Fish individuals lacking at least some of the function of sterilizing proteins

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

Technical Field

[0001] The present invention relates to a fish individual in which at least a part of the function of a sterilizing protein is defective, a method for producing the fish individual, a polynucleotide, a method for transplanting germ cells, and a method for identifying a genotype.

Background Art

[0002] The Dead End (dnd) gene is known as one of the sterilizing genes related to sterilization. It is known that an individual lacking the function of a protein encoded by a sterilizing gene (hereinafter also referred to as "sterilizing protein") does not form germ cells and becomes sterile.

Summary of the Invention

Problems to be Solved by the Invention

[0003] In a conventional method for artificially producing a germ cell-deficient individual by utilizing the loss of function of a sterilizing protein, as shown in FIG. 1, heterozygotes in which the function of the sterilizing protein is heterozygously defective are crossed to produce a homozygote in which the function of the sterilizing gene is homozygously defective. The next generation born from crossing heterozygotes is one of wild type, homozygous type, and heterozygous type, but it was impossible to identify these genotypes by appearance. For this reason, in order to identify the genotype, it was necessary to collect a part of the fish body and perform gene analysis.

[0004] As shown in FIG. 2, an object of the present invention is to provide a fish individual capable of identifying the genotype of a sterilizing protein by appearance without collecting a part of the fish body when producing a germ cell-deficient individual.

Means for Solving the Problems

[0005] Item 1. A fish individual in which at least part of the function of a sterilization protein is lost, wherein at least one of the sterilization gene alleles encoding a sterilization protein that exhibits infertility due to a deficiency in at least part of its function has the capability to express a polynucleotide sequence encoding a fluorescent protein. Section 2. The fish individual described in item 1, wherein both alleles of the sterilization gene have polynucleotide sequences encoding the fluorescent protein, and the individual is deficient in reproductive capacity. Section 3. The fish individual according to item 2, wherein both alleles of the sterilization gene have polynucleotide sequences encoding a fluorescent protein having the same fluorescence wavelength band. Section 4. The fish individual according to item 2, wherein each of the two alleles of the sterilization gene has a polynucleotide sequence encoding a fluorescent protein having a different fluorescence wavelength band. Section 5. The fish individual described in item 1, wherein the aforementioned sterilization gene is a Dead End (dnd) gene. Section 6. The fish individual described in item 1, wherein the expression site of the fluorescent protein is the eye. Section 7. A method for producing fish individuals in which at least a portion of the function of a sterilizing protein that exhibits infertility due to a deficiency in at least a part of its function is lost, This includes introducing a polynucleotide sequence encoding a fluorescent protein into at least one of the infertility gene alleles encoding an infertility protein in an expressible manner, Manufacturing method. Section 8. The method for producing the product as described in item 7, wherein the introduction is performed by a genome editing method or a knock-in method using a gene site-specific recombination method. Section 9. The method for creating the genome according to Section 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 TAL effector nuclease (TALEN) system. Section 10. The method for producing according to item 8, wherein the gene site-directed 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. Section 11. A polynucleotide comprising a sequence within a portion of a sterilization gene encoding a sterilization protein that exhibits infertility due to a deficiency in at least part of its function, and which contains a polynucleotide sequence capable of expressing a polynucleotide sequence encoding a fluorescent protein. Section 12. This includes transplanting germ cells of a different fish species into a fish individual as described in item 2. A method of transplanting germ cells. Section 13. A method for identifying a genotype, comprising: mating male and female fish individuals, among those described in item 1, in which only one of the sterilization gene alleles is capable of expressing a polynucleotide sequence encoding a fluorescent protein; and observing the expression of the fluorescent protein in the embryo or adult fish obtained by mating. [Effects of the Invention]

[0006] In creating germ cell-deficient individuals, it is possible to provide fish individuals in which the genotype of the sterilizing protein can be identified by their appearance. [Brief explanation of the drawing]

[0007] [Figure 1] The conventional method is shown. [Figure 2] Shows the outline of the present invention. [Figure 3] Shows the gene transfer map of tilapia. [Figure 4] Shows the expression of fluorescent protein in the fertilized eggs, embryos and adult fish eyes of tilapia. [Figure 5] Shows the positions of primers designed to confirm that the cassette polynucleotide was correctly introduced into the dnd gene of tilapia. [Figure 6] Shows the results of PCR using the primers shown in Figure 5. [Figure 7] Shows the sequences of the cassette polynucleotide introduction sites of tilapia dndgfp individuals. (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. [Figure 8] Shows the sequences of the cassette polynucleotide introduction sites of tilapia dndrfp individuals. (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. [Figure 9] Shows the gene transfer map of fugu. [Figure 10] Shows the expression of fluorescent protein in the fertilized eggs, embryos and adult fish eyes of fugu. [Figure 11] Shows the positions of primers designed to confirm that the cassette polynucleotide was correctly introduced into the dnd gene of fugu. [Figure 12] Shows the results of PCR using the primers shown in Figure 11. [Figure 13] Shows the sequences of the cassette polynucleotide introduction sites of fugu dndgfp individuals. (A) shows the 5'-side sequence analyzed using the Talb_dnd_5-prime_Fw primer. (B) shows the 3'-side sequence analyzed using the Talb_dnd_3-prime_Rv primer. [Figure 14] The sequence of the cassette polynucleotide introduction site of the dndrfp individuals of pufferfish is shown. (A) shows the 5'-side sequence analyzed using the Talb_dnd_5-prime_Fw primer. (B) shows the 3'-side sequence analyzed using the Talb_dnd_3-prime_Rv primer.

Mode for Carrying Out the Invention

[0008] 1. A fish individual in which at least a part of the function of the sterilizing protein is defective One embodiment relates to a fish individual (hereinafter, also simply referred to as "fish individual") in which at least a part of the function of a sterilizing protein is defective, which shows sterility due to at least a part of its function being defective. The fish individual has a polynucleotide sequence encoding a fluorescent protein expressibly in at least one of the sterilizing gene alleles encoding the sterilizing protein.

[0009] "A fish individual in which at least a part of the function of the sterilizing protein is defective" may include, as a genotype, a heterozygous type in which the polynucleotide sequence encoding the fluorescent protein exists only in one allele of the fish individual, and a homozygous type in which the polynucleotide sequence encoding the fluorescent protein exists in both alleles.

[0010] In the heterozygous type, the function of the sterilizing protein encoded by the one allele is defective by introducing the polynucleotide sequence encoding the fluorescent protein into the allele. The heterozygous type is, as a phenotype, an individual that retains at least a part of the germ cells (also referred to as "heterozygous defective individual" in this specification), and is fertile both in male and female.

[0011] Homozygous individuals are characterized by the introduction of polynucleotide sequences encoding fluorescent proteins into the alleles of both sterilization genes, resulting in a loss of function in both alleles of the sterilization proteins. Homozygous individuals are characterized phenotypic individuals with a substantial lack of germ cells (also referred to herein as "homozygous deficient individuals"), and both males and females are infertile. Germ cell deficiency can be confirmed by histological observation. For example, if the number of germ cells (primordial germ cells, spermatogonia, oogonia, spermatocytes, oocytes, spermatocytes, egg cells, eggs, or sperm) is reduced to 50%, 30%, 20%, 10%, 5%, 3%, or 1% compared to a wild-type individual, it can be determined that germ cells are deficient.

[0012] In this specification, fish are not limited. The term "fish" is not particularly limited. In this specification, "fish" may include saltwater fish, freshwater fish, brackish water fish, circadian migratory fish, and the like.

[0013] The aforementioned fish include, for example, 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, and Carangidae. Examples of fish include those belonging to the Carangidae family, Lateolabrax, Moronidae, Latidae, Rachycentridae, Cynoglossidae, Anguillidae, and Congridae families.

[0014] The aforementioned fish of the family Paralichthyidae may include, for example, Paralichthys olivaceus (TEMMINCK et SCHLEGEL), flounder belonging to the genus Paralichthys (such as the Japanese flounder, the southern flounder, the swan flounder, the starry flounder, the spotted flounder, the Taiwanese flounder, etc.), flounder belonging to the genus Paralichthys (such as the Japanese flounder), flounder belonging to the genus Paralichthys (such as the Japanese flounder), flounder belonging to the genus Paralichthys (such as the Japanese flounder), flounder belonging to the genus Paralichthys (such as the Japanese flounder), etc. Preferably, Paralichthys olivaceus (TEMMINCK et SCHLEGEL).

[0015] Examples of the aforementioned pufferfish include species of the genus *Takifugu*, such as *Takifugu rubripes*, *Takifugu porphyreus*, and *Takifugu niphobles*; and species of the genus *Lagocephalus*, such as *Lagocephalus wheeleri*. The aforementioned boxfish of the family Ostracidae may include, for example, fish of the genus Ostracion, such as the boxfish (Ostracion immaculatus).

[0016] The aforementioned fish of the Sparidae family include, for example, the genus Pagrus, such as the red sea bream (Pagrus major) and the Australian red sea bream (Pagrus auratus); the genus Acanthopagrus, such as the black sea bream (Acanthopagrus schlegelii) and the yellowfin sea bream (Acanthopagrus latus); and the genus Dentex, such as the yellowback sea bream (Dentex tumifrons). This may include fish of the genus Sparus, such as the European sea bream (Sparus aurata).

[0017] The aforementioned salmonid fish include, for example, fish of the genus Oncorhynchus such as rainbow trout (Oncorhynchus mykiss), king salmon (Oncorhynchus tshawytscha), cherry salmon (Oncorhynchus masou), Satsuki salmon (Oncorhynchus masou), kunimasu (Oncorhynchus kawamurae), pink salmon (Oncorhynchus gorbuscha), and salmon (Oncorhynchus keta); fish of the genus Salmo 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 char (Salvelinus This may include fish of the genus Salvelinus, such as Salvelinus leucomaenis, Salvelinus fontinalis, and Salvelinus namaycush; and fish of the genus Hucho, such as Parahucho perryi.

[0018] The aforementioned carp family fish may include, for example, Gnathopogon caerulescens, Hypophthalmichthys molitrix, Cyprinus carpio, Ctenopharyngodon idellus, Hypophthalmichthys nobilis, Carassius carassius, Cyprinus catla, Mylopharyngodon piceus, Cirrhinoceros molitorella, Cirrhinoceros cirrhosus, Catla catla, Labeo rohita, Megalobrama amblycephala, and others.

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

[0020] The aforementioned fish of the superfamily Siluroidea may include, for example, the Japanese catfish (Silurus asotus), the Biwa catfish (Silurus biwaensis), the Iwatoko catfish (Silurus lithophilus), the European catfish (Silurus glanis), the fin catfish (Clarias fuscus), the walking catfish (Clarias batrachus), and others.

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

[0022] The aforementioned grouper species include, for example, the common grouper (Epinephelus septemfasciatus), the longtooth grouper (Epinephelus bruneus), the yellowtail grouper (Epinephelus akaara), the spotted grouper (Epinephelus malabaricus), the white grouper (Epinephelus aeneus), the black-spotted blue grouper (Epinephelus amblycephalus), the large-spotted grouper (Epinephelus areolatus), the yellow-spotted grouper (Epinephelus bleekeri), the white grouper (Epinephelus bontoides), (Epinephelus chlorostigma), the brown-spotted grouper (Epinephelus coiodes), the red grouper (Epinephelus fasciatus), the red-spotted grouper (Epinephelus fuscoguttatus), and the starry grouper (Epinephelus... Epinephelus species such as 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., Epinephelus species such as Cromileptes altivelis, Plectropomus This may include fish of the genus Plectropomus, such as *Plectropomus leopardus*, and hybrids of fish within the Serranidae family.

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

[0024] The aforementioned fish of the family Adrianichthyidae may include, for example, fish of the genus Oryzias, such as the Japanese rice fish (Oryzias latipes, Oryzias sakaizumii) and the Javan rice fish (Oryzias javanicus).

[0025] The aforementioned fish of the family Monacanthidae may include, for example, fish of the genus Stephanolepis, such as the triggerfish (Stephanolepis cirrhifer), and fish of the genus Thamnaconus, such as the filefish (Thamnaconus modestus).

[0026] The aforementioned Osmeridae fish may include, for example, fish from the Plecoglossinae subfamily such as the ayu (Plecoglossus altivelis), the Hypomesinae subfamily such as the smelt (Hypomesus nipponensis) and pond smelt (Hypomesus japonicus), and the Osmerinae subfamily such as the smelt (Osmerus mordax dentex), capelin (Spirinchus lanceolatus), and icefish (Spirinchus lanceolatus).

[0027] The aforementioned fish of the family Scombridae include, for example, the genus Scombrini such as Pacific mackerel (Scomber japonicus), Atlantic mackerel (Scomber scombrus), and spotted mackerel (Scomber australasicus); the genus Thunnini such as Pacific bluefin tuna (Thunnus orientalis), Atlantic bluefin tuna (Thunnus mackerel), southern bluefin tuna (Thunnus maccoyii), bigeye tuna (Thunnus obesus), yellowfin tuna (Thunnus albacares), albacore tuna (Thunnus alalunga), and longtail tuna (Thunnus tonggol); the genus Euthynnus such as skipjack tuna (Euthynnus affinis) and Atlantic yellowtail (Euthynnus alletteratus); and skipjack tuna (Katsuwonus) This may include fish of the genus Katsuwonus (such as pelamis), fish of the genus Scomberomorini, fish of the genus Auxis, fish of the genus Sardini, and fish of the genus Gymnosarda.

[0028] The aforementioned flatfish may include, for example, the flatfish (Pseudopleuronectes herzensteini), the marbled flatfish (Pleuronectes yokohamae), the stone flounder (Kareius bicoloratus), the Pacific halibut (Hippoglossus stenolepis), and the Japanese flounder (Verasper moseri).

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

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

[0031] The aforementioned 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 aforementioned fish of the family Rachycentridae may include, for example, fish such as amberjack (Rachycentron canadum).

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

[0033] The aforementioned eel species may include, for example, the Japanese eel (Anguilla japonica) and the European eel (Anguilla anguilla). The aforementioned conger eel species may include, for example, the Japanese conger eel (Conger myriaster) and the black conger eel (Conger japonicus). Preferably, the fish are from the families Cichlidae and Tetraodontidae, specifically tilapia and grass pufferfish.

[0034] In this specification, the fish may be a fixed species or a hybrid. The hybrid may include, for example, a hybrid resulting from intergeneric hybridization.

[0035] "Fish" preferably refers to farmed fish. Furthermore, "farmed fish" may include fish that are farmed for purposes such as food, breeding, or ornamental purposes.

[0036] One example of a gene that causes infertility is the Dead End (dnd) gene. The Gene IDs registered with the NCBI for major fish infertility 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 light: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:Salmo trutta:115172323 8036:Salvelinus alpinus:111965708 8038: Salvelinus fountain: 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: River perch: 120566341 8175:Sparus aurata:115568425 8177:Acanthopagrus latus:119007972 8187: Late calcarifer: 108895524 8208:Notothenia coriiceps:104953557 8218: Gymnodraco acuticeps:117550442 8236: Thunnus albacares: 122990905 8240:Thunnus maccoyii:121902261 8245:Xiphias sword:120794382 8245:Xiphias sword:120784879 8255: Paralichthys olivaceus: 109627263 8255: Paralichthys olivaceus: 109629018 8262: Pleuronectes platessa: 128436393 8262: Pleuronectes platessa: 128446010 8267:Hippoglossus hippoglossus:1177690828267:Hippoglossus hippoglossus:11776969813013:Clarias gariepinus:128512679 13489:Dicentrarchus labrax:127375252 13676: Japanese mackerel: 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 red: 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:120720608 43700: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 clupeaiformis: 121542650 63155:Archocentrus centrarchus:11578722563155:Archocentrus centrarchus:11578721864144:Anabas testudineus:113150433 64144:Anabas testudineus:113160572 66913:Ictalurus forcatus:128618702 69293: Gasterosteus aculeatus: 120816881 70543:Myxocyprinus asiaticus:127417548 70543:Myxocyprinus asiaticus:127412725 72105:Sharkfish:119493801 74940:Oncorhynchus tshawytscha:11222092675038:Scatophagus argus:124067992 75329: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 gibelius:127971074 101364:Carassius gibelius:128027787 105023:Nothobranchius furzeri:107373510106582:Maylandia zebra:101485786 109280: Count Hippocampus: 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:Boleophthalmus pectinirostris: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:Southern catfish:124392454 181472:Salarias fasciatus:115401214 188132:Poeciliopsis prolifica:129353865195615:Hippoglossus stenolepis:118118622195615:Hippoglossus stenolepis:118112154205130:Mastacembelus armatus:113144127 208333:Girardinichthys multiradiatus:124860564210632:Parambassis range:114442990 215358:Larimichthys crocea:104935689 229290:Anoplopoma fimbria:129090164 241271: Cheilinus undulatus: 121515885 244447:Cynoglossus semilaevis:103390515270530:Synchiropus splendidus:128767449278164:Alosa alosa:125286341 283035: Sander pikeperch: 116064383 293821:Epinephelus fuscoguttatus:125894322299321:Denticeps clupeoides:114768310 300413:Epinephelus moara:126389345 303518:Pundamilia nyererei:102198131 307959:Sinocyclocheilus rhinocerous:107758536307959:Sinocyclocheilus rhinocerous: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:Gadus chalcogrammus: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] There are no restrictions on the fluorescent proteins introduced into the infertility gene alleles. For example, 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; and red fluorescent proteins such as TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRed-Monomer, AsRed2, mStrawberry, Dendra2, Kaede, EosFP, KikumeGR. Examples of proteins include farred fluorescent proteins such as TurboFP602, mRFP1, JRed, KillerRed, mCherry, HcRed, KeimaRed, mRaspberry, and mPlum.

[0038] In the case of homozygous fertility, polynucleotide sequences encoding fluorescent proteins with the same fluorescence wavelength band may be introduced into both alleles of the fertility gene, or polynucleotide sequences encoding fluorescent proteins with different fluorescence wavelength bands may be introduced into each of the two alleles of the fertility gene.

[0039] Fluorescent proteins having the same fluorescence wavelength band are defined as two fluorescent proteins whose peak fluorescence wavelengths differ by, for example, within 30 nm, preferably within 20 nm, when irradiated with excitation light of an appropriate wavelength.

[0040] Fluorescent proteins having different fluorescence wavelength bands are defined as two fluorescent proteins whose peak fluorescence wavelengths differ from each other when irradiated with excitation light of an appropriate wavelength, for example, by 20 nm or more, preferably 30 nm or more, and more preferably 50 nm or more. The appropriate excitation wavelength refers to the excitation light recommended for each fluorescent protein.

[0041] "Ability to express polynucleotide sequences encoding fluorescent proteins" means that fluorescent proteins can be expressed from polynucleotide sequences encoding fluorescent proteins under the control of a promoter.

[0042] The polynucleotide sequence encoding the fluorescent protein is directly or indirectly ligated to the 3' downstream region of the promoter sequence for fluorescent protein expression. "Indirectly" means that a spacer sequence of approximately 1 to 20 nucleotides (nt) may be included.

[0043] The promoter is not limited as long as it exhibits promoter activity in the fish individual. The promoter may regulate tissue-specific expression or systemic expression. Tissue-specific fluorescent protein expression is preferably observed in areas observable from the body surface, such as the eyes, scales, skin, and fins. Examples of promoters that regulate tissue-specific expression include gamma-crystallin, alpha-crystallin, beta-crystallin, delta-crystallin, rhodopsin, RPE65, IRBP, or the arrestin gene, which regulate expression in the eye. Examples of promoters that regulate tissue-specific expression include sp7, keratin, or the KRT5 gene. Examples of promoters that regulate systemic expression include the actin promoter.

[0044] Heterozygous and homozygous phenotypes can be confirmed by checking the expression of fluorescent proteins. Fluorescent protein expression can be determined by irradiating the embryo or adult fish body surface with appropriate excitation light and observing the fluorescence. Fluorescence observation may be performed by a human with the naked eye, or using a fluorescence microscope or microscope. Alternatively, it may be observed by a computer. In the case of heterozygosity, a human or computer can determine the presence or absence of fluorescent protein; if fluorescent protein expression is present, it can be determined to be heterozygous. In the case of homozygosity, if both alleles of the infertility gene have polynucleotide sequences encoding fluorescent proteins with the same fluorescence wavelength band, it can be determined to be homozygous if the fluorescence intensity is stronger than that of heterozygosity. Furthermore, if both alleles of the infertility gene have polynucleotide sequences encoding fluorescent proteins with different fluorescence wavelength bands, it can be determined to be homozygous if both fluorescent proteins are expressed. In this case, the embryo or adult fish can be kept alive.

[0045] 2. Method for producing fish individuals lacking at least some of the functions of sterilizing proteins. One embodiment relates to a method for producing fish individuals lacking the function of at least some of the sterilization proteins (hereinafter also simply referred to as the "production method"). The production method includes introducing a polynucleotide sequence encoding a fluorescent protein (hereinafter also simply referred to as the "fluorescent protein sequence") into at least one of the sterilization gene alleles encoding the sterilization protein in an expressible manner. In this section, the definitions of terms described in item 1 above shall apply hereafter.

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

[0047] Genome editing methods include introducing proteins and nucleic acids that constitute genome editing technology, or vectors encoding them, into a fertilized egg. An example of such a protein is the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) enzyme. Specifically, examples of the 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. The nucleic acids mentioned above include, for example, crRNA and tracrRNA, or single-stranded nucleic acids in which these are linked via a linker. In this case, the nucleic acids are designed such that, for example, the base sequence that anneals with the target sequence in the crRNA is a base sequence complementary to the base sequence encoding the infertility gene. The nucleic acids may be used individually or in combination of two or more types. An example of a genome editing system using the CRISPR enzyme is 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. Preferably, the CRISPR / Cas9 system introduces Cas9 as mRNA or protein, and introduces gRNA in the form of sgRNA or crRNA and tracrRNA.Furthermore, 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 the same vector. The promoter for activating CRISPR is not particularly limited, but the U6 promoter is preferred. The promoter for activating Cas9 is not particularly limited, but a promoter expressed in mammalian cells, such as the cytomegalovirus promoter, is preferred. A commercially available vector such as the pX330-U6-Chimeric_BB-CBh-hSpCas9 vector can be used as the CRISPR / Cas9 system.

[0048] The sequence that targets the infertility gene and is incorporated into the CRISPR sequence (hereinafter also referred to as the "target sequence") is not limited as long as it is a sequence that can be incorporated into guide RNA (also called gRNA, sgRNA, or crRNA) by the CRISPR / Cas9 system and transcribed, or introduced into cells as a guide RNA containing a sequence complementary to the target sequence, thereby recombining the infertility gene. Generally, it is said that the target sequence can be selected from a sequence of approximately 20 bases in the upstream 5' region of the base sequence "NGG" (PAM sequence: N is one of the partial clayotide A, G, T, or C) present in the infertility gene. Target sequences can be designed using publicly available design tools such as the 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 / (China Central Agricultural University)), and CRISPR RGEN Tools (http: / / www.rgenome.net / (Seoul National University)).

[0049] Furthermore, preferably, if single nucleotide polymorphisms (SNPs) are present in the PAM sequence, it is preferable to avoid such sequences. With respect to the target sequence, if the SNPs of an individual are known, it is preferable to optimize the sequence to match each SNP. The 5' terminal region of the target sequence may be 1, 2, 3, or 4 bases shorter, preferably 1, 2, or 3 bases shorter.

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

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

[0052] The genome editing system can be injected into the cytoplasm of a fertilized egg, preferably a one-cell stage fertilized egg, by microinjection. For example, when introducing the Cas9 protein, the amount injected per fertilized egg can range from 5 pg to 100 pg, preferably 10 pg to 80 pg, and more preferably 10 pg to 50 pg. At this time, the guide RNA can be injected in the range of 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 mutations using genome editing technology, see, for example, Example 1 described later.

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

[0054] 3. Method for identifying genotypes One embodiment relates to a method for identifying genotypes (hereinafter also simply referred to as the "identification method"). This method includes mating a male and female fish individual, each having a polynucleotide sequence capable of expressing a fluorescent protein in only one of the sterilization gene alleles of the fish individual described in 1. above, and observing the expression of the fluorescent protein in the embryo or adult fish obtained by mating. In the identification method, the male and female to be mated may be of the same species or of different species capable of hybridization.

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

[0056] The method for observing the expression of the fluorescent protein and determining whether the organism is heterozygous or homozygous is as described in section 1 above. In the wild type, no expression of the fluorescent protein is observed.

[0057] 4. Method of transplanting germ cells One embodiment relates to a germ cell transplantation method, which includes transplanting germ cells of a different fish species into a homozygous fish individual described in 1. above. Here, the fish species that are different from the homozygous fish individuals described in 1. above are not limited as long as they are compatible with the body of the homozygous fish individuals.

[0058] The transplantation method is well known. For example, there is a method for producing gametes in fish using surrogate broodstock techniques. The undifferentiated germ cells of donors used in surrogate broodstock techniques include primordial germ cells obtained from the gonads before sex differentiation, spermatogonial cells obtained from the testes, and oogonia obtained from the ovaries.

[0059] To obtain undifferentiated germ cells from a donor, they can be collected using standard methods from the donor tissue corresponding to the differentiation stage of the desired undifferentiated germ cells. For example, undifferentiated germ cells can be obtained by excising pre-sex differentiated gonads or post-sex differentiated tissue, such as testes or ovaries, from a donor, and then dispersing the tissue into individual cells by physical dissection or treatment with proteolytic enzymes. The dispersed individual cells can then be isolated, for example, using marker antibodies or a cell sorter.

[0060] Undifferentiated germ cells can be obtained from frozen specimens or live individuals. To increase the success rate of surrogate parent fish technology, it is preferable to obtain undifferentiated germ cells from live individuals.

[0061] When introducing donor cells into a recipient, it is preferable to introduce them into an embryo or larval fish individual before the recipient's immune system is fully functional. The introduction can be performed using a manipulator such as a micromanipulator, electrosurgical unit, or laser scalpel. The introduction may be performed in any tissue or site of the recipient, such as the epidermis or peritoneal cavity. There are no particular restrictions on the number of cells introduced into an embryo or larval fish individual; for example, 1 to 100,000 cells can be introduced. [Examples]

[0062] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the examples. The sequences corresponding to the sequence numbers used in the examples are shown in the sequence list below.

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

[0064] II. Examples using tilapia as the test species 1. Preparation of the donor construct To label germ cell-deficient individuals using genome editing with tilapia as the test species, a donor construct was created 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 constructed, comprising 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, gRNA#1 shown in SEQ ID NO: 10, the Ori sequence which is the origin of plasmid replication 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. In addition, an Onil Crystallin promoter-mCherry-SV40 polyA vector was constructed by introducing Crystallin promoter-mCherry-SV40 polyA (nucleotide sequence shown in SEQ ID NO: 14) shown in SEQ ID NO: 6 instead of Crystallin promoter-EGFP-SV40 polyA shown in SEQ ID NO: 4.

[0067] 2. Introduction of cassette polynucleotides into the dnd gene region and confirmation testing (1) Introduction of cassette polynucleotides 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, cassette polynucleotides were introduced into the tilapira genome using a knock-in technique of genome editing with 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 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) that cleaves the construct. (2) Confirmation of fluorescent protein expression

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

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

[0071] The PCR primers were designed to be located outside the homology region during knock-in using CRISPR-Cas9 and between the cassette polynucleotides, as shown in Figure 5. The asterisks in Figure 5 indicate the confirmation sites. For confirmation of the 5' side (upstream side), PCR was performed using Onil_dnd_5-prime_Fw (SEQ ID NO: 17) and cryP_Rv (SEQ ID NO: 18). For confirmation of the 3' side (downstream side), dnd gfp The samples are GFP_Fw (sequence number 19) and Onil_dnd_3-prime_Rv (sequence number 20), dnd rfpPCR was performed using RFP_Fw (SEQ ID NO: 21) and Onil_dnd_3-prime_Rv (SEQ ID NO: 20). The PCR products were subjected to electrophoresis on a 1.5% agarose gel, and the presence and size of the bands were checked to confirm that the desired amplification product was obtained (Figure 6).

[0072] Furthermore, 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.

[0073] dnd gfp Figure 7 shows the sequences of the cassette polynucleotide introduction sites in the individual organisms. 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 Figure 8 shows the sequences of the cassette polynucleotide introduction sites in the individual organisms. 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 was correctly inserted at the target site.

[0075] III. Examples using the pufferfish as the test species 1. Donor Construct To label germ cell-deficient individuals using genome editing with the pufferfish (Takifugu rubripes) as the test species, a donor construct was created to introduce the cassette polynucleotide described in Section 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 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 constructed, comprising 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 sequence 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 (nucleotide sequence shown in SEQ ID NO: 26) was constructed by introducing Crystallin promoter-mCherry-SV40 polyA shown in SEQ ID NO: 6 instead of Crystallin promoter-EGFP-SV40 polyA shown in SEQ ID NO: 4. 2. Introduction of cassette polynucleotides into the dnd gene region and confirmation testing. (1) Introduction of cassette polynucleotides 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, cassette polynucleotides were introduced into the pufferfish genome using a knock-in technique with a CRISPR-Cas9 genome editing method.

[0079] 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 pufferfish dnd gene; 2.5 ng / μl of a Talb Crystallin promoter - EGFP - SV40 polyA vector or a Talb Crystallin promoter - mCherry - SV40 polyA vector; and 50 ng / μl of gRNA#1 (Sequence 10) to cleave the construct were microinjected into pufferfish fertilized eggs.

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

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

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

[0083] Furthermore, the above PCR products were subcloned, and Sanger sequencing analysis was performed using Talb_dnd_5-prime_Fw primer and Talb_dnd_3-prime_Rv primer, respectively.

[0084] dnd gfp Figure 13 shows the sequences of the cassette polynucleotide introduction sites in the individual organisms. 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 Figure 8 shows the sequence of the cassette polynucleotide introduction site in the individual. 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 was correctly inserted at the target site.

Claims

1. A fish individual lacking the function of the dnd protein, having polynucleotide sequences encoding a fluorescent protein expressibly within the coding regions of both alleles of the Dead End (dnd) gene, The expression of the aforementioned fluorescent protein is induced by a promoter specific to at least one selected from the eyes, scales, skin, and fins. The aforementioned fish individual.

2. The fish individual according to claim 1, wherein each of the two alleles of the dnd gene has a polynucleotide sequence encoding a fluorescent protein having a different fluorescence wavelength band.

3. The Dead End (dnd) gene has expressible polynucleotide sequences encoding a first fluorescent protein within the coding region of at least one allele, and the expression of the first fluorescent protein is induced by a promoter specific to at least one selected from the eye, scales, skin, and fins. The dnd gene is crossed with another fish individual that is capable of expressing a polynucleotide sequence encoding a second fluorescent protein having the same fluorescence wavelength band as the first fluorescent protein or a different fluorescence wavelength band from the first fluorescent protein, and the expression of the second fluorescent protein is induced by a promoter specific to at least one selected from the eye, scales, skin, and fins. A fish individual used to obtain the fish individual described in claim 1.

4. A method for producing a fish individual to obtain the fish individual described in claim 1, A method for producing a product, comprising introducing an expressible polynucleotide sequence encoding a fluorescent protein into the coding region of at least one allele of a Dead End (dnd) gene allele, wherein the expression of the fluorescent protein is induced by a promoter specific to at least one selected from the eye, scale, skin, and fin, and the function of the dnd protein encoded by the allele into which the polynucleotide sequence encoding the fluorescent protein has been introduced is lost.

5. The method for producing according to claim 4, wherein the introduction is performed by a knock-in method using genome editing or a gene site-specific recombination method.

6. The method for creating a genome according to claim 5, 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 TAL effector nuclease (TALEN) system.

7. The method for producing according to claim 5, 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.

8. A method for producing a fish individual according to claim 1, This includes crossing a first fish individual with a second fish individual that is capable of mating with the first fish individual, The first fish individual is the fish individual described in claim 3, The second fish individual is a fish individual having in the coding region of at least one allele of the dnd gene a polynucleotide sequence capable of expressing a second fluorescent protein which has the same fluorescence wavelength band as the first fluorescent protein expressed in the fish individual described in claim 3, or a different fluorescence wavelength band from the first fluorescent protein, wherein the expression of the second fluorescent protein is induced by a promoter specific to at least one selected from the eye, scales, skin, and fins. The aforementioned manufacturing method.

9. A polynucleotide for producing a fish individual according to claim 1, comprising a sequence within a portion of the coding region of the Dead End (dnd) gene that is capable of expressing a polynucleotide sequence encoding a fluorescent protein, wherein The expression of the aforementioned fluorescent protein is induced by a promoter specific to at least one selected from the eyes, scales, skin, and fins. The aforementioned polynucleotide.

10. The method includes transplanting germ cells of a different fish species into the fish individual described in claim 1. A method of transplanting germ cells.

11. This includes observing the tissue-specific expression of a fluorescent protein in an embryo or individual obtained by crossing a first fish individual with a second fish individual capable of mating with the first fish individual, in at least one selected from the eyes, scales, skin, and fins. The first fish individual is the fish individual described in claim 3, The second fish individual is a fish individual having in the coding region of at least one allele of the dnd gene a polynucleotide sequence capable of expressing a second fluorescent protein which has the same fluorescence wavelength band as the first fluorescent protein expressed in the fish individual described in claim 3, or a different fluorescence wavelength band from the first fluorescent protein, wherein the expression of the second fluorescent protein is induced by a promoter specific to at least one selected from the eye, scales, skin, and fins. Methods for identifying genotypes.

Images