飼料を摂取させる場合、当該飼料中の雄性化剤含有量は、例えば0.00001～50質量％である。当該含有量の上限・下限は、例えば0.0001質量％、0.001質量％、0.01質量％、0.1質量％、0.2質量％、0.5質量％、1質量％、2質量％、5質量％、10質量％、20質量％、30質量％、40質量％等であり得る。 The dose of the virilizing agent is not particularly limited as long as the amount of sperm transfer to the ovarian cavity occurs, and the presence or absence of the phenomenon can be appropriately set as an index. For example, when the maleizing agent is dissolved in the water for raising fish, the concentration of the maleizing agent in the water is, for example, 1 to 1000 ng / ml, preferably 5 to 500 ng / ml, more preferably 10 to 200 ng / ml, and further preferably. It is 20 to 100 ng / ml. For example, when a feed containing a virilizing agent is ingested, the content of the virilizing agent in the feed is, for example, 0.00001 to 50% by mass. The upper and lower limits of the content are, for example, 0.0001% by mass, 0.001% by mass, 0.01% by mass, 0.1% by mass, 0.2% by mass, 0.5% by mass, 1% by mass, 2% by mass, 5% by mass, 10% by mass, It can be 20% by mass, 30% by mass, 40% by mass, and the like.
　雄性化剤の投与の回数は、特に制限されるものではないが、好ましくは雄性化剤投与は持続投与である。すなわち、雄性化剤を、継続して、又は一定間隔（例えば1日1～3回、2～3日に1回等）で、持続的に投与することが好ましい。 The number of administrations of the virilizing agent is not particularly limited, but the administration of the virilizing agent is preferably continuous administration. That is, it is preferable to administer the virilizing agent continuously or continuously at regular intervals (for example, 1 to 3 times a day, once every 2 to 3 days, etc.).
　雄性化剤の投与期間は、卵巣腔への精子の移行が起こる程度の期間である限り特に制限されず、当該現象の有無を指標として、適宜設定することができる。当該期間は、例えば1～200日間、好ましくは5～100日間、より好ましくは10～50日間、さらに好ましくは20～40日間である。この期間は、魚類の成熟までの期間に応じて変化し得る。 The administration period of the virilizing agent is not particularly limited as long as the period of sperm transfer to the ovarian cavity occurs, and the presence or absence of the phenomenon can be appropriately set as an index. The period is, for example, 1 to 200 days, preferably 5 to 100 days, more preferably 10 to 50 days, and even more preferably 20 to 40 days. This period can vary depending on the time to maturity of the fish.
　雄性化剤投与後は、必要に応じて、雄性化剤非投与環境下で飼育することができる。これにより、卵巣腔への精子の移行をより多くすることも可能である。この飼育期間は、特に制限されず、雄性化剤投与期間1に対して、例えば0.1～10、好ましくは0.3～5、より好ましくは0.7～3である。当該飼育期間は、より具体的には、例えば1～400日間、好ましくは5～200日間、より好ましくは10～100日間、さらに好ましくは20～80日間である。 After administration of the virilizing agent, it can be bred in an environment where the maleizing agent is not administered, if necessary. This also allows for more sperm transfer into the ovarian cavity. This breeding period is not particularly limited, and is, for example, 0.1 to 10, preferably 0.3 to 5, and more preferably 0.7 to 3 with respect to the maleizing agent administration period 1. More specifically, the breeding period is, for example, 1 to 400 days, preferably 5 to 200 days, more preferably 10 to 100 days, and even more preferably 20 to 80 days.
　本発明の製造方法1で得られる魚類（本明細書において、「本発明の魚類」と示すこともある。）は、自家受精能を有する。すなわち、卵巣実質部から外へ（例えば卵巣腔や体腔へ）精子の一部が移行しているので、成熟卵が卵巣実質部から外へ移行した際に、自身の精子と受精することが可能である。 The fish obtained by the production method 1 of the present invention (sometimes referred to as "the fish of the present invention" in the present specification) has a self-fertilizing ability. That is, since a part of sperm is transferred from the parenchyma of the ovary to the outside (for example, to the ovarian cavity or body cavity), when a mature egg is transferred from the parenchyma of the ovary to the outside, it is possible to fertilize with its own sperm. Is.
　3．自家受精卵の製造方法A
　本発明は、その一態様において、（2）本発明の魚類に排卵誘発処理する工程を含む、自家受精卵の製造方法（本明細書において、「本発明の製造方法2A」と示すこともある。）に関する。以下に、これについて説明する。 3. 3. Manufacturing method of self-fertilized egg A
In one aspect of the present invention, a method for producing a self-fertilized egg including (2) a step of inducing ovulation in the fish of the present invention may be referred to as "the production method 2A of the present invention" in the present specification. .). This will be described below.
　排卵誘発処理としては、特に制限されず、例えば雄魚類との交配、排卵誘発剤の投与等が挙げられる。雄魚類は、自家受精卵のみを得るためには、不稔雄魚類であることが好ましい。不稔雄魚類は、例えば、不稔に関与する遺伝子（例えばnanos遺伝子）変異により、作製することができる。また、排卵誘発剤としては、例えばクロミフェン、ゴナドトロピン、絨毛性ゴナドトロピン、GnRHアゴニスト、GnRHアンタゴニスト等が挙げられる。 The ovulation induction treatment is not particularly limited, and examples thereof include mating with male fish and administration of an ovulation inducer. Male fish are preferably sterile male fish in order to obtain only self-fertilized eggs. Sterile male fish can be produced, for example, by mutation of a gene involved in sterility (for example, nanos gene). Examples of the ovulation inducer include clomiphene, gonadotropin, chorionic gonadotropin, GnRH agonist, GnRH antagonist and the like.
　排卵誘発処理により、排卵される際に、自己精子との受精が起こり、自家受精卵を得ることができる。得られた自家受精卵は、適切な環境下で培養して発生させて魚類個体を得ることができる。 By the ovulation induction process, fertilization with self-fertilized sperm occurs when ovulation occurs, and a self-fertilized egg can be obtained. The obtained self-fertilized egg can be cultivated and generated in an appropriate environment to obtain an individual fish.
　4．自家受精卵の製造方法B
　本発明は、その一態様において、（1）生殖細胞中のfoxl3遺伝子の機能及び／又は発現が欠損又は低下した魚類に雄性化剤投与する工程を含む、自家受精卵の製造方法（本明細書において、「本発明の製造方法2B」と示すこともある。）に関する。以下に、これについて説明する。 Four. Manufacturing method of self-fertilized egg B
In one aspect of the present invention, a method for producing a self-fertilized egg, which comprises (1) administering a virilizing agent to fish in which the function and / or expression of the foxl3 gene in germ cells is deficient or decreased (the present specification). In the above, it may be referred to as "the production method 2B of the present invention"). This will be described below.
　「生殖細胞中のfoxl3遺伝子の機能及び／又は発現が欠損又は低下した魚類」及び「雄性化剤投与」については、本発明の製造方法1と同様である。 "Fishes in which the function and / or expression of the foxl3 gene in germ cells is deficient or decreased" and "administration of a virilizing agent" are the same as in the production method 1 of the present invention.
　雄性化剤投与により、卵巣腔へ精子の一部が移行しているので、成熟卵が卵巣腔へ移行した際に自身の精子と受精する。よって、これにより自家受精卵を得ることができる。 Since part of the sperm has been transferred to the ovarian cavity by administration of the maleizing agent, when the mature egg is transferred to the ovarian cavity, it fertilizes with its own sperm. Therefore, this makes it possible to obtain a self-fertilized egg.
　本発明の製造方法2Bにおいては、さらに、（3）生殖細胞中のfoxl3遺伝子の機能及び／又は発現が欠損又は低下した魚類を排卵誘発処理する工程を含むことが好ましい。当該工程により、自家受精がより起こり易くなり、自家受精卵の製造効率を高めることができる。排卵誘発処理については、本発明の製造方法2Aと同様である。 The production method 2B of the present invention preferably further includes (3) a step of inducing ovulation in fish in which the function and / or expression of the foxl3 gene in germ cells is deficient or reduced. By this step, self-fertilization is more likely to occur, and the production efficiency of self-fertilized eggs can be improved. The ovulation induction treatment is the same as the production method 2A of the present invention.
　得られた自家受精卵は、適切な環境下で培養して発生させて魚類個体を得ることができる。 The obtained self-fertilized egg can be cultivated and generated in an appropriate environment to obtain an individual fish.
　以下に、実施例に基づいて本発明を詳細に説明するが、本発明はこれらの実施例によって限定されるものではない。 Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples.
　参考例1．不稔雄メダカの作製
　nanos遺伝子の両方のアレルが破壊されたnanosホモ接合変異体（nanos -/-）の不稔雄メダカをTALEN技術を利用して次のようにして作製した。nanos3遺伝子のTALENターゲットサイトを、TALEN Targeterプログラム（https://tale-nt.cac.cornell.edu/node/add/talen）を使用して検索した。TALENプラスミドをNotI消化により線形化し、mMESSAGE mMACHINE（登録商標）T7転写キット（Thermo Fisher）を用いたin vitro RNA合成のテンプレートとして使用した。TALEN mRNA（250 ng/μlレフト及びライト）を1細胞期または2細胞期の胚に注入した。F0ファウンダーを、野生型のメダカと交配した。得られたnanosヘテロ接合変異体（nanos +/-）の雄と雌を交配して、nanosホモ接合変異体（nanos -/-）の不稔雄メダカを得た。 Reference example 1. Preparation of sterile male medaka A sterile male medaka of a nanos homozygous mutant (nanos-/-) in which both alleles of the nanos gene were disrupted was prepared as follows using TALEN technology. The TALEN target site for the nanos3 gene was searched using the TALEN Targeter program (https://tale-nt.cac.cornell.edu/node/add/talen). The TALEN plasmid was linearized by NotI digestion and used as a template for in vitro RNA synthesis using the mMESSAGE mMACHINE® T7 Transcription Kit (Thermo Fisher). TALEN mRNA (250 ng / μl left and right) was injected into 1-cell or 2-cell stage embryos. The F0 founder was crossed with a wild-type medaka. Males and females of the obtained nanos heterozygous mutant (nanos +/-) were crossed to obtain a sterile male medaka of the nanos homozygous mutant (nanos-/-).
　試験例1．自家受精能を有する魚類及び自家受精卵の製造
　foxl3遺伝子の一方のアレルが破壊されたfoxl3ヘテロ接合変異体（foxl3 +/-）のメダカ（特許文献１）同士を交配させて、foxl3ホモ接合変異体（foxl3 -/-）の雌染色体型（XX型）メダカを得た。foxl3 -/-メダカを常法に従って水槽で飼育し、孵化から90～120日から、雄性化剤（11-ケトテストステロン）を50 ng/mlの濃度で含む雄性化剤水溶液中で30日間飼育した。この間、雄性化剤水溶液は、24時間に1回の頻度で交換した。雄性化剤水溶液中での飼育後、雄性化剤を含まない通常の水中で、少なくとも30日間（30～60日間）飼育した。水槽に不稔雄メダカ（参考例1）を入れ、30日間飼育して交配させた。この間、産卵された卵を回収し、受精の有無を、卵膜の活性化及び胞胚期までの通常の発生を指標として判断した。 Test example 1. Production of self-fertilizing fish and self-fertilized eggs Foxl3 homozygous mutation by mating medaka (Patent Document 1) of foxl3 heterozygous mutant (foxl3 +/-) in which one allele of the foxl3 gene is disrupted A female chromosome type (XX type) medaka of the body (foxl3-/-) was obtained. foxl3-/-Medaka was bred in an aquarium according to a conventional method, and from 90 to 120 days after hatching, it was bred in a virilizing agent aqueous solution containing a virilizing agent (11-ketotestosterone) at a concentration of 50 ng / ml for 30 days. .. During this time, the virilizing agent aqueous solution was changed once every 24 hours. After breeding in an aqueous virilizing agent, the animals were bred in normal water containing no maleizing agent for at least 30 days (30 to 60 days). A sterile male medaka fish (Reference Example 1) was placed in an aquarium and bred for 30 days for mating. During this period, the laid eggs were collected and the presence or absence of fertilization was judged using the activation of the egg membrane and the normal development up to the blastogenic stage as indicators.
　その結果、個体1については、33個の卵が産卵され、その内の8個の卵が受精卵であり、さらにその内の2個の卵が正常に孵化した。また、個体2については、29個の卵が産卵され、その内の8個の卵が受精卵であり、さらにその内の2個の卵が正常に孵化した。一方、雄性化剤水溶液中での飼育に代えて雄性化剤を含まない水中での飼育を行う以外は同様の方法で試験した場合、受精卵は確認されなかった。 As a result, for individual 1, 33 eggs were laid, 8 of them were fertilized eggs, and 2 of them hatched normally. For individual 2, 29 eggs were laid, 8 of them were fertilized eggs, and 2 of them hatched normally. On the other hand, no fertilized eggs were confirmed when the test was conducted by the same method except that the eggs were bred in water containing no virilizing agent instead of being bred in the aqueous virilizing agent.
　試験例2．卵巣の組織学的解析
　試験例1の不稔雄メダカとの交配開始前のfoxl3 -/-雌メダカの生殖腺を解剖し、Buin溶液で4℃で一晩固定した。Technovit（登録商標）8100（Heraeus Kulzer）を使用して、厚さ4μmのプラスチック切片を調製した。切片を、常法に従って過ヨウ素酸シッフとヘマトキシリで染色した。 Test example 2. Histological analysis of ovaries The gonads of foxl3-/-female medaka before the start of mating with sterile male medaka in Test Example 1 were dissected and fixed overnight at 4 ° C with Buin solution. A 4 μm thick plastic section was prepared using Technovit® 8100 (Heraeus Kulzer). Sections were stained with periodic acid Schiff and hematoxyli according to a conventional method.
　結果を図２に示す。成熟卵が排卵される卵巣腔の中に精子（点線部の中の黒いドット）が存在することが観察された。 The results are shown in Fig. 2. It was observed that sperm (black dots in the dotted line) were present in the ovarian cavity where mature eggs were ovulated.

Claims (15)

  1. （1）生殖細胞中のfoxl3遺伝子の機能及び／又は発現が欠損又は低下した魚類に雄性化剤投与する工程を含む、自家受精能を有する魚類の製造方法。 (1) A method for producing a fish having self-fertilizing ability, which comprises a step of administering a virilizing agent to a fish in which the function and / or expression of the foxl3 gene in germ cells is deficient or decreased.
  2. 前記魚類が雌である、請求項１に記載の製造方法。 The production method according to claim 1, wherein the fish is a female.
  3. 前記魚類が卵形成期である、請求項１又は２に記載の製造方法。 The production method according to claim 1 or 2, wherein the fish is in the oogenesis stage.
  4. 前記雄性化剤投与が持続投与である、請求項１～３のいずれかに記載の製造方法。 The production method according to any one of claims 1 to 3, wherein the administration of the virilizing agent is continuous administration.
  5. 前記雄性化剤投与の期間が5～100日間である、請求項１～４のいずれかに記載の製造方法。 The production method according to any one of claims 1 to 4, wherein the period of administration of the virilizing agent is 5 to 100 days.
  6. 請求項１～５のいずれかに記載の製造方法で得られる、自家受精能を有する魚類。 A fish having a self-fertilizing ability, which is obtained by the production method according to any one of claims 1 to 5.
  7. （2）請求項６に記載の魚類に排卵誘発処理する工程を含む、自家受精卵の製造方法。 (2) A method for producing a self-fertilized egg, which comprises the step of inducing ovulation in the fish according to claim 6.
  8. 前記排卵誘発処理が雄魚類との交配又は排卵誘発剤の投与である、請求項７に記載の製造方法。 The production method according to claim 7, wherein the ovulation induction treatment is mating with a male fish or administration of an ovulation inducer.
  9. 前記排卵誘発処理が不稔の雄魚類との交配である、請求項８に記載の製造方法。 The production method according to claim 8, wherein the ovulation induction treatment is mating with a sterile male fish.
  10. （1）生殖細胞中のfoxl3遺伝子の機能及び／又は発現が欠損又は低下した魚類に雄性化剤投与する工程を含む、自家受精卵の製造方法。 (1) A method for producing a self-fertilized egg, which comprises a step of administering a virilizing agent to a fish in which the function and / or expression of the foxl3 gene in germ cells is deficient or decreased.
  11. さらに、（3）生殖細胞中のfoxl3遺伝子の機能及び／又は発現が欠損又は低下した魚類を排卵誘発処理する工程を含む、請求項１０に記載の製造方法。 The production method according to claim 10, further comprising (3) a step of inducing ovulation in a fish in which the function and / or expression of the foxl3 gene in germ cells is deficient or decreased.
  12. 前記工程1終了後に前記工程2を行う、請求項１１に記載の製造方法。 The manufacturing method according to claim 11, wherein the step 2 is performed after the step 1 is completed.
  13. 前記排卵誘発処理が雄魚類との交配又は排卵誘発剤の投与である、請求項１１又は１２に記載の製造方法。 The production method according to claim 11 or 12, wherein the ovulation induction treatment is mating with a male fish or administration of an ovulation inducer.
  14. 請求項７～１３のいずれかに記載の製造方法で得られる、自家受精卵。 A self-fertilized egg obtained by the production method according to any one of claims 7 to 13.
  15. 請求項１４に記載の自家受精卵が発生してなる、魚類。 A fish in which the self-fertilized egg according to claim 14 is generated.
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