Fish individual having dysfunction in at least portion of infertility proteins

Abstract

An object of the present invention is to provide a fish individual which, when producing a germ cell deficient individual as shown in FIG. 2, is capable of recognizing the genotype of an infertility protein by the appearance even without collecting a portion of the fish body. The present invention is characterized by having a polynucleotide sequence that encodes a fluorescent protein in an expressible manner in at least one of infertility gene alleles that encode an infertility protein that exhibits infertility due to at least a part of the function being impaired, said polynucleotide sequence being characterized by having a nucleotide sequence that encodes a fluorescent protein that can be expressed in the alleles of an infertility gene that encodes an infertility protein that exhibits infertility. The problem is solved by a fish individual in which at least a part of infertility proteins have a defect in function.

Description

Technical Field

[0001] It relates to a fish individual in which at least a part of the infertility protein has a functional defect, a method for producing the above fish individual, a polynucleotide, a method for transplanting germ cells, and a method for identifying a genotype. Background Art

[0002] As one of the infertility genes related to infertility, the Dead End (dnd) gene is known. It is known that an individual in which the function of the protein encoded by the infertility gene (hereinafter also referred to as "infertility protein") is defective does not form germ cells and is infertile. Summary of the Invention

[0003] Problems to be Solved by the Invention

[0004] In the conventional method for artificially producing a germ cell-deficient individual with a defective germ cell by using the functional defect of the infertility protein, as Figure 1 shown, heterozygotes in which the function of the infertility protein is defective in a heterozygous manner are mated with each other to produce a homozygote in which the function of the infertility gene is defective in a homozygous manner. The next generation produced by mating heterozygotes with each other is any one of wild type, homozygous type, and heterozygous type, and these genotypes cannot be identified by appearance. Therefore, in order to identify the genotype, a part of the fish body needs to be collected for gene analysis.

[0005] One object of the present invention is to provide a fish individual in which the genotype of the infertility protein can be identified by appearance even without collecting a part of the fish body when producing a germ cell-deficient individual as Figure 2 shown.

[0006] Solutions to the Problems

[0007] Item 1.

[0008] A fish individual in which at least a part of the infertility protein has a functional defect, wherein at least one of the alleles of the infertility gene encoding the infertility protein that shows infertility due to at least a part of the function being defective has a polynucleotide sequence encoding a fluorescent protein in an expressible manner.

[0009] Item 2.

[0010] The fish individual according to Item 1, wherein the polynucleotide sequence encoding the fluorescent protein is present in two alleles of the infertility gene, and the fish individual has a defective reproductive function.

[0011] Item 3.

[0012] The fish individual according to item 2, wherein among the two alleles of the above-mentioned infertility gene, there is a polynucleotide sequence encoding a fluorescent protein having the same fluorescence wavelength band.

[0013] Item 4.

[0014] The fish individual according to item 2, wherein among the two alleles of the above-mentioned infertility gene, there are respectively polynucleotide sequences encoding fluorescent proteins having different fluorescence wavelength bands.

[0015] Item 5.

[0016] The fish individual according to item 1, wherein the above-mentioned infertility gene is the dead end (dnd) gene.

[0017] Item 6.

[0018] The fish individual according to item 1, wherein the expression site of the above-mentioned fluorescent protein is the eye.

[0019] Item 7.

[0020] A method for producing a fish individual in which at least a part of an infertility protein that shows infertility due to a defect in at least a part of its function has a functional defect,

[0021] which includes the step of introducing a polynucleotide sequence encoding a fluorescent protein into at least one of the alleles of the infertility gene encoding the infertility protein in an expressible manner.

[0022] Item 8.

[0023] The production method according to item 7, wherein the above-mentioned introduction is carried out by a gene knock-in method using a genome editing method or a gene site-specific recombination method.

[0024] Item 9.

[0025] The production method according to item 8, wherein the above-mentioned genome editing method is a method using at least one system selected from the Clustered regularly interspaced short palindromic repeats / CRISPR associated protein 9 (CRISPR / Cas9) system, the CompoZr Zinc Finger Nuclease (ZFN) system, and the Transcription Activator-Like Effector Nuclease (TALEN) system.

[0026] Item 10.

[0027] The production method according to item 8, wherein the gene locus-specific recombination method is a method using at least one system selected from the Cre / loxP system, the Flp / FRT system, the Dre / rox system, and the PhiC31 integrase system.

[0028] Item 11.

[0029] A polynucleotide, wherein within a part of the sequence of a sterility gene encoding a sterility protein that exhibits sterility due to a defect in at least a part of its function, the polynucleotide sequence contains a polynucleotide sequence encoding a fluorescent protein in an expressible manner.

[0030] Item 12.

[0031] A method for transplanting germ cells, which includes the step of transplanting germ cells of a fish of a variety different from that of the fish individual described in item 2 into the fish individual.

[0032] Item 13.

[0033] A method for identifying a genotype, which includes the following steps: mating a female and a male of the fish individuals described in item 1, in which only one sterility gene allele has a polynucleotide sequence encoding a fluorescent protein in an expressible manner; and observing the expression of the fluorescent protein in the embryos or adult fish obtained by the mating.

[0034] Effects of the invention

[0035] It is possible to provide fish individuals in which the genotype of the sterility protein can be visually recognized when producing germ cell-deficient individuals. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Shows the conventional method.

[0037] Figure 2 Shows the outline of the present invention.

[0038] Figure 3 Shows the gene introduction map of tilapia.

[0039] Figure 4 Shows the expression of fluorescent protein in the eyes of fertilized eggs, embryos, and adult fish of tilapia.

[0040] Figure 5 Shows the positions of primers designed to confirm the correct introduction of the cassette polynucleotide into the dnd gene of tilapia.

[0041] Figure 6 Shows the use of Figure 5 The results of PCR using the primers shown.

[0042] Figure 7 Showing dnd of tilapia gfp Sequences of the cassette polynucleotide introduction sites of individuals. (A) shows the 5'-side sequence obtained by analysis using the Onil_dnd_5-prime_Fw primer. (B) shows the 3'-side sequence obtained by analysis using the Onil_dnd_3-prime_Rv primer.

[0043] Figure 8 Showing dnd of tilapia rfp Sequences of the cassette polynucleotide introduction sites of individuals. (A) shows the 5'-side sequence obtained by analysis using the Onil_dnd_5-prime_Fw primer. (B) shows the 3'-side sequence obtained by analysis using the Onil_dnd_3-prime_Rv primer.

[0044] Figure 9 Showing the gene transfer map of Takifugu alboplumbeus

[0045] Figure 10 Showing the fluorescence protein expression in the eyes of fertilized eggs, embryos and adult fish of Takifugu alboplumbeus

[0046] Figure 11 Showing the positions of primers designed to confirm the correct introduction of the cassette polynucleotide into the dnd gene of Takifugu alboplumbeus

[0047] Figure 12 Showing the use of Figure 11 The results of PCR using the primers shown

[0048] Figure 13 Showing dnd of Takifugu alboplumbeus gfp Sequences of the cassette polynucleotide introduction sites of individuals. (A) shows the 5'-side sequence obtained by analysis using the Talb_dnd_5-prime_Fw primer. (B) shows the 3'-side sequence obtained by analysis using the Talb_dnd_3-prime_Rv primer.

[0049] Figure 14 Showing dnd of Takifugu alboplumbeus rfp Sequences of the cassette polynucleotide introduction sites of individuals. (A) shows the 5'-side sequence obtained by analysis using the Talb_dnd_5-prime_Fw primer. (B) shows the 3'-side sequence obtained by analysis using the Talb_dnd_3-prime_Rv primer. Detailed implementation mode

[0050] 1. Fish individuals in which at least a part of the infertility protein has a functional defect

[0051] One embodiment relates to a fish individual (hereinafter also simply referred to as "fish individual") in which at least a part of a sterility protein that exhibits sterility due to a defect in at least a part of its function has a functional defect. The fish individual has a polynucleotide sequence encoding a fluorescent protein in an expressible manner in at least one of the sterility gene alleles encoding the sterility protein.

[0052] In the "fish individual in which at least a part of the sterility protein has a functional defect", as a genotype, it may include a heterozygous type in which the polynucleotide sequence encoding the fluorescent protein is present only in one allele of the fish individual and a homozygous type in which the polynucleotide sequence encoding the fluorescent protein is present in both alleles.

[0053] In the heterozygous type, since the polynucleotide sequence encoding the fluorescent protein is introduced into one allele of the sterility gene, the function of the sterility protein encoded by the one allele is defective. As a phenotype, the heterozygous type is an individual that retains at least a part of the germ cells (also denoted as "heterozygous defective individual" in this specification), and both females and males can reproduce.

[0054] In the homozygous type, since the polynucleotide sequence encoding the fluorescent protein is introduced into both alleles of the sterility gene, the functions of the sterility proteins of both alleles are defective. As a phenotype, the homozygous type is an individual in which the germ cells are substantially defective (also denoted as "homozygous defective individual" in this specification), and the reproductive functions of both females and males are defective. The defect of the germ cells can be confirmed by histological observation. For example, when the number of germ cells (the number of primordial germ cells, spermatogonia, oogonia, spermatocytes, oocytes, spermatids, ova, eggs or sperm) decreases to 50% or less, 30% or less, 20% or less, 10% or less, 5% or less, 3% or less, 1% or less compared with that of wild-type individuals, it can be determined that the germ cells are defective.

[0055] In this specification, there is no limitation on the fish.

[0056] There is no particular limitation on the "fish". In this specification, the above-mentioned fish may include marine fish, freshwater fish, brackish water fish, migratory fish, etc.

[0057] Examples of the above fish include, for example, fish of the families Paralichthys, Tetraodontidae (puffers), Ostraciidae (boxfishes), Sparidae (sea breams and porgies), Salmonidae, Cyprinidae, Ictaluroidea, Siluroidea, Bagroidea, Serranidae (sea basses), Cichlidae, Oryziidae (medakas), Monacanthidae, Osmeridae, Scombridae, Pleuronectidae, Carangidae, Lateolabrax, Moronidae, Latidae, Rachycentridae, Cynoglossidae, Anguillidae, Congridae, etc.

[0058] The fish of the family Paralichthyidae may include, for example, Paralichthys olivaceus (TEMMINCK et SCHLEGEL), Paralichthys of the genus Pseudorhombus (Pseudorhombus dupliciocellatus, Pseudorhombus oligodon, Pseudorhombus ctenosquamis, Pseudorhombus arsius, Pseudorhombus pentophthalmus, Pseudorhombus cinnamoneus, Pseudorhombus levisquamis, etc.), Paralichthys of the genus Tarphops (Tarphops oligolepis, etc.), Paralichthys of the genus Asterorhombus (Asterorhombus intermedius), Paralichthys of the genus Lepidopsetta (Lepidopsetta mochigarei), Paralichthys of the genus Taeniopsetta (Taeniopsetta ocellata), etc. Paralichthys olivaceus (TEMMINCK et SCHLEGEL) is preferred.

[0059] The fish of the family Tetraodontidae may include, for example, Takifugu rubripes, Takifugu porphyreus, Takifugu niphobles, etc. of the genus Takifugu; Lagocephalus wheeleri of the genus Lagocephalus; and other fish.

[0060] The fish of the family Ostraciidae may include, for example, Ostracion immaculatus and other fish of the genus Ostracion.

[0061] The above-mentioned fish of the Sparidae family may include, for example, fish of the genus Pagrus such as Pagrus major, Pagrus auratus, etc.; fish of the genus Acanthopagrus such as Acanthopagrus schlegelii, Acanthopagrus latus, etc.; fish of the genus Dentex such as Dentex tumifrons (also known as Lianzi Dentex); fish of the genus Sparus such as Sparus aurata, etc.

[0062] The above-mentioned fish of the Salmonidae family may include, for example, fish of the genus Oncorhynchus in the Pacific such as Oncorhynchus mykiss, Oncorhynchus tshawytscha, Oncorhynchus masou, Oncorhynchus masou ishikawae, Oncorhynchus kawamurae, Oncorhynchus gorbuscha, Oncorhynchus keta, etc.; fish of the genus Salmo in the Atlantic such as Salmo trutta, Oncorhynchus nerka, Oncorhynchus kisutch, Salmo salar, etc.; fish of the genus Salvelinus such as Salvelinus malma, Salvelinus alpinus, Salvelinus leucomaenis, Salvelinus fontinalis, Salvelinus namaycush, etc.; fish of the genus Parahucho such as Parahucho perryi, etc.

[0063] The above-mentioned cyprinid fish may include, for example, Gnathopogon caerulescens, Hypophthalmichthys molitrix, Cyprinus carpio, Ctenopharyngodon idellus, Hypophthalmichthys nobilis, Carassius carassius, Cyprinus catla, Mylopharyngodon piceus, Cirrhinus molitorella, Cirrhinus cirrhosus, Catla catla, Labeo rohita, Megalobrama amblycephala, etc.

[0064] The above-mentioned fish of the North American catfish superfamily (Ictaluroidea) may include, for example, Ictalurus punctatus, Ictalurus furcatus, etc.

[0065] The above-mentioned fish of the catfish family (Siluroidea) may include, for example, Silurus asotus, Silurus biwaensis, Silurus lithophilus, Silurus glanis, Clarias fuscus, Clarias batrachus, etc.

[0066] The above-mentioned fish of the Bagroidea family may include, for example, Pseudobagrus fulvidraco, Pangasianodon gigas, Pangasius bocourti, Pangasianodon hypophthalmus, etc.

[0067] The above-mentioned Serranidae fish may include, for example, Epinephelus septemfasciatus, Epinephelus bruneus, Epinephelus akaara, Epinephelus malabaricus, Epinephelus aeneus, Epinephelus amblycephalus, Epinephelus areolatus, Epinephelus bleekeri, Epinephelus bontoides, Epinephelus chlorostigma, Epinephelus coiodes, Epinephelus fasciatus, Epinephelus fuscoguttatus, Epinephelus labriformis, Epinephelus lanceolatus, Epinephelus maculatus, Epinephelus malabricus, Epinephelus marginatus, Epinephelus ongus, Epinephelus polyphekadion, Epinephelus quoyanus, Epinephelus sexfasciatus, Epinephelus striatus, Epinephelus tauvina, Epinephelus tukula, etc. of the genus Epinephelus, Cromileptes altivelis of the genus Cromileptes, Plectropomus leopardus of the genus Plectropomus, etc., and hybrids between such fish and Serranidae fish.

[0068] The above-mentioned fish of the Cichlidae family may include, for example, fish of the genus Oreochromis such as Oreochromis niloticus, Oreochromis mossambicus, Oreochromis aureus, etc., and mouthbrooding tilapia.

[0069] The above-mentioned fish of the Adrianichthyidae family may include, for example, fish of the genus Oryzias such as Oryzias latipes, Oryzias sakaizumii, Oryzias javanicus, etc.

[0070] The above-mentioned fish of the Monacanthidae family are, for example, fish of the genus Stephanolepis such as Stephanolepis cirrhifer, and fish of the genus Thamnaconus such as Thamnaconus modestus.

[0071] The above-mentioned fish of the Osmeridae family may include, for example, fish of the subfamily Plecoglossinae such as Plecoglossus altivelis, fish of the subfamily Hypomesinae such as Hypomesus nipponensis, Hypomesus japonicus, etc., and fish of the subfamily Osmerinae such as Osmerus mordax dentex, Spirinchus lanceolatus, Spirinchus lanceolatus, etc.

[0072] The fish of the Scombridae family may include, for example, fish of the genus Scombrini such as Scomber japonicus, Scomber scombrus, Scomber australasicus, etc., fish of the genus Thunnini such as Thunnus orientalis, Thunnus thynnus, Thunnus maccoyii, Thunnus obesus, Thunnus albacares, Thunnus alalunga, Thunnus tonggol, etc., fish of the genus Euthynnus such as Euthynnus affinis, Euthynnus alletteratus, etc., fish of the genus Katsuwonus such as Katsuwonus pelamis, fish of the genus Scomberomorini, fish of the genus Auxis, fish of the genus Sardini, fish of the genus Gymnosarda, etc.

[0073] The fish of the Pleuronectidae family may include, for example, fish such as Pseudopleuronectes herzensteini, Pleuronectes yokohamae, Kareius bicoloratus, Hippoglossus stenolepis, Verasper moseri, etc.

[0074] The fish of the Carangidae family may include, for example, fish of the genus Seriola such as Seriola dumerili, Seriola lalandi, Seriola rivoliana, Seriola quinqueradiata, etc., fish of the genus Pseudocaranx such as Trachurus japonicus, Pseudocaranx dentex, etc., fish of the genus Trachinotus such as Trachinotus ovatus, etc.

[0075] The fish of the family Lateolabrax may include, for example, Lateolabrax latus, Lateolabrax maculatus, etc. The fish of the family Moronidae may include fish such as Dicentrarchus labrax.

[0076] The fish of the family Latidae may include fish such as Lates calcarifer, Lates niloticus, etc. of the genus Lates. The fish of the family Rachycentridae are, for example, Rachycentron canadum and other fish.

[0077] The fish of the family Cynoglossidae may include, for example, Cynoglossus joyneri, Cynoglossus semilaevis and other fish.

[0078] The fish of the family Anguillidae may include, for example, Anguilla japonica, Anguilla anguilla and other fish.

[0079] The fish of the family Congridae may include, for example, Conger myriaster, Conger japonicus and other fish.

[0080] As fish, fish of the families Cichlidae and Tetraodontidae are preferred, which are tilapia and Fugu alboplumbeus.

[0081] In this specification, the above fish may be purebred varieties or hybrid varieties. The above hybrid varieties may include, for example, hybrids derived from intergeneric hybridization.

[0082] As "fish", cultured fish are preferred. In addition, "cultured fish" may include, for example, fish cultured for purposes such as food, breeding, appreciation, etc.

[0083] As a sterility gene, for example, the dead end (dnd) gene can be cited.

[0084] The gene IDs of the main registered fish sterility genes in NCBI are as follows:

[0085] Taxonomy ID: Organism name: Gene ID

[0086] 7906: Acipenser ruthenus: 131699448

[0087] 7906: Sterlet: 117412941

[0088] 7913: Paddlefish (Polyodon spathula): 121323773

[0089] 7913: Paddlefish: 121297601

[0090] 7918: Spotted gar (Lepisosteus oculatus): 102698012

[0091] 7936: European eel: 118224086

[0092] 7950: Atlantic herring (Clupea harengus): 105903488

[0093] 7955: Zebrafish (Danio rerio): 373074

[0094] 7957: Crucian carp (Carassius auratus): 113057682

[0095] 7957: Crucian carp: 113113732

[0096] 7959: Grass carp: 127494684

[0097] 7962: Common carp: 109089193

[0098] 7962: Common carp: 109108346

[0099] 7994: Mexican tetra (Astyanax mexicanus): 103031423

[0100] 7998: Channel catfish: 108274524

[0101] 8005: Electric eel (Electrophorus electricus): 118242670 8005: Electric eel: 118242669 8005: Electric eel: 113581746 8010: Northern pike (Esox lucius): 105029177

[0102] 8017: Pink salmon (Oncorhynchus gorbuscha): 123993384

[0103] 8018: Chinook salmon: 118377783

[0104] 8019: Oncorhynchus kisutch: 109864762

[0105] 8022: Oncorhynchus mykiss: 100136693

[0106] 8023: Oncorhynchus nerka: 115130677

[0107] 8030: Salmo salar: 101448053

[0108] 8030: Salmo salar: 106611692

[0109] 8032: Salmo trutta: 115172323

[0110] 8036: Salvelinus alpinus: 111965708

[0111] 8038: Salvelinus fontinalis: 129858071

[0112] 8040: Salvelinus namaycush: 120046990

[0113] 8049: Gadus morhua: 115551867

[0114] 8078: Fundulus heteroclitus: 105920226

[0115] 8081: Poecilia reticulata: 103471153

[0116] 8083: Xiphophorus maculatus: 102223082

[0117] 8084: Xiphophorus hellerii: 116714911

[0118] 8090: Cyprinodon variegatus: 100302723

[0119] 8103: Cyclopterus lumpus: 117737633

[0120] 8128: Oreochromis niloticus: 100712141

[0121] 8153: Haplochromis burtoni: 102308484

[0122] 8154: Astatotilapia calliptera: 1130301828167: Perca flavescens: 114562870

[0123] 8168: Perca fluviatilis: 120566341

[0124] 8175: Atlantic porgy: 115568425

[0125] 8177: Sparus latus: 119007972

[0126] 8187: Lates calcarifer: 108895524

[0127] 8208: Notothenia coriiceps: 104953557

[0128] 8218: Gymnodraco acuticeps: 117550442

[0129] 8236: Yellowfin tuna: 122990905

[0130] 8240: Southern bluefin tuna: 121902261

[0131] 8245: Swordfish (Xiphias gladius): 120794382

[0132] 8245: Swordfish: 120784879

[0133] 8255: Japanese flounder (Paralichthys olivaceus): 109627263

[0134] 8255: Japanese flounder: 109629018

[0135] 8262: European plaice (Pleuronectes platessa): 128436393

[0136] 8262: European plaice: 128446010

[0137] 8267: Atlantic halibut (Hippoglossus hippoglossus): 117769082 8267: Atlantic halibut: 117769698 13013: African catfish (Clarias gariepinus): 128512679

[0138] 13489: European perch: 127375252

[0139] 13676: Japanese mackerel: 128363493

[0140] 27687: Reedfish (Erpetoichthys calabaricus): 11466000027706: Largemouth bass (Micropterus salmoides): 119884054

[0141] 27706: Largemouth bass: 119889311

[0142] 28743: Sheepshead minnow (Cyprinodon variegatus): 107097352

[0143] 28829: Senegal sole (Solea senegalensis): 122760098

[0144] 29144: Milkfish (Chanos chanos): 115804993

[0145] 30732: Marine medaka (Oryzias melastigma): 112153172

[0146] 31033: Tiger pufferfish: 101063254

[0147] 32473: Couch's swordtail (Xiphophorus couchianus): 114152529

[0148] 32507: Neolamprologus brichardi: 10277558333528: Mosquito fish (Gambusia affinis): 122836594

[0149] 34773: American shad (Alosa sapidissima): 121694293

[0150] 34816: Striped bass (Morone saxatilis): 118333920

[0151] 37003: Mangrove rivulus (Kryptolebias marmoratus): 10824965740690: Antarctic toothfish (Trematomus bernacchii): 117470848

[0152] 41447: Seriola dumerili: 111224560

[0153] 41447: Seriola dumerili: 111230405

[0154] 42514: Pygocentrus nattereri: 108442481

[0155] 42526: Colossoma macropomum: 118811266

[0156] 42526: Colossoma macropomum: 118801938

[0157] 42636: Brienomyrus brachyistius: 12575088143689: Simochromis diagramma: 120720608

[0158] 43700: Monopterus albus: 109966434

[0159] 47969: Oreochromis aureus: 116317994

[0160] 48193: Mugil cephalus: 125008417

[0161] 48698: Poecilia formosa: 103154733

[0162] 48699: Poecilia latipinna: 106940460

[0163] 48701: Poecilia mexicana: 106910880

[0164] 52239: Pseudochaenichthys georgianus: 11745418952670: Austrofundulus limnaeus: 10652439152904: Scophthalmus maximus: 118319583

[0165] 52904: Scophthalmus maximus: 118312266

[0166] 54343: Orangespotted Darter (Etheostoma spectabile): 116697245

[0167] 55291: Senegal Bichir (Polypterus senegalus): 120543184

[0168] 56716: Saddleback Sculpin (Cottoperca gobio): 115014268

[0169] 56723: Ballan Wrasse (Labrus bergylta): 109994465

[0170] 59861: Lake Herring (Coregonus clupeaformis): 121585904

[0171] 59861: Lake Herring: 121542650

[0172] 63155: Nicaraguan Midas Cichlid (Archocentrus centrarchus): 115787225 63155: Nicaraguan Midas Cichlid: 115787218 64144: Climbing Perch (Anabas testudineus): 113150433

[0173] 64144: Climbing Perch: 113160572

[0174] 66913: Longfin Bullhead Catfish: 128618702

[0175] 69293: Three-spined Stickleback (Gasterosteus aculeatus): 120816881

[0176] 70543: Chinese Sucker (Myxocyprinus asiaticus): 127417548

[0177] 70543: Chinese Sucker: 127412725

[0178] 72105: Dusky Rockfish (Sebastes umbrosus): 119493801

[0179] 74940: Lake Trout: 112220926 75038: Spotted Scat (Scatophagus argus): 124067992

[0180] 75329: Loach (Misgurnus anguillicaudatus): 12942252075352: Bluntnose black bream: 12525910275366: Golden-line barbel (Sinocyclocheilus grahami): 10758708975366: Golden-line barbel: 10755929277115: White Sands pupfish (Cyprinodon tularosa): 119773491

[0181] 80966: Spiny chromis (Acanthochromis polyacanthus): 11097021780966: Spiny chromis: 11094679780972: Ocellaris clownfish (Amphiprion ocellaris): 111570639

[0182] 84645: Rohu (Labeo rohita): 127175905

[0183] 90069: European sole (Solea solea): 131472944

[0184] 90988: Fathead minnow (Pimephales promelas): 120460971

[0185] 101364: Gibel carp (Carassius gibelio): 127971074

[0186] 101364: Gibel carp: 128027787

[0187] 105023: Turquoise killifish (Nothobranchius furzeri): 107373510106582: Zebra mbuna (Maylandia zebra): 101485786

[0188] 109280: Tiger tail seahorse (Hippocampus comes): 109525804

[0189] 109293: Dwarf seahorse (Hippocampus zosterae): 127599348

[0190] 109905: Longnose hawkfish (Chelmon rostratus): 121611674

[0191] 113540: Asian arowana (Scleropages formosus): 108936068

[0192] 113540: Golden Arowana: 108923024

[0193] 118141: Tarpon (Megalops cyprinoides): 118790659

[0194] 119488: Mandarin fish (Siniperca chuatsi): 122880547

[0195] 134920: Nine-spined stickleback (Pungitius pungitius): 119210979

[0196] 137520: Transpacific smelt (Hypomesus transpacificus): 124470547 144197: Reef chromis (Stegastes partitus): 103363223

[0197] 147949: Smallmouth bass (Micropterus dolomieu): 123958144

[0198] 150288: Black-spotted mudskipper (Boleophthalmus pectinirostris): 110159042 154827: Texas highfin chub (Xyrauchen texanus): 127663360

[0199] 154827: Texas highfin chub: 127659470

[0200] 158456: Siamese fighting fish (Betta splendens): 114864992

[0201] 160734: Leopard coral grouper: 121948100 161448: Intestine pipefish (Corythoichthys intestinalis): 130926506 161448: Intestine pipefish: 130927109 161450: Blue-banded pipefish (Doryrhamphus excisus): 131110374

[0202] 161453: Knobby pipefish (Dunckerocampus dactyliophorus): 129189966 161584: Spined pipefish (Syngnathus acus): 119121031

[0203] 161590: Gulf pipefish (Syngnathus scovelli): 125967294

[0204] 173247: Remora (Echeneis naucrates): 115037193

[0205] 173247: Remora: 115049621

[0206] 175797: Southern catfish (Silurus meridionalis): 124392454

[0207] 181472: Zebra blenny (Salarias fasciatus): 115401214

[0208] 188132: Prolific molly (Poeciliopsis prolifica): 129353865195615: Greenland halibut: 118118622195615: Greenland halibut: 118112154205130: Spiny eel (Mastacembelus armatus): 113144127

[0209] 208333: Many-rayed gambusia (Girardinichthys multiradiatus): 124860564210632: Indian glassy fish (Parambassis ranga): 114442990

[0210] 215358: Large yellow croaker (Larimichthys crocea): 104935689

[0211] 229290: Sablefish (Anoplopoma fimbria): 129090164

[0212] 241271: Humphead wrasse (Cheilinus undulatus): 121515885

[0213] 244447: Half-smooth tongue sole: 103390515270530: Mandarinfish (Synchiropus splendidus): 128767449278164: Allis shad (Alosa alosa): 125286341

[0214] 283035: Pikeperch (Sander lucioperca): 116064383

[0215] 293821: Brown-marbled grouper: 125894322299321: Tooth herring (Denticeps clupeoides): 114768310

[0216] 300413: Epinephelus moara: 126389345

[0217] 303518: Pundamilia nyererei: 102198131

[0218] 307959: Sinocyclocheilus rhinocerous: 107758536307959: Sinocyclocheilus rhinocerous: 107731723310571: Epinephelus lanceolatus: 117260757310915: Pangasianodon hypophthalmus: 113529581337641: Hemibagrus wyckioides: 131365133

[0219] 369639: Onychostoma macrolepis: 131553405375764: Sphaeramia orbicularis: 115416804375764: Sphaeramia orbicularis: 115427540409849: Periophthalmus magnuspinnatus: 117377737417921: Etheostoma cragini: 117952205

[0220] 433405: Anarrhichthys ocellatus: 116400932433684: Takifugu flavidus: 130531798

[0221] 441366: Gouania willdenowi: 114471152

[0222] 451745: Nematolebias whitei: 119408307

[0223] 586833: Myripristis murdjan: 115371519

[0224] 586833: Myripristis murdjan: 115366093

[0225] 941984: Toxotes jaculatrix: 121193628

[0226] 941984: Archerfish: 121193618

[0227] 941984: Archerfish: 121188811

[0228] 992332: Triplophysa rosa: 130564032

[0229] 1042646: Gadus chalcogrammus: 130390898

[0230] 1142201: Danio aesculapii: 130240883

[0231] 1203425: Notolabrus celidotus: 117817455

[0232] 1234273: Tachysurus fulvidraco: 113654857 1250792: Melanotaenia boesemani: 121643126 1582913: Triplophysa dalaica: 130438038

[0233] 1606681: Puntigrus tetrazona: 122357788

[0234] 1608454: Sinocyclocheilus anshuiensis: 107680907 1608454: Sinocyclocheilus anshuiensis: 107660197 1676925: Paramormyrops kingsleyae: 111852987 1841481: Seriola lalandi dorsalis: 111660041 1841481: Seriola lalandi dorsalis: 111663599 2059687: Pseudoliparis swirei: 130209981

[0235] 2546036: Lampris incognitus: 130110548

[0236] 2546036: Lampris incognitus: 130110540

[0237] 2871759: Yellowtail amberjack (Seriola aureovittata): 130163207

[0238] 2871759: Yellowtail amberjack: 130173671

[0239] 3034132: Rhinichthys klamathensis goyatoka: 130084764.

[0240] There is no restriction on the fluorescent protein in the infertility-causing gene alleles. Examples include green fluorescent proteins such as TurboGFP, AcGFP, TagGFP, Azami-Green, ZsGreen, EmGFP, EGFP, GFP2, HyPerGFP, PS-CFP, Dendra2, Kaede, EosFP, KikumeGR; blue fluorescent proteins such as Sirius, EBFP; cyan fluorescent proteins such as ECFP, mTurquoise, TagCFP, AmCyan, mTFP1, MidoriishiCyan, CFP, PS-CFP; yellow fluorescent proteins such as TagYFP, EYFP, Venus, YFP, PhiYFP, PhiYFP-m, TurboYFP, ZsYellow, mBanana; orange fluorescent proteins such as KusabiraOrange, mOrange; red fluorescent proteins such as TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRed-Monomer, AsRed2, mStrawberry, Dendra2, Kaede, EosFP, KikumeGR; far-red fluorescent proteins such as TurboFP602, mRFP1, JRed, KillerRed, mCherry, HcRed, KeimaRed, mRasberry, mPlum, etc.

[0241] In the case of the homozygous type, a polynucleotide sequence encoding a fluorescent protein having the same fluorescence wavelength band can be introduced into both alleles of the infertility-causing gene, or polynucleotide sequences encoding fluorescent proteins having different fluorescence wavelength bands can be introduced into both alleles of the infertility-causing gene, respectively.

[0242] Fluorescent proteins having the same fluorescence wavelength band mean that when two fluorescent proteins are irradiated with an excitation light of an appropriate wavelength, the difference in the peak wavelengths of the fluorescence emitted by the two fluorescent proteins is, for example, within 30 nm, preferably within 20 nm.

[0243] Fluorescent proteins having different fluorescence wavelength bands mean that when two fluorescent proteins are irradiated with an excitation light of an appropriate wavelength, the difference in the peak wavelengths of the fluorescence emitted by the two fluorescent proteins is, for example, 20 nm or more, preferably 30 nm or more, more preferably 50 nm or more.

[0244] The excitation light of an appropriate wavelength means the excitation light recommended for each fluorescent protein.

[0245] "A polynucleotide sequence encoding a fluorescent protein in an expressible manner..." means that a fluorescent protein can be expressed from the polynucleotide sequence encoding the fluorescent protein under the control of a promoter.

[0246] The polynucleotide sequence encoding the fluorescent protein is directly or indirectly linked to the 3'-downstream region of the promoter sequence for expressing the fluorescent protein. "Indirectly" means that it may contain a spacer sequence of about 1 base (nt) to 20 nt.

[0247] The promoter is not limited as long as it exhibits promoter activity in fish individuals. The promoter can be a promoter that controls tissue-specific expression or a promoter that controls systemic expression. The expression of tissue-specific fluorescent proteins preferably occurs in parts observable from the body surface such as, for example, the eye, scales, skin, fins, etc. As promoters that control tissue-specific expression, promoters such as those of γ-crystallin, α-crystallin, β-crystallin, δ-crystallin, rhodopsin, RPE65, IRBP, or arrestin genes that control expression in the eye can be cited. As promoters that control tissue-specific expression, promoters such as sp7, keratin, or KRT5 genes can be cited. As promoters that control systemic expression, promoters such as actin can be cited.

[0248] The confirmation of the phenotypes of the heterozygous and homozygous types can be carried out by confirming the expression of the fluorescent protein. The expression of the fluorescent protein can be carried out by irradiating the embryo or adult fish body surface of the fish individual with an appropriate excitation light and observing the fluorescence. The observation of the fluorescence can be carried out by a person with the naked eye or using a fluorescence microscope or a microscope. Alternatively, it can be observed by a computer. In the case of the heterozygous type, whether there is a fluorescent protein is determined by a person or a computer, and if there is an expression of the fluorescent protein, it can be determined as the heterozygous type. In the case of the homozygous type, when the two alleles of the infertility gene have a polynucleotide sequence encoding a fluorescent protein having the same fluorescence wavelength band, if the fluorescence is stronger compared to the intensity of the heterozygous type, it can be determined as the homozygous type. Additionally, when the two alleles of the infertility gene have a polynucleotide sequence encoding a fluorescent protein having different fluorescence wavelength bands, if there is an expression of each fluorescent protein, it can be determined as the homozygous type. At this time, the embryo or adult fish of the fish individual can be maintained in a viable state.

[0249] 2. Method for Producing a Fish Individual in Which at Least a Portion of a Sterilization Protein Has a Defective Function

[0250] One embodiment relates to a method for producing a fish individual in which at least a portion of a sterilization protein has a defective function (hereinafter also simply referred to as "production method"). The production method includes introducing a polynucleotide sequence encoding a fluorescent protein (hereinafter also simply referred to as "fluorescent protein sequence") in an expressible manner into at least one of the alleles of the sterilization gene encoding the sterilization protein,

[0251] In this item, the description of the terms described in the above 1. is incorporated herein by reference.

[0252] For example, as described in the above 1., the fluorescent protein sequence is introduced into the allele of the sterilization gene as a donor cassette polynucleotide directly or indirectly linked to the 3'-downstream region of the promoter sequence. The fluorescent protein sequence is preferably the cDNA sequence of the fluorescent protein. The donor cassette polynucleotide preferably further has a polyA signal in addition to the promoter sequence and the fluorescent protein sequence.

[0253] The introduction of the donor cassette polynucleotide into the allele of the sterilization gene can be carried out by a known method.

[0254] As an introduction method, for example, a gene knock-in method using a genome editing method or a gene site-specific recombination method can be cited.

[0255] Examples of genome editing methods include methods of introducing proteins and nucleic acids that make up genome editing technologies, or vectors encoding these, into fertilized eggs. Examples of the above proteins include, for example, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) enzymes. Specifically, examples of the above CRISPR enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, etc. Examples of the above nucleic acids include, for example, crRNA and tracrRNA, or single-stranded nucleic acids formed by ligating these with a linker. At this time, regarding the above nucleic acids, for example, the base sequence annealing to the target sequence in crRNA is designed to be a base sequence complementary to the base sequence encoding the infertility gene. The above nucleic acids can be used alone as one kind, or two or more kinds can be used in combination. Examples of genome editing systems using CRISPR enzymes include the Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR-associated protein 9 (CRISPR / Cas9) system. In addition, as other genome editing systems, examples include at least one method selected from the CompoZr zinc finger nuclease (ZFN) system and the transcription activator-like effector nuclease (TALEN) system. Preferably, the CRISPR / Cas9 system in which Cas9 is introduced in the form of mRNA or protein and gRNA is introduced in the state of sgRNA or crRNA and tracrRNA. In addition, in the CRISPR / Cas9 system using a vector, the nucleic acid encoding CRISPR and the nucleic acid encoding Cas9 can be on different vectors or on one vector. The promoter for making CRISPR function is not particularly limited, and the U6 promoter is preferred. The promoter for making Cas9 function is not particularly limited, and promoters expressed in mammalian cells such as the cytomegalovirus promoter are preferred. As the CRISPR / Cas9 system, commercially available vectors such as the pX330-U6-Chimeric_BB-CBh-hSpCas9 vector can be preferably used.

[0256] The sequence targeting the infertility gene integrated into the CRISPR sequence (hereinafter also referred to as "target sequence") is not limited as long as it is integrated into the guide RNA (also referred to as gRNA, sgRNA, crRNA) through the CRISPR / Cas9 system and transcribed, or imported into cells in the form of a guide RNA containing a sequence complementary to the target sequence and capable of recombining the infertility gene. It has been reported that as the target sequence, a sequence of about 20 bases in the 5'-side upstream region of the base sequence "NGG" (PAM sequence: N is any one of the nucleotides A, G, T, C) existing in the infertility gene is usually selectable. The target sequence can be designed using publicly known design tools disclosed in optimized CRISPR design tools (the website of the Massachusetts Institute of Technology, Zhang Lab (http: / / crispr.mit.edu / )), E-CRISP (http: / / www.e-crisp.org / E-CRISP / (German Cancer Research Center)), ZiFiT Targeter (http: / / zifit.partners.org / ZiFit / (Zing Finder Consortium)), Cas9 design (http: / / cas9.cbi.pku.edu.cn (Peking University)), CRISPRdirect (http: / / crispr.dbcls.jp (The University of Tokyo)), CRISPR-P (http: / / cbi.hzau.edu.cn / crispr / (Huazhong Agricultural University)), CRISPR RGEN Tools (http: / / www.rgenome.net / (Seoul National University)), etc.

[0257] In addition, preferably, when there are single nucleotide polymorphisms (SNPs) in the PAM sequence, it is preferable to avoid such sequences. Regarding the target sequence, when the SNPs of an individual are known, it is preferable to optimize the sequence according to each SNP.

[0258] The 5'-terminal region of the target sequence can be truncated by 1, 2, 3, or 4 bases, preferably 1, 2, or 3 bases.

[0259] In addition, the CRISPR / Cas9 system can introduce genes into cells in the form of a vector, and can introduce into cells a combination of gRNA, crRNA, trans-activating crRNA (tracrRNA) and RNA encoding Cas9 synthesized by artificial synthesis or in vitro transcription. Alternatively, the Cas9 protein and the guide RNA can be combined and introduced into cells.

[0260] Furthermore, the above genome editing system can co-introduce donor oligonucleotide DNAs such as single-stranded oligomers (ssODNs). The ssODNs can be designed by known methods.

[0261] The genome editing system can be injected into the cytoplasm of a fertilized egg, preferably a fertilized egg at the single cell stage, by microinjection. For example, when introducing the Cas9 protein, it can be injected in a range of about 5 pg to 100 pg, preferably 10 pg to 80 pg, more preferably 10 pg to 50 pg per fertilized egg. At this time, the guide RNA can be injected in a range of about 0.1 pg to 50 pg, preferably 0.5 pg to 20 pg, more preferably 1 pg to 5 pg. The method for introducing mutations using genome editing technology can be referred to, for example, in Example 1 described below.

[0262] As the gene site-specific recombination method, for example, a method using at least one system selected from the Cre / loxP system, Flp / FRT system, Dre / rox system, PhiC31 integrase system, or variants of the above gene site-specific recombination methods can be cited.

[0263] 3. Method for identifying genotype

[0264] One embodiment relates to a method for identifying genotype (hereinafter also simply referred to as "identification method"). It includes the following steps: mating a female and a male of the fish individuals described in 1. above, in which only one of the infertility gene alleles has a polynucleotide sequence encoding a fluorescent protein in an expressible manner; and observing the expression of the fluorescent protein in the embryos or adult fish obtained by the mating.

[0265] In the identification method, the female and male for mating can be of the same species or different crossable species.

[0266] When mating heterozygous female and male fish individuals among the fish individuals described in 1. above, according to Mendel's law of inheritance, the genotypes of their next generation can be wild type, heterozygous, and homozygous. These can be identified by the expression of the fluorescent protein.

[0267] The observation of the expression of the fluorescent protein and the method for determining whether it is heterozygous or homozygous are as described in 1. above. In the case of the wild type, the expression of the fluorescent protein is not observed.

[0268] 4. Method for transplanting germ cells

[0269] One embodiment relates to a method for transplanting germ cells, which includes the step of transplanting germ cells of a fish of a different variety from the above fish individuals into the homozygous fish individuals described in 1. above.

[0270] Here, there is no limitation as long as the fish species different from the homozygous fish individuals described in the above 1. is adapted to the fish body of the above homozygous fish individuals.

[0271] The transplantation method is well-known. For example, a method for producing fish gametes using a surrogate parent fish method. Undifferentiated germ cells used as donors in surrogate parent fish technology include primordial germ cells obtained from the gonads before sexual differentiation, spermatogonia obtained from the testis, oogonia obtained from the ovary, and the like.

[0272] In order to obtain undifferentiated germ cells from a donor, they can be collected from a donor tissue corresponding to the differentiation stage of the undifferentiated germ cells as the target by a conventional method. For example, the gonads before sexual differentiation or tissues after sexual differentiation, such as the testis or ovary, can be removed from the donor, and the tissues can be dispersed into single cells by physical dissection or treatment with proteolytic enzymes, etc., thereby obtaining undifferentiated germ cells. The single cells thus dispersed can be isolated, for example, using an antibody as a marker or a cell sorter.

[0273] Undifferentiated germ cells can be obtained from frozen bodies or living individuals. In order to improve the success rate of the surrogate parent fish technology, it is preferable to obtain undifferentiated germ cells from living individuals.

[0274] When introducing the donor cells into the recipient, it is preferable to introduce them into an embryo or a fry-stage individual before the immune system of the recipient fully functions. The introduction can be carried out using an operating instrument such as a micromanipulator, an electroknife, or a laser knife. The introduction can be performed on any tissue or part of the recipient. As the tissue or part to be the introduction target, for example, the epidermis or the abdominal cavity can be cited. There is no particular limitation on the number of cells when introducing them into an embryo or a fry-stage individual. For example, 1 cell to 100,000 cells can be introduced.

[0275] Examples

[0276] The following examples illustrate the present invention in more detail. However, the present invention is not construed as being limited to the examples. It should be noted that the sequences corresponding to the sequence numbers used in the examples are shown in the sequence listing described later.

[0277] I. Preparation of Fluorescent Protein Expression Cassette Polynucleotide

[0278] The first cassette polynucleotide and the second cassette polynucleotide capable of expressing a fluorescent protein under the control of a crystallin promoter were prepared. The first cassette polynucleotide has an EGFP cDNA sequence (SEQ ID NO: 2) downstream of the mouse γ-crystallin promoter (SEQ ID NO: 1), and further has a polyA signal of simian virus 40 (SV40) (SEQ ID NO: 3) downstream thereof. Hereinafter, this cassette polynucleotide will be referred to as Crystallin promoter-EGFP-SV40 polyA, and the polynucleotide sequence is shown in SEQ ID NO: 4. The second cassette polynucleotide has an mCherry cDNA sequence (SEQ ID NO: 5) downstream of the mouse γ-crystallin promoter (SEQ ID NO: 1), and further has an SV40 polyA signal (SEQ ID NO: 3) downstream thereof. Hereinafter, this donor cassette polynucleotide will be referred to as Crystallin promoter-mCherry-SV40 polyA, and the polynucleotide sequence is shown in SEQ ID NO: 6.

[0279] II. Example using tilapia as the test species

[0280] 1. Preparation of donor construct

[0281] Using tilapia as the test species, in order to label germ cell-deficient individuals by genome editing, a donor construct for introducing the cassette polynucleotide described in I. above into tilapia was prepared. The sequence map of the donor construct is shown in Figure 3 .

[0282] (1) Isolate 5-prime Onil dnd homology having the sequence shown in SEQ ID NO: 7 and 3-prime Onil dnd homology having the sequence shown in SEQ ID NO: 8 from tilapia dnd (SEQ ID NO: 9).

[0283] (2) An Onil Crystallin promoter-EGFP-SV40 polyA vector (the nucleotide sequence is shown in SEQ ID NO: 13) was prepared, which has the gRNA#1 guide RNA sequence shown in SEQ ID NO: 10 for carrier cutting, the 5-prime Onil dnd homology sequence shown in SEQ ID NO: 7, the Crystallin promoter-EGFP-SV40 polyA sequence shown in SEQ ID NO: 4, the 3-prime Onil dnd homology sequence shown in SEQ ID NO: 8, the gRNA#1 shown in SEQ ID NO: 10, the replication origin Ori sequence of the plasmid in Escherichia coli shown in SEQ ID NO: 11, and the ampicillin resistance gene AmpR sequence shown in SEQ ID NO: 12. In addition, an Onil Crystallin promoter-mCherry-SV40 polyA vector was prepared by introducing the Crystallin promoter-mCherry-SV40 polyA shown in SEQ ID NO: 6 (the nucleotide sequence is shown in SEQ ID NO: 14) to replace the Crystallin promoter-EGFP-SV40 polyA shown in SEQ ID NO: 4.

[0284] 2. Introduction of cassette polynucleotide into the dnd gene region and confirmation test

[0285] (1) Introduction of cassette polynucleotide into the dnd gene region

[0286] Using the Onil Crystallin promoter-EGFP-SV40 polyA vector shown in SEQ ID NO: 13 and the Onil Crystallin promoter-mCherry-SV40 polyA vector shown in SEQ ID NO: 14, the cassette polynucleotide was introduced into the tilapia genome by the gene knock-in method of the genome editing method using the CRISPR-Cas9 system.

[0287] 250 ng / μl of Cas9 protein, 50 ng / μl of gRNA#3 (SEQ ID NO: 16) targeting exon 3 (SEQ ID NO: 15) of the tilapia dnd gene, 2.5 ng / μl of the Onil Crystallin promoter-EGFP-SV40 polyA vector or the Onil Crystallin promoter-mCherry-SV40 polyA vector, and 50 ng / μl of gRNA#1 (SEQ ID NO: 10) for cutting the construct were microinjected into tilapia fertilized eggs.

[0288] (2) Confirmation of the expression of fluorescent protein

[0289] The microinjected tilapia fertilized eggs were cultured at 28 °C, and the expression of GFP or mCherry in the 4- to 6-day-old embryos was observed under a stereomicroscope. In addition, the expression of fluorescent proteins in the eyes of embryos and adult fish developed from the microinjected tilapia fertilized eggs was confirmed( Figure 4 ).

[0290] To confirm that the cassette polynucleotide was correctly introduced into the dnd region, genomic DNA was extracted from individuals that showed fluorescence in the above observations, and the genomic sequence was confirmed by PCR and nucleotide sequence sequencing.

[0291] The PCR primers were designed, as shown Figure 5 , outside the homologous regions and between the cassette polynucleotides when using CRISPR-Cas9 for gene knock-in. Figure 5 The asterisks in show the confirmation sites. For the 5'-side (upstream side) confirmation, PCR was performed using Onil_dnd_5-prime_Fw (SEQ ID NO: 17) and cryP_Rv (SEQ ID NO: 18). In addition, for the 3'-side (downstream side) confirmation, the dnd gfp sample was subjected to PCR using GFP_Fw (SEQ ID NO: 19) and Onil_dnd_3-prime_Rv (SEQ ID NO: 20), and the dnd rfp sample was subjected to PCR using RFP_Fw (SEQ ID NO: 21) and Onil_dnd_3-prime_Rv (SEQ ID NO: 20). For each PCR product, electrophoresis was performed using 1.5% agarose gel to confirm the presence or absence and size of the bands, and it was confirmed that the target amplification product was obtained( Figure 6 ).

[0292] In addition, the above PCR products were subcloned, and Sanger sequencing analysis was performed using Onil_dnd_5-prime_Fw primer and Onil_dnd_3-prime_Rv primer, respectively.

[0293] The sequence of the cassette polynucleotide insertion site of the dnd gfp individual is shown in Figure 7 . Figure 7 (A) of shows the 5'-side sequence obtained by analysis using the Onil_dnd_5-prime_Fw primer. Figure 7 (B) of shows the 3'-side sequence obtained by analysis using the Onil_dnd_3-prime_Rv primer.

[0294] The sequence of the cassette polynucleotide insertion site of the dnd rfp individual is shown in Figure 8 . Figure 8(A) shows the sequence of the 5'-side obtained by analysis using the Onil_dnd_5-prime_Fw primer. Figure 8 (B) shows the sequence of the 3'-side obtained by analysis using the Onil_dnd_3-prime_Rv primer.

[0295] By PCR method and sequencing, it was shown that the cassette polynucleotide was correctly inserted at the target site.

[0296] III. Example with Takifugu alboplumbeus as the test species

[0297] 1. Donor construct

[0298] Using Takifugu alboplumbeus as the test species, in order to label germ cell-deficient individuals by genome editing method, a donor construct for introducing the cassette polynucleotide described in I. above into Takifugu alboplumbeus was prepared. The sequence map of the donor construct is shown in Figure 9 .

[0299] (1) Isolate 5-prime Talb dnd homology (SEQ ID NO: 22) and 3-prime Talb dnd homology (SEQ ID NO: 23) from Takifugu alboplumbeus dnd (SEQ ID NO: 24).

[0300] (2) Prepared a Talb Crystallin promoter-EGFP-SV40 polyA vector (showing the nucleotide sequence in SEQ ID NO: 25) having the gRNA#1 guide RNA sequence shown in SEQ ID NO: 10 for vector cleavage, the 5-prime Talb dnd homology sequence shown in SEQ ID NO: 22, the Crystallin promoter-EGFP-SV40 polyA sequence shown in SEQ ID NO: 4, the 3-prime Talb dnd homology sequence shown in SEQ ID NO: 23, the gRNA#1 shown in SEQ ID NO: 10, the Ori sequence shown in SEQ ID NO: 11, and the AmpR sequence shown in SEQ ID NO: 12. In addition, a Talb Crystallin promoter-mCherry-SV40 polyA vector (showing the nucleotide sequence in SEQ ID NO: 26) was prepared by introducing Crystallin promoter-mCherry-SV40 polyA shown in SEQ ID NO: 6 in place of Crystallin promoter-EGFP-SV40 polyA shown in SEQ ID NO: 4.

[0301] 2. Introduction of cassette polynucleotide into the dnd gene region and confirmation test

[0302] (1) Introduction of cassette polynucleotide into the dnd gene region

[0303] Using the Talb Crystallin promoter-EGFP-SV40 polyA vector shown in SEQ ID NO: 25 or the Talb Crystallin promoter-mCherry-SV40 polyA vector shown in SEQ ID NO: 26, a cassette polynucleotide is introduced into the genome of Takifugu alboplumbeus by gene knock-in using a genome editing method of the CRISPR-Cas9 system.

[0304] Inject 500 ng / μl of Cas9 protein, 50 ng / μl of gRNA#3 (SEQ ID NO: 28) targeting exon 4 (SEQ ID NO: 27) of the Takifugu alboplumbeus dnd gene, 2.5 ng / μl of the Talb Crystallin promoter-EGFP-SV40 polyA vector or the Talb Crystallin promoter-mCherry-SV40 polyA vector, and 50 ng / μl of gRNA#1 (Sequence 10) for cleaving the construct into fertilized eggs of Takifugu alboplumbeus by microinjection.

[0305] (2) Confirmation of expression of fluorescent protein

[0306] Cultivate the microinjected fertilized eggs of Takifugu alboplumbeus at 20°C, and observe the expression of GFP or RFP in embryos at 3 to 6 days post-fertilization under a stereomicroscope. In addition, confirm the expression of fluorescent protein in the eyes of embryos and adult fish developed from the microinjected fertilized eggs of Takifugu alboplumbeus.

[0307] To confirm that the cassette polynucleotide was correctly introduced into the dnd region, genomic DNA is extracted from individuals that showed fluorescence in the above observation, and the genomic sequence is confirmed by PCR and nucleotide sequence sequencing.

[0308] The PCR primers are as Figure 11 shown, and are designed outside the homologous region and between the cassette polynucleotides during gene knock-in using CRISPR-Cas9. Figure 11 The asterisks in Figure 12 indicate the confirmation sites. For confirmation of the 5'-side (upstream side), PCR is performed using Talb_dnd_5-prime_Fw (SEQ ID NO: 29) and cryP_Rv (SEQ ID NO: 18). In addition, for confirmation of the 3'-side (downstream side), PCR is performed using polyA-Fw (SEQ ID NO: 30) and Talb_dnd_3-prime_Rv (SEQ ID NO: 31). For each PCR product, electrophoresis is performed using a 1.5% agarose gel to confirm the presence or absence and size of the bands, and it is confirmed that the target amplification product (

[0309] In addition, the above PCR products were subcloned, and Sanger sequencing analysis was performed using the Talb_dnd_5-prime_Fw primer and the Talb_dnd_3-prime_Rv primer, respectively.

[0310] The sequence of the cassette polynucleotide insertion site of the dnd gfp individual is shown in Figure 13 . Figure 13 (A) of shows the 5'-side sequence obtained by analysis using the Talb_dnd_5-prime_Fw primer. Figure 13 (B) of shows the 3'-side sequence obtained by analysis using the Talb_dnd_3-prime_Rv primer.

[0311] The sequence of the cassette polynucleotide insertion site of the dnd rfp individual is shown in Figure 8 . Figure 14 (A) of shows the 5'-side sequence obtained by analysis using the Talb_dnd_5-prime_Fw primer. Figure 14 (B) of shows the 3'-side sequence obtained by analysis using the Talb_dnd_3-prime_Rv primer.

[0312] PCR and sequencing showed that the cassette polynucleotide was correctly inserted at the target site.

Claims

1. A fish individual in which at least a part of a sterility protein has a functional defect, wherein, At least one of the infertility-causing gene alleles encoding an infertility-causing protein that exhibits infertility due to a defect in at least a part of its function has a polynucleotide sequence encoding a fluorescent protein in an expressible manner.

2. The fish individual according to claim 1, wherein, The fish individual has a defective reproductive function, and both of the two alleles of the infertility-causing gene have the polynucleotide sequence encoding the fluorescent protein.

3. The fish individual according to claim 2, wherein, Both of the two alleles of the infertility-causing gene have a polynucleotide sequence encoding a fluorescent protein having the same fluorescence wavelength band.

4. The fish individual according to claim 2, wherein, Each of the two alleles of the infertility-causing gene has a polynucleotide sequence encoding a fluorescent protein having a different fluorescence wavelength band.

5. The fish individual according to claim 1, wherein, The infertility-causing gene is the dead end (dnd) gene.

6. The fish individual according to claim 1, wherein, The expression site of the fluorescent protein is the eye.

7. A method for producing a fish individual in which at least a part of an infertility-causing protein that exhibits infertility due to a defect in at least a part of its function has a defective function, which comprises the step of introducing, in an expressible manner, a polynucleotide sequence encoding a fluorescent protein into at least one of the infertility-causing gene alleles encoding the infertility-causing protein.

8. The manufacturing method according to claim 7, wherein, The introduction is carried out by a gene knock-in method using a genome editing method or a gene site-specific recombination method.

9. The manufacturing method according to claim 8, wherein, The genome editing method is a method using at least one system selected from the Clustered regularly interspaced short palindromic repeats / CRISPR associated protein 9 (CRISPR / Cas9) system, the CompoZr Zinc Finger Nuclease (ZFN) system, and the Transcription Activator-Like Effector Nuclease (TALEN) system.

10. The manufacturing method according to claim 8, wherein, The gene site-specific recombination method is a method using at least one system selected from the Cre / loxP system, the Flp / FRT system, the Dre / rox system, and the PhiC31 integrase system.

11. A polynucleotide, wherein, Within a partial sequence of an infertility-causing gene encoding an infertility-causing protein that exhibits infertility due to a defect in at least a part of its function, a sequence containing a polynucleotide sequence encoding a fluorescent protein in an expressible manner is contained within the polynucleotide sequence.

12. A method for transplanting germ cells, which comprises the step of transplanting germ cells of a fish of a variety different from that of the fish individual according to claim 2 into the fish individual.

13. A method for identifying a genotype, which comprises the following steps: mating a female and a male of a fish individual according to claim 1, in which only one of the infertility-causing gene alleles has a polynucleotide sequence encoding a fluorescent protein in an expressible manner; and observing the expression of the fluorescent protein in the embryos or adult fish obtained by the mating.

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