Fish individual deficient in at least part of function of sterilization protein

Abstract

One problem addressed by the present invention is to provide a fish individual which, as illustrated in fig. 2, makes it possible, in producing a gamete-deficient individual, to identify a genotype of a sterilization protein by appearance without collection of part of the body of the fish. This problem is solved by a fish individual deficient in at least part of a function of a sterilization protein, said fish individual comprising, in at least one of sterilization gene alleles coding for a sterilization protein which indicates sterility by at least part of a function thereof being deficient, a polynucleotide sequence coding for a fluorescent protein in such a manner as to be capable of being expressed.

Description

Fish individuals lacking at least part of the function of a sterilization protein

[0001] The present invention relates to a fish individual lacking at least a partial function of a sterilization protein, a method for producing the fish individual, a polynucleotide, a method for transplanting germ cells, and a method for identifying genotypes.

[0002] The Dead End (dnd) gene is known to be one of the sterility genes involved in sterility. It is known that individuals lacking the function of the protein encoded by this sterility gene (hereinafter referred to as the "sterility protein") are infertile due to the lack of germ cell formation.

[0003] In the conventional method of artificially creating germ cell-deficient individuals by utilizing the functional deficiency of a sterilization protein, heterozygotes with a heterozygote deficiency in the sterilization protein are crossed with each other to create homozygotes with a homozygote deficiency in the sterilization gene, as shown in Figure 1. The next generation produced by crossing heterozygotes with each other is either wild type, homozygote, or heterozygote, but it is impossible to distinguish these genotypes by appearance. Therefore, to distinguish the genotype, it is necessary to collect a portion of the fish and perform genetic analysis.

[0004] As shown in Figure 2, one objective of the present invention is to provide fish individuals whose sterilization protein genotype can be identified by appearance without having to collect part of the fish body when producing germ cell-deficient individuals.

[0005] Item 1. A fish individual lacking at least a portion of the function of a sterilization protein, wherein at least one allele of a sterilization gene encoding a sterilization protein that exhibits sterility due to the loss of at least a portion of the function has a polynucleotide sequence encoding a fluorescent protein in an expressible state. Item 2. A fish individual according to Item 1, wherein both alleles of the sterilization gene have polynucleotide sequences encoding the fluorescent protein, and the fish individual is deficient in reproductive ability. Item 3. A fish individual according to Item 2, wherein both alleles of the sterilization gene have polynucleotide sequences encoding fluorescent proteins having the same fluorescence wavelength band. Item 4. A fish individual according to Item 2, wherein both alleles of the sterilization gene each have polynucleotide sequences encoding fluorescent proteins having different fluorescence wavelength bands. Item 5. A fish individual according to Item 1, wherein the sterilization gene is a Dead End (dnd) gene. Item 6. A fish individual according to Item 1, wherein the fluorescent protein is expressed in the eye. Item 7. Item 8. A method for producing a fish individual lacking at least part of the function of a sterilization protein that exhibits sterility due to the loss of at least part of the function, the method comprising expressibly introducing a polynucleotide sequence encoding a fluorescent protein into at least one of the sterilization gene alleles that encode the sterilization protein. Item 9. The method according to Item 8, wherein the introduction is performed by a genome editing method or a knock-in method using a gene site-specific recombination method. Item 9. The method according to Item 8, wherein the genome editing method uses 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 TAL effector nuclease (TALEN) system. Item 10. The method according to Item 8, wherein the gene site-specific recombination method uses at least one system selected from the Cre / loxP system, the Flp / FRT system, the Dre / rox system, and the PhiC31 integrase system.Item 11. A polynucleotide comprising a sequence within a partial sequence of a sterilization gene encoding a sterilization protein that exhibits sterility due to the loss of at least a portion of its function, wherein the polynucleotide sequence has an expressible polynucleotide sequence encoding a fluorescent protein. Item 12. A method of transplanting germ cells, comprising transplanting germ cells of a fish species different from the fish individual described in Item 2 into said fish individual. Item 13. A method of identifying genotypes, comprising mating male and female fish individuals described in Item 1 that have an expressible polynucleotide sequence encoding a fluorescent protein in only one sterilization gene allele, and observing the expression of the fluorescent protein in embryos or adult fish obtained by mating.

[0006] When producing germ cell-deficient individuals, it is possible to provide fish individuals whose sterilization protein genotype can be identified by appearance.

[0007] 1 shows a conventional method; 2 shows an outline of the present invention; 3 shows a gene transfer map of tilapia; 4 shows the expression of fluorescent protein in fertilized eggs, embryos, and eyes of adult tilapia; 5 shows the positions of primers designed to confirm that the cassette polynucleotide was correctly introduced into the dnd gene of tilapia; 6 shows the results of PCR using the primers shown in FIG. 7. gfp The sequences of the cassette polynucleotide introduction site of the individual are shown. (A) shows the 5' sequence analyzed using the Onil_dnd_5-prime_Fw primer. (B) shows the 3' sequence analyzed using the Onil_dnd_3-prime_Rv primer. Tilapia dnd rfpThe sequence of the cassette polynucleotide introduction site in an individual is shown. (A) shows the 5'-side sequence analyzed using the Onil_dnd_5-prime_Fw primer. (B) shows the 3'-side sequence analyzed using the Onil_dnd_3-prime_Rv primer. A gene introduction map of the pufferfish is shown. Expression of fluorescent protein in the fertilized eggs, embryos, and eyes of adult pufferfish is shown. The positions of primers designed to confirm that the cassette polynucleotide was correctly introduced into the dnd gene of the pufferfish are shown. The results of PCR using the primers shown in Figure 11 are shown. The dnd gene of the pufferfish gfp The sequences of the cassette polynucleotide introduction site of the individual are shown. (A) shows the 5' sequence analyzed using the Talb_dnd_5-prime_Fw primer. (B) shows the 3' sequence analyzed using the Talb_dnd_3-prime_Rv primer. rfp The sequences of the cassette polynucleotide introduction site in each individual are shown. (A) shows the 5' sequence analyzed using the Talb_dnd_5-prime_Fw primer. (B) shows the 3' sequence analyzed using the Talb_dnd_3-prime_Rv primer.

[0008] 1. Fish Individuals Deficient in At Least Partial Function of Sterilization Protein One embodiment relates to fish individuals (hereinafter simply referred to as "fish individuals") deficient in at least partial function of a sterilization protein, which exhibits sterility due to the loss of at least part of the function. The fish individuals have a polynucleotide sequence encoding a fluorescent protein in at least one of the sterilization gene alleles encoding the sterilization protein in an expressible manner.

[0009] A "fish individual lacking at least some of the functions of a sterilization protein" may include, as genotypes, a heterozygous fish individual in which the polynucleotide sequence encoding the fluorescent protein is present in only one allele, and a homozygous fish individual in which the polynucleotide sequence encoding the fluorescent protein is present in both alleles.

[0010] In a heterozygous individual, a polynucleotide sequence encoding a fluorescent protein is introduced into one allele of a sterilization gene, resulting in a loss of function of the sterilization protein encoded by that allele. A heterozygous individual is an individual that retains at least some of its germ cells as a phenotype (also referred to herein as a "heterozygous individual") and is capable of reproduction in both sexes.

[0011] In a homozygote, the function of both sterilization protein alleles is lost due to the introduction of a polynucleotide sequence encoding a fluorescent protein into both sterilization gene alleles. A homozygote is an individual that is substantially germ cell-deficient (also referred to herein as a "homozygote-deficient individual") phenotypically, and both males and females are deficient in fertility. The germ cell deficiency can be confirmed by histological observation. For example, a germ cell deficiency can be determined when the number of germ cells (primordial germ cells, spermatogonia, oogonia, spermatocytes, oocytes, sperm cells, egg cells, eggs, or sperm) is reduced to 50% or less, 30% or less, 20% or less, 10% or less, 5% or less, 3% or less, or 1% or less compared to a wild-type individual.

[0012] In the present specification, the fish is not limited. The "fish" is not particularly limited. In the present specification, the fish may include saltwater fish, freshwater fish, brackish water fish, diadromous fish, etc.

[0013] Examples of the fish include 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, and Carangidae. Examples of fish include fish from the families Carangidae, Lateolabrax, Moronidae, Latidae, Rachycentridae, Cynoglossidae, Anguillidae, and Congridae.

[0014] Examples of fish of the family Paralichthys include Paralichthys olivaceus (TEMMINCK et SCHLEGEL), flounders belonging to the genus Paralichthys (such as large sole, Pacific flounder, Japanese flounder, Japanese maple flounder, and Taiwan flounder), flounders belonging to the genus Paralichthys (such as Paralichthys), flounders belonging to the genus Paralichthys (such as Paralichthys), flounders belonging to the genus Paralichthys (such as Japanese flounder, Japanese flounder, and Japanese flounder), and flounders belonging to the genus Paralichthys (such as Japanese flounder). Preferably, Paralichthys olivaceus (TEMMINCK et SCHLEGEL) is used.

[0015] Examples of the fish of the Tetraodontidae family include fish of the genus Takifugu, such as the tiger pufferfish (Takifugu rubripes), the red pufferfish (Takifugu porphyreus), and the grass pufferfish (Takifugu niphobles), and fish of the genus Lagocephalus, such as the white mackerel pufferfish (Lagocephalus wheeleri). The fish of the Piscidae family may include fish of the genus Piscidae, such as the boxfish (Ostracion immaculatus).

[0016] Examples of fish of the Sparidae family include fish of the Pagrus genus, such as red sea bream (Pagrus major) and gilthead sea bream (Pagrus auratus); fish of the Acanthopagrus genus, such as black porgy (Acanthopagrus schlegelii) and yellowtail sea bream (Acanthopagrus latus); fish of the Dentex genus, such as yellow sea bream (Dentex tumifrons); and fish of the Sparus genus, such as gilthead sea bream (Sparus aurata).

[0017] Examples of the fish of the Salmonidae family include fish of the genus Salmon, such as rainbow trout (Oncorhynchus mykiss), king salmon (Oncorhynchus tshawytscha), cherry salmon (Oncorhynchus masou), satsukimasu (Oncorhynchus masou), kunimasu (Oncorhynchus kawamurae), pink salmon (Oncorhynchus gorbuscha), and salmon (Oncorhynchus keta); fish of the genus Salmon, such as brown trout (Salmo trutta), sockeye salmon (Oncorhynchus nerka), coho salmon (Oncorhynchus kisutch), and Atlantic salmon (Salmo salar); Dolly Varden trout (Salvelinus malma), Arctic char (Salvelinus alpinus), and Japanese char (Salvelinus leucomaenis), brook trout (Salvelinus fontinalis), lake trout (Salvelinus namaycush), and other fish of the genus Salvelinus; and fish of the genus Sakhalin, such as the huchen (Parahucho perryi).

[0018] Examples of the fish of the Cyprinidae family include Honmoroko (Gnathopogon caerulescens), Silver carp (Hypophthalmichthys molitrix), Common carp (Cyprinus carpio), Grass carp (Ctenopharyngodon idellus), Bighead carp (Hypophthalmichthys nobilis), European crucian carp (Carassius carassius), Catla (Cyprinus catla), Japanese bluefin tuna (Mylopharyngodon piceus), Kenhi (Cirrhinus molitorella), Mrigal carp (Cirrhinus cirrhosus), Catla (Catla catla), Rohita (Labeo rohita), and Slenderhead bream (Megalobrama amblycephala).

[0019] The fish of the superfamily Ictaluroidea may include, for example, channel catfish (Ictalurus punctatus), blue catfish (Ictalurus furcatus), and the like.

[0020] The fish of the superfamily Siluroidea may include, for example, catfish (Silurus asotus), Biwa catfish (Silurus biwaensis), rock catfish (Silurus lithophilus), European catfish (Silurus glanis), longfin catfish (Clarias fuscus), walking catfish (Clarias batrachus), and the like.

[0021] The fish of the superfamily Bagroidea may include, for example, the Korean catfish (Pseudobagrus fulvidraco), the Mekong giant catfish (Pangasianodon gigas), the pangasius (Pangasius bocourti), and the sea bass (Pangasianodon hypophthalmus).

[0022] Examples of the fish of the family Grouper include the white spotted grouper (Epinephelus septemfasciatus), grouper (Epinephelus bruneus), red spotted grouper (Epinephelus akaara), yellow spotted grouper (Epinephelus malabaricus), white grouper (Epinephelus aeneus), black spotted grouper (Epinephelus amblycephalus), spotted grouper (Epinephelus areolatus), yellow spotted grouper (Epinephelus bleekeri), white spotted grouper (Epinephelus bontoides), (Epinephelus chlorostigma), orange spotted grouper (Epinephelus coiodes), red spotted grouper (Epinephelus fasciatus), red spotted grouper (Epinephelus fuscoguttatus), starry grouper (Epinephelus labriformis), and grouper (Epinephelus The species may include fish of the grouper genus Epinephelus, such as the yellow spotted grouper (Epinephelus lanceolatus), white spotted grouper (Epinephelus maculatus), yellow spotted grouper (Epinephelus malabricus), dusky grouper (Epinephelus marginatus), striated grouper (Epinephelus ongus), spotted grouper (Epinephelus polyphekadion), yellow spotted grouper (Epinephelus quoyanus), black fin grouper (Epinephelus sexfasciatus), Nassau grouper (Epinephelus striatus), single-spotted grouper (Epinephelus tauvina), and potato grouper (Epinephelus tukula); fish of the grouper genus Cromileptes, such as the yellow spotted grouper (Cromileptes altivelis); and Plectropomus, such as the striped grouper (Plectropomus leopardus), as well as hybrids between grouper species.

[0023] The fish of the Cichlidae family may include, for example, fish of the Oreochromis genus, such as Nile tilapia (Oreochromis niloticus), cichlid fish (Oreochromis mossambicus), and blue tilapia (Oreochromis aureus).

[0024] The fish of the medaka family may include, for example, fish of the genus Oryzias, such as medaka (Oryzias latipes, Oryzias sakaizumii) and Java medaka (Oryzias javanicus).

[0025] The fish of the Filefish family may include, for example, fish of the Stephanolepis genus, such as the filefish (Stephanolepis cirrhifer), and fish of the Thamnaconus genus, such as the black-spotted filefish (Thamnaconus modestus).

[0026] The fish of the Smeltidae family may include, for example, fish of the Plecoglossinae subfamily (Plecoglossinae) such as sweetfish (Plecoglossus altivelis), smelt (Hypomesus nipponensis) and Japanese whiting (Hypomesus japonicus) and other fish of the Hypomesinae subfamily (Hypomesinae) such as smelt (Osmerus mordax dentex), shishamo (Spirinchus lanceolatus), and icefish (Spirinchus lanceolatus) and other fish of the Osmerinae subfamily (Osmerinae).

[0027] Examples of the fish of the Scombridae family include the genus Scombrini, such as chub mackerel (Scomber japonicus), Atlantic mackerel (Scomber scombrus), and yellow mackerel (Scomber australasicus); the genus Thunnini, such as Pacific bluefin tuna (Thunnus orientalis), Atlantic bluefin tuna (Thunnus orientalis), southern bluefin tuna (Thunnus maccoyii), bigeye tuna (Thunnus obesus), yellowfin tuna (Thunnus albacares), albacore tuna (Thunnus alalunga), and longfin tuna (Thunnus tonggol); the genus Euthynnus, such as Japanese amberjack (Euthynnus affinis) and Atlantic amberjack (Euthynnus alletteratus); and the genus Katsuwonus. The species may include Katsuwonus, such as pelamis, Scomberomorini, Auxis, Sardini, Gymnosarda, and the like.

[0028] The Pleuronectidae fish may include, for example, fish such as brown flounder (Pseudopleuronectes herzensteini), marbled flounder (Pleuronectes yokohamae), stone flounder (Kareius bicoloratus), Pacific halibut (Hippoglossus stenolepis), and barfin flounder (Verasper moseri).

[0029] The fish of the Carangidae family may include, for example, fish of the Seriola genus, such as Seriola dumerili, Seriola lalandi, Seriola rivoliana, and Seriola quinqueradiata; fish of the Pseudocaranx genus, such as horse mackerel (Trachurus japonicus) and striped jack (Pseudocaranx dentex); and fish of the Trachinotus genus, such as the Japanese jack mackerel.

[0030] The fish of the family Lateolabrax may include, for example, blackfin bass (Lateolabrax latus) and amberjack bass (Lateolabrax maculatus), and the fish of the family Moronidae may include, for example, European sea bass (Dicentrarchus labrax).

[0031] The fish of the family Latidae may include, for example, fish of the genus Lates, such as barramundi (Lates calcarifer) and Nile perch (Lates niloticus).The fish of the family Rachycentridae may include, for example, fish of the cedar (Rachycentron canadum).

[0032] The fish of the family Cynoglossidae may include, for example, fish such as red tongue sole (Cynoglossus joyneri) and semilaevis tongue sole (Cynoglossus semilaevis).

[0033] The fish of the family Anguillididae may include, for example, the Japanese eel (Anguilla japonica), the European eel (Anguilla anguilla), etc. The fish of the family Conger eel may include, for example, the Japanese conger eel (Conger myriaster), the Japanese black conger eel (Conger japonicus), etc. Preferred fish are fish of the families Cichlidae and Tetraodontidae, such as tilapia and pufferfish.

[0034] As used herein, the fish may be a fixed species or a hybrid, which may include, for example, a hybrid resulting from intergeneric crossing.

[0035] The "fish" is preferably a farmed fish. Furthermore, the "farmed fish" may include fish farmed for purposes such as food, breeding, and ornamental purposes.

[0036] An example of a sterilization gene is the Dead End (dnd) gene. The Gene IDs registered with NCBI for the main fish sterility genes are as follows: Taxonomy ID:Organism name:Gene ID 7906:Acipenser ruthenus:131699448 7906:Acipenser ruthenus:117412941 7913:Polyodon spathula:121323773 7913:Polyodon spathula:121297601 7918:Lepisosteus oculatus:102698012 7936:Anguilla anguilla:118224086 7950:Clupea harengus:105903488 7955:Danio rerio:373074 7957:Carassius auratus:113057682 7957:Carassius auratus:113113732 7959:Ctenopharyngodon idella:127494684 7962:Cyprinus carpio:109089193 7962:Cyprinus carpio:109108346 7994:Astyanax mexicanus:103031423 7998:Ictalurus punctatus:108274524 8005:Electrophorus electricus:1182426708005:Electrophorus electricus:1182426698005:Electrophorus electricus:1135817468010:Esox lucius:105029177 8017:Oncorhynchus gorbuscha:123993384 8018:Oncorhynchus keta:118377783 8019:Oncorhynchus kisutch:109864762 8022:Oncorhynchus mykiss:100136693 8023:Oncorhynchus nerka:115130677 8030:Salmo salar:101448053 8030:Salmo salar:106611692 8032:Salmotrout:115172323 8036:Salvelinus alpinus:111965708 8038:Salvelinus fontinalis:129858071 8040:Salvelinus namaycush:120046990 8049:Gadus morhua:115551867 8078:Fundulus heteroclitus:105920226 8081:Poecilia reticulata:103471153 8083:Xiphophorus maculatus:102223082 8084:Xiphophorus hellerii:116714911 8090:Oryzias latipes:100302723 8103:Cyclopterus lumpus:117737633 8128:Oreochromis niloticus:100712141 8153:Haplochromis burtoni:102308484 8154:Astatotilapia calliptera:1130301828167:Perca flavescens:114562870 8168:Perca fluviatilis:120566341 8175:Sparus aurata:115568425 8177:Acanthopagrus latus:119007972 8187:Lates calcarifer:108895524 8208:Notothenia coriiceps:104953557 8218:Gymnodraco acuticeps:117550442 8236:Thunnus albacares:122990905 8240:Thunnus maccoyii:121902261 8245:Xiphias gladius:120794382 8245:Xiphias gladius:120784879 8255:Paralichthys olivaceus:109627263 8255:Paralichthys olivaceus:109629018 8262:Pleuronectes platessa:128436393 8262:Pleuronectes platessa:1284460108267:Hippoglossus hippoglossus:1177690828267:Hippoglossus hippoglossus:11776969813013:Clarias gariepinus:128512679 13489:Dicentrarchus labrax:127375252 13676:Scomber japonicus:128363493 27687: Erpetoichthys calabaricus: 11466000027706: Micropterus salmoides: 119884054 27706: Micropterus salmoides: 119889311 28743: Cyprinodon variegatus: 107097352 28829: Solea senegalensis: 122760098 29144: Chanos chanos:115804993 30732:Oryzias melastigma:112153172 31033:Takifugu rubripes:101063254 32473:Xiphophorus couchianus:114152529 32507:Neolamprologus brichardi:10277558333528:Gambusia affinis:122836594 34773:Alosa sapidissima:121694293 34816:Morone saxatilis:118333920 37003:Kryptolebias marmoratus:10824965740690:Trematomus bernacchii:117470848 41447:Seriola dumerili:111224560 41447:Seriola dumerili:111230405 42514:Pygocentrus nattereri:108442481 42526:Colossoma macropomum:118811266 42526:Colossoma macropomum:118801938 42636:Brienomyrus brachyistius:12575088143689:Simochromis diagramma:12072060843700:Monopterus albus:109966434 47969:Oreochromis aureus:116317994 48193:Mugil cephalus:125008417 48698:Poecilia formosa:103154733 48699:Poecilia latipinna:106940460 48701:Poecilia mexicana:106910880 52239:Pseudochaenichthys georgianus:11745418952670:Austrofundulus limnaeus:10652439152904:Scophthalmus maximus:118319583 52904:Scophthalmus maximus:118312266 54343:Etheostoma spectabile:116697245 55291:Polypterus senegalus:120543184 56716:Cottoperca gobio:115014268 56723:Labrus bergylta:109994465 59861:Coregonus clupeaformis:121585904 59861:Coregonus clupeaformis:121542650 63155:Archocentrus centrarchus:11578722563155:Archocentrus centrarchus:11578721864144:Anabas testudineus:113150433 64144:Anabas testudineus:113160572 66913:Ictalurus furcatus:128618702 69293:Gasterosteus aculeatus:120816881 70543:Myxocyprinus asiaticus:127417548 70543:Myxocyprinus asiaticus:127412725 72105:Sebastes umbrosus:119493801 74940:Oncorhynchus tshawytscha:11222092675038:Scatophagus argus:12406799275329:Misgurnus anguillicaudatus:12942252075352:Megalobrama amblycephala:12525910275366:Sinocyclocheilus grahami:10758708975366:Sinocyclocheilus grahami:10755929277115:Cyprinodon tularosa:119773491 80966:Acanthochromis polyacanthus:11097021780966:Acanthochromis polyacanthus:11094679780972:Amphiprion ocellaris:111570639 84645:Labeo rohita:127175905 90069:Solea solea:131472944 90988:Pimephales promelas:120460971 101364:Carassius gibelio:127971074 101364:Carassius gibelio:128027787 105023:Nothobranchius furzeri:107373510106582:Maylandia zebra:101485786 109280:Hippocampus comes:109525804 109293:Hippocampus zosterae:127599348 109905:Chelmon rostratus:121611674 113540:Scleropages formosus:108936068 113540:Scleropages formosus:108923024 118141:Megalops cyprinoides:118790659 119488:Siniperca chuatsi:122880547 134920:Pungitius pungitius:119210979 137520:Hypomesus transpacificus:124470547144197:Stegastes partitus:103363223 147949:Micropterus dolomieu:123958144 150288:Boleophthalmuspectinirostris:110159042154827:Xyrauchen texanus:127663360 154827:Xyrauchen texanus:127659470 158456:Betta splendens:114864992 160734:Plectropomus leopardus:121948100161448:Corythoichthys intestinalis:130926506161448:Corythoichthys intestinalis:130927109161450:Doryrhamphus excisus:131110374 161453:Dunckerocampus dactyliophorus:129189966161584:Syngnathus acus:119121031 161590:Syngnathus scovelli:125967294 173247:Echeneis naucrates:115037193 173247:Echeneis naucrates:115049621 175797:Silurus meridionalis:124392454 181472:Salarias fasciatus:115401214 188132:Poeciliopsis prolifica:129353865195615:Hippoglossus stenolepis:118118622195615:Hippoglossus stenolepis:118112154205130:Mastacembelus armatus:113144127 208333:Girardinichthys multiradiatus:124860564210632:Parambassis ranga:114442990 215358:Larimichthys crocea:104935689 229290:Anoplopoma fimbria:129090164 241271:Cheilinus undulatus:121515885 244447:Cynoglossus semilaevis:103390515270530:Synchiropus splendidus:128767449278164:Alosaalosa:125286341 283035:Sander light perch:116064383 293821:Epinephelus fuscoguttatus:125894322299321:Denticeps clupeoides:114768310 300413:Epinephelus moara:126389345 303518:Pundamilia nyererei:102198131 307959:Sinocyclocheilus rhinocerus:107758536307959:Sinocyclocheilus rhinocerus:107731723310571:Epinephelus lanceolatus:117260757310915:Pangasianodon hypophthalmus:113529581337641:Hemibagrus wyckioides:131365133 369639:Onychostoma macrolepis:131553405375764:Sphaeramia orbicularis:115416804375764:Sphaeramia orbicularis:115427540409849:Periophthalmus magnuspinnatus:117377737417921:Etheostoma cragini:117952205 433405:Anarrhichthys ocellatus:116400932433684:Takifugu flavidus:130531798 441366:Gouania willdenowi:114471152 451745:Nematolebias whitei:119408307 586833:Myripristis murdjan:115371519 586833:Myripristis murdjan:115366093 941984:Toxotes jaculatrix:121193628 941984:Toxotes jaculatrix:121193618 941984:Toxotes jaculatrix:121188811 992332:Triplophysa rosa:130564032 1042646:Gaduschalcogrammus:130390898 1142201:Danio aesculapii:130240883 1203425:Notolabrus celidotus:117817455 1234273:Tachysurus fulvidraco:1136548571250792:Melanotaenia boesemani:1216431261582913:Triplophysa dalaica:130438038 1606681:Puntigrus tetrazona:122357788 1608454:Sinocyclocheilus anshuiensis:1076809071608454:Sinocyclocheilus anshuiensis:1076601971676925:Paramormyrops kingsleyae:1118529871841481:Seriola lalandi dorsalis:1116600411841481:Seriola lalandi dorsalis:1116635992059687:Pseudoliparis swirei:130209981 2546036:Lampris incognitus:130110548 2546036:Lampris incognitus:130110540 2871759:Seriola aureovittata:130163207 2871759:Seriola aureovittata:130173671 3034132:Rhinichthys klamathensis goyatoka:130084764.

[0037] The fluorescent protein introduced into the sterilization gene allele is not limited. For example, green fluorescent proteins such as TurboGFP, AcGFP, TagGFP, Azami-Green, ZsGreen, EmGFP, EGFP, GFP2, HyPerGFP, PS-CFP, Dendra2, Kaede, EosFP, and KikumeGR; blue fluorescent proteins such as Sirius and EBFP; cyan fluorescent proteins such as ECFP, mTurquoise, TagCFP, AmCyan, mTFP1, MidoriishiCyan, CFP, and PS-CFP; yellow fluorescent proteins such as TagYFP, EYFP, Venus, YFP, PhiYFP, PhiYFP-m, TurboYFP, ZsYellow, and mBanana; orange fluorescent proteins such as KusabiraOrange and mOrange; and red fluorescent proteins such as TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRed-Monomer, AsRed2, mStrawberry, Dendra2, Kaede, EosFP, and KikumeGR. Proteins include far-red fluorescent proteins such as TurboFP602, mRFP1, JRed, KillerRed, mCherry, HcRed, KeimaRed, mRasberry, and mPlum.

[0038] In the case of a homozygous type, a polynucleotide sequence encoding a fluorescent protein having the same fluorescence wavelength band may be introduced into both alleles of the sterilization gene, or a polynucleotide sequence encoding a fluorescent protein having a different fluorescence wavelength band may be introduced into each of the two alleles of the sterilization gene.

[0039] Fluorescent proteins having the same fluorescence wavelength band mean that when two types of fluorescent proteins are irradiated with excitation light of an appropriate wavelength, the difference in peak wavelength of the fluorescence emitted by the two fluorescent proteins is, for example, within 30 nm, preferably within 20 nm.

[0040] "Fluorescent proteins with different fluorescence wavelength bands" refers to those in which, when two types of fluorescent proteins are irradiated with excitation light of an appropriate wavelength, the difference in peak wavelength of the fluorescence emitted by the two fluorescent proteins is, for example, 20 nm or more, preferably 30 nm or more, and more preferably 50 nm or more. "Excitation light of an appropriate wavelength" refers to excitation light recommended for each fluorescent protein.

[0041] The phrase "capable of expressing a polynucleotide sequence encoding a fluorescent protein" means that the fluorescent protein can be expressed from the polynucleotide sequence encoding the fluorescent protein under the control of a promoter.

[0042] 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. The term "indirectly" means that a spacer sequence of about 1 nucleotide (nt) to 20 nt may be included.

[0043] The promoter is not limited as long as it exerts promoter activity in individual fish. The promoter may be one that controls tissue-specific expression or one that controls systemic expression. Tissue-specific expression of fluorescent proteins preferably occurs in sites that can be observed from the body surface, such as eyes, scales, skin, and fins. Examples of promoters that control tissue-specific expression include promoters of gamma-crystallin, alpha-crystallin, beta-crystallin, delta-crystallin, rhodopsin, RPE65, IRBP, or arrestin genes, which control expression in the eye. Examples of promoters that control tissue-specific expression include promoters of sp7, keratin, or KRT5 genes. Examples of promoters that control systemic expression include promoters of actin, etc.

[0044] Heterozygous and homozygous phenotypes can be confirmed by confirming the expression of a fluorescent protein. The expression of a fluorescent protein can be confirmed by irradiating the surface of an embryo or adult fish with appropriate excitation light and observing the fluorescence. Fluorescence can be observed with the naked eye, using a fluorescence microscope, or a computer. In the case of a heterozygous phenotype, a human or computer determines the presence or absence of the fluorescent protein, and if the fluorescent protein is expressed, the phenotype can be determined to be heterozygous. In the case of a homozygous phenotype, if both alleles of the sterilization gene contain polynucleotide sequences encoding fluorescent proteins with the same fluorescence wavelength band, the phenotype can be determined to be homozygous if the fluorescence is stronger than that of the heterozygous phenotype. Furthermore, if both alleles of the sterilization gene contain polynucleotide sequences encoding fluorescent proteins with different fluorescence wavelength bands, the phenotype can be determined to be homozygous if the respective fluorescent proteins are expressed. In this case, the embryo or adult fish of the individual fish can be maintained alive.

[0045] 2. Method for Producing Individual Fish in Which At Least Part of the Function of a Sterilization Protein is Lost One embodiment relates to a method for producing individual fish in Which at least part of the function of a sterilization protein is lost (hereinafter also simply referred to as the "production method"). The production method comprises expressibly introducing a polynucleotide sequence encoding a fluorescent protein (hereinafter also simply referred to as the "?uorescent protein sequence") into at least one sterilization gene allele that encodes the sterilization protein. In this section, the explanations of the terms described in 1 above are incorporated herein by reference.

[0046] The fluorescent protein sequence can be introduced into the sterilization gene allele, for example, as a donor cassette polynucleotide linked directly or indirectly to the 3' downstream region of the promoter sequence, as described in 1 above. The fluorescent protein sequence is preferably a cDNA sequence of the fluorescent protein. The donor cassette polynucleotide preferably comprises a polyA signal in addition to the promoter sequence and the fluorescent protein sequence. The donor cassette polynucleotide can be introduced into the sterilization gene allele by known methods. Examples of introduction methods include genome editing or knock-in techniques using site-specific recombination.

[0047] Genome editing techniques include introducing proteins and nucleic acids constituting genome editing technology, or vectors encoding these, into fertilized eggs. Examples of the proteins include CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) enzymes. Specific examples of the CRISPR enzyme 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, and Csf4. Examples of the nucleic acid include crRNA and tracrRNA, or single-stranded nucleic acids in which these are linked via a linker. In this case, the nucleic acid is designed so that the base sequence that anneals to the target sequence in the crRNA is complementary to the base sequence encoding the sterilization gene. One type of nucleic acid may be used alone, or two or more types may be used in combination. Examples of genome editing systems that use CRISPR enzymes include the Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR-associated Protein 9 (CRISPR / Cas9) system. Other genome editing systems include at least one method selected from the CompoZr Zinc Finger Nuclease (ZFN) system and the TAL effector nuclease (TALEN) system. A preferred CRISPR / Cas9 system introduces Cas9 as mRNA or protein, and introduces gRNA as sgRNA or crRNA and tracrRNA.In a CRISPR / Cas9 system using a vector, the nucleic acid encoding CRISPR and the nucleic acid encoding Cas9 may be on different vectors or on a single vector. The promoter for CRISPR function is not particularly limited, but the U6 promoter is preferred. The promoter for Cas9 function is not particularly limited, but a promoter expressed in mammalian cells, such as the cytomegalovirus promoter, is preferred. Commercially available vectors, such as the pX330-U6-Chimeric_BB-CBh-hSpCas9 vector, can be used as the CRISPR / Cas9 system.

[0048] The sequence that targets the sterilization gene (hereinafter also referred to as the "target sequence") to be incorporated into the CRISPR sequence is not limited, as long as it can be incorporated into a guide RNA (also referred to as gRNA, sgRNA, or crRNA) by the CRISPR / Cas9 system and transcribed, or introduced into a cell as a guide RNA containing a sequence complementary to the target sequence to recombine the sterilization gene. Generally, it is said that the target sequence can be a sequence of about 20 bases upstream of the 5'-side of the base sequence "NGG" (PAM sequence: N is any of the partial nucleotides A, G, T, or C) present in the sterilization gene. Target sequences can be designed using known design tools such as Optimized CRISPR design tool (Massachusetts Institute of Technology, ZhangLab webpage (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 Finger Consortium)), Cas9 design (http: / / cas9.cbi.pku.edu.cn (Peking University)), CRISPRdirect (http: / / crispr.dbcls.jp (University of Tokyo)), CRISPR-P (http: / / cbi.hzau.edu.cn / crispr / (Huazhou Agricultural University)), and CRISPR RGEN Tools (http: / / www.rgenome.net / (Seoul National University)).

[0049] In addition, if single-nucleotide polymorphisms (SNPs) exist in the PAM sequence, it is preferable to avoid such sequences. If the SNPs of an individual are known for the target sequence, it is preferable to optimize the sequence for each SNP. The 5'-terminal region of the target sequence may be shortened by 1, 2, 3, or 4 bases, preferably 1, 2, or 3 bases.

[0050] The CRISPR / Cas9 system may be introduced into cells as a vector, or may be introduced into cells in combination with artificially synthesized or in vitro transcribed gRNA, crRNA, trans-activating crRNA (tracrRNA), and RNA encoding Cas9. Alternatively, the Cas9 protein and guide RNA may be introduced into cells in combination.

[0051] Furthermore, in the genome editing system, donor oligo DNA such as single-stranded oligonucleotides (ssODNs) may be co-introduced. ssODNs can be designed according to known methods.

[0052] The genome editing system can be injected by microinjection into the cytoplasm of a fertilized egg, preferably a one-cell stage fertilized egg. For example, when introducing Cas9 protein, the amount injected per fertilized egg can be approximately 5 pg to 100 pg, preferably 10 pg to 80 pg, and more preferably 10 pg to 50 pg. In this case, the guide RNA can be injected in the range of approximately 0.1 pg to 50 pg, preferably 0.5 pg to 20 pg, and more preferably 1 pg to 5 pg. For a method of introducing a mutation using genome editing technology, see, for example, Example 1 described below.

[0053] Examples of gene site-specific recombination methods include methods using at least one system selected from the Cre / loxP system, the Flp / FRT system, the Dre / rox system, and the PhiC31 integrase system, or variants of the above gene site-specific recombination methods.

[0054] 3. Genotype Identification Method One embodiment relates to a genotype identification method (hereinafter also referred to simply as "identification method"). This method includes mating male and female fish individuals described in 1 above, each of which has a polynucleotide sequence encoding a fluorescent protein in only one sterilization gene allele, and observing the expression of the fluorescent protein in embryos or adult fish obtained by mating. In the identification method, the male and female fish mated may be of the same species or of interbreedable different species.

[0055] When the heterozygous fish individuals described in 1. above are mated with each other, the next generation can have three genotypes: wild type, heterozygous, and homozygous, according to Mendel's laws of inheritance. These genotypes can be identified by the expression of fluorescent proteins.

[0056] The method for observing the expression of the fluorescent protein and determining whether it is a heterozygote or a homozygote is as described above in 1. In the case of the wild type, no expression of the fluorescent protein is observed.

[0057] 4. Method for Transplanting Germ Cells One embodiment relates to a method for transplanting germ cells, comprising transplanting germ cells of a fish species different from the homozygous fish individual described in 1 above into the homozygous fish individual. Here, the fish species different from the homozygous fish individual described in 1 above is not limited as long as it is compatible with the fish body of the homozygous fish individual.

[0058] Transplantation methods are known. For example, there is a method for producing fish gametes using a surrogate parent fish technique. The donor undifferentiated germ cells used in the surrogate parent fish technique include primordial germ cells obtained from the gonads before sexual differentiation, spermatogonia obtained from the testes, and oogonia obtained from the ovaries.

[0059] Undifferentiated germ cells can be obtained from a donor by conventional methods from donor tissues appropriate for the differentiation stage of the desired undifferentiated germ cells. For example, undifferentiated germ cells can be obtained by removing pre-sex differentiation gonads or post-sex differentiation tissues, such as testes or ovaries, from the donor and dissociating the tissue into individual cells by physical abrasion or treatment with protease. The dissociated individual cells can be isolated, for example, using marker antibodies or a cell sorter.

[0060] The undifferentiated germ cells can be obtained from frozen or live individuals, but to increase the success rate of the surrogate broodstock technique, it is preferable to obtain the undifferentiated germ cells from live individuals.

[0061] When donor cells are introduced into a recipient, they are preferably introduced into an individual at the embryonic or larval stage before the recipient's immune system is fully functional. Introduction can be performed using a manipulator such as a micromanipulator, an electric scalpel, or a laser scalpel. Introduction can be performed into any tissue or site of the recipient, and examples of the tissue or site to be introduced include the epidermis or peritoneal cavity. There are no particular limitations on the number of cells introduced into an individual at the embryonic or larval stage; for example, 1 to 100,000 cells can be introduced.

[0062] The present invention will be described in more detail below with reference to examples. However, the present invention should not be construed as being limited to these examples. The sequences corresponding to the SEQ ID NOs used in the examples are shown in the sequence list below.

[0063] I. Preparation of Cassette Polynucleotides for Fluorescent Protein Expression First and second cassette polynucleotides capable of expressing fluorescent proteins under the control of a crystallin promoter were prepared. The first cassette polynucleotide contained an EGFP cDNA sequence (SEQ ID NO: 2) downstream of the mouse gamma-crystallin promoter (SEQ ID NO: 1), followed by a Simian virus 40 (SV40) polyA signal (SEQ ID NO: 3) downstream of that. Hereinafter, this cassette polynucleotide is referred to as Crystallin promoter-EGFP-SV40 polyA, and its polynucleotide sequence is shown in SEQ ID NO: 4. The second cassette polynucleotide contained an mCherry cDNA sequence (SEQ ID NO: 5) downstream of the mouse gamma-crystallin promoter (SEQ ID NO: 1), followed by an SV40 polyA signal (SEQ ID NO: 3) downstream of that. Hereinafter, this donor cassette polynucleotide is referred to as Crystallin promoter-mCherry-SV40 polyA, and its polynucleotide sequence is shown in SEQ ID NO: 6.

[0064] II. Example Using Tilapia as a Test Species 1. Preparation of Donor Construct In order to label germ cell-deficient individuals using tilapia as a test species by genome editing, a donor construct was prepared for introducing the cassette polynucleotide described in I. above into tilapia. The sequence map of the donor construct is shown in Figure 3.

[0065] (1) A 5-prime Onil dnd homology having the sequence shown in SEQ ID NO:7 and a 3-prime Onil dnd homology having the sequence shown in SEQ ID NO:8 were isolated from tilapia dnd (SEQ ID NO:9).

[0066] (2) An Onil Crystallin promoter-EGFP-SV40 polyA vector (nucleotide sequence shown in SEQ ID NO: 13) was prepared, which contains the gRNA#1 guide RNA sequence shown in SEQ ID NO: 10 for vector cleavage, 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 Ori sequence which is the origin of replication of the plasmid in E. coli shown in SEQ ID NO: 11, and the AmpR sequence which is the ampicillin resistance gene shown in SEQ ID NO: 12. Additionally, an Onil Crystallin promoter-mCherry-SV40 polyA vector was prepared by replacing the Crystallin promoter-EGFP-SV40 polyA shown in SEQ ID NO: 4 with the Crystallin promoter-mCherry-SV40 polyA shown in SEQ ID NO: 6 (nucleotide sequence shown in SEQ ID NO: 14).

[0067] 2. Introduction of a cassette polynucleotide into the dnd gene region and confirmation test (1) Introduction of a cassette polynucleotide into the dnd gene region 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, a cassette polynucleotide was introduced into the Tirapila genome by the knock-in method, a genome editing method using the CRISPR-Cas9 system.

[0068] Tilapia fertilized eggs were microinjected with 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 onil crystallin promoter-mCherry-SV40 polyA vector, and 50 ng / μl of gRNA#1 (SEQ ID NO: 10) cleaving the construct. (2) Confirmation of fluorescent protein expression

[0069] Microinjected tilapia fertilized eggs were cultured at 28°C, and the expression of GFP or mCherry was observed under a stereomicroscope at 4- to 6-day-old embryos. Expression of the fluorescent proteins was also confirmed in the embryonic bodies and eyes of adult fish that developed from the microinjected tilapia fertilized eggs (Figure 4).

[0070] To confirm that the cassette polynucleotide had been correctly introduced into the dnd region, genomic DNA was extracted from individuals in which fluorescence was observed in the above observation, and the genomic sequence was confirmed by PCR and nucleotide sequencing.

[0071] As shown in Figure 5, PCR primers were designed outside the homology region and between the cassette polynucleotides during knock-in using CRISPR-Cas9. The asterisks in Figure 5 indicate the confirmed sites. PCR was performed using Onil_dnd_5-prime_Fw (SEQ ID NO: 17) and cryP_Rv (SEQ ID NO: 18) to confirm the 5' side (upstream side). Also, PCR was performed using dnd gfp The samples were GFP_Fw (SEQ ID NO: 19), Onil_dnd_3-prime_Rv (SEQ ID NO: 20), and dnd rfp PCR was performed using RFP_Fw (SEQ ID NO: 21) and Onil_dnd_3-prime_Rv (SEQ ID NO: 20) as samples. Each PCR product was electrophoresed on a 1.5% agarose gel to confirm the presence and size of bands, confirming that the desired amplification products were obtained (Figure 6).

[0072] In addition, the above PCR products were subcloned and subjected to Sanger sequencing analysis using the Onil_dnd_5-prime_Fw and Onil_dnd_3-prime_Rv primers, respectively.

[0073] dnd gfp The sequences of the cassette polynucleotide introduction sites of the individuals are shown in Figure 7. Figure 7(A) shows the 5' sequence analyzed using the Onil_dnd_5-prime_Fw primer. Figure 7(B) shows the 3' sequence analyzed using the Onil_dnd_3-prime_Rv primer.

[0074] dnd rfp The sequences of the cassette polynucleotide introduction sites in the individual plants are shown in Figure 8. Figure 8(A) shows the 5' sequence analyzed using the Onil_dnd_5-prime_Fw primer. Figure 8(B) shows the 3' sequence analyzed using the Onil_dnd_3-prime_Rv primer. PCR and sequencing demonstrated that the cassette polynucleotide had been correctly inserted into the target site.

[0075] III. Example using the pufferfish as a test species 1. Donor construct In order to label germ cell-deficient individuals using the pufferfish as a test species by genome editing, a donor construct was created to introduce the cassette polynucleotide described in I. above into the pufferfish. The sequence map of the donor construct is shown in Figure 9.

[0076] (1) 5-prime Talb dnd homology (SEQ ID NO: 22) and 3-prime Talb dnd homology (SEQ ID NO: 23) were isolated from the pufferfish dnd (SEQ ID NO: 24).

[0077] (2) A Talb Crystallin promoter-EGFP-SV40 polyA vector (nucleotide sequence shown in SEQ ID NO: 25) was prepared, which contained 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. Additionally, a Talb Crystallin promoter-mCherry-SV40 polyA vector (nucleotide sequence shown in SEQ ID NO: 26) was prepared, in which the Crystallin promoter-EGFP-SV40 polyA shown in SEQ ID NO: 4 was replaced with the Crystallin promoter-mCherry-SV40 polyA shown in SEQ ID NO: 6. 2. Introduction of a cassette polynucleotide into the dnd gene region and confirmation test (1) Introduction of a cassette polynucleotide into the dnd gene region

[0078] 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 was introduced into the Kusafugu genome by a knock-in method, a genome editing method using the CRISPR-Cas9 system.

[0079] Fertilized eggs of Kusafugu were microinjected with 500 ng / μl of Cas9 protein; 50 ng / μl of gRNA#3 (SEQ ID NO: 28) targeting exon 4 of the Kusafugu dnd gene (SEQ ID NO: 27); 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 (SEQ ID NO: 10) cleaving the construct.

[0080] (2) Confirmation of fluorescent protein expression Microinjected pufferfish fertilized eggs were cultured at 20°C, and the expression of GFP or RFP was observed under a stereomicroscope in embryos 3 to 6 days old. In addition, the expression of fluorescent proteins was confirmed in the embryonic bodies and eyes of adult fish that developed from the microinjected pufferfish fertilized eggs.

[0081] To confirm that the cassette polynucleotide had been correctly introduced into the dnd region, genomic DNA was extracted from individuals in which fluorescence was observed in the above observation, and the genomic sequence was confirmed by PCR and nucleotide sequencing.

[0082] As shown in Figure 11, PCR primers were designed outside the homology region and between the cassette polynucleotides used in CRISPR-Cas9 knock-in. The asterisks in Figure 11 indicate the confirmation sites. PCR was performed using Talb_dnd_5-prime_Fw (SEQ ID NO: 29) and cryP_Rv (SEQ ID NO: 18) to confirm the 5' end (upstream side). PCR was also performed using polyA-Fw (SEQ ID NO: 30) and Talb_dnd_3-prime_Rv (SEQ ID NO: 31) to confirm the 3' end (downstream side). Each PCR product was electrophoresed on a 1.5% agarose gel to confirm the presence and size of bands, confirming that the desired amplification products were obtained (Figure 12).

[0083] In addition, the above PCR products were subcloned and subjected to Sanger sequencing analysis using the Talb_dnd_5-prime_Fw and Talb_dnd_3-prime_Rv primers, respectively.

[0084] dnd gfp The sequences of the cassette polynucleotide introduction sites of the individuals are shown in Figure 13. Figure 13(A) shows the 5' sequence analyzed using the Talb_dnd_5-prime_Fw primer. Figure 13(B) shows the 3' sequence analyzed using the Talb_dnd_3-prime_Rv primer.

[0085] dnd rfp The sequence of the cassette polynucleotide introduction site in each individual is shown in Figure 8. Figure 14(A) shows the 5' sequence analyzed using the Talb_dnd_5-prime_Fw primer. Figure 14(B) shows the 3' sequence analyzed using the Talb_dnd_3-prime_Rv primer. PCR and sequencing demonstrated that the cassette polynucleotide had been correctly inserted into the target site.

Claims

1. A fish individual in which at least part of the function of a sterilization protein is lost, the fish having at least one allele of a sterilization gene encoding a sterilization protein that exhibits sterility due to the loss of at least part of its function, and which has a polynucleotide sequence encoding a fluorescent protein in an expressible state.

2. An individual fish as described in claim 1, which has polynucleotide sequences encoding the fluorescent protein in both alleles of the sterility gene and is reproductively deficient.

3. An individual fish as described in claim 2, wherein both alleles of the sterilization gene have polynucleotide sequences encoding fluorescent proteins having the same fluorescent wavelength band.

4. An individual fish as described in claim 2, wherein each of the two alleles of the sterilization gene has a polynucleotide sequence encoding a fluorescent protein having a different fluorescent wavelength band.

5. The fish individual described in claim 1, wherein the sterilization gene is a Dead End (dnd) gene.

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

7. A method for producing a fish individual lacking at least a portion of the function of a sterilization protein that exhibits sterility due to the loss of at least a portion of that function, the method comprising expressibly introducing a polynucleotide sequence encoding a fluorescent protein into at least one of the sterilization gene alleles that encode the sterilization protein.

8. The method of claim 7, wherein the introduction is performed by a knock-in technique using genome editing or gene site-specific recombination.

9. The method of claim 8, wherein the genome editing method uses 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 TAL effector nuclease (TALEN) system.

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

11. A polynucleotide comprising an array having, within a portion of the sequence of a sterility gene encoding a sterility protein that exhibits sterility due to the loss of at least a portion of its function, a polynucleotide sequence that expressibly has a polynucleotide sequence encoding a fluorescent protein.

12. A method for transplanting germ cells, comprising transplanting germ cells of a fish species different from the fish individual according to claim 2 into said fish individual.

13. A method for identifying genotypes comprising mating male and female fish individuals of the fish described in claim 1, which have a polynucleotide sequence capable of expressing a fluorescent protein in only one of the sterilization gene alleles, and observing the expression of the fluorescent protein in the embryo or adult fish obtained by mating.

Images