JP4581083B2 - Method for breeding fish by germ cell transplantation using triploid recipients - Google Patents
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/02—Breeding vertebrates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/40—Fish
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
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- Biodiversity & Conservation Biology (AREA)
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
本発明は、ドナー魚類由来の分離始原生殖細胞（２ｎ）を、異種のレシピエント魚類（３倍体）の初期胚に移植して、生殖細胞系列への分化誘導方法や、かかる分化誘導方法を利用したドナー魚類由来の卵子及び／又は精子を特異的に形成せしめるドナー魚類の増殖方法に関する。 The present invention relates to a method for inducing differentiation into a germ cell line, and a method for inducing such differentiation by transplanting a separated primordial germ cell (2n) derived from a donor fish into an early embryo of a different recipient fish (triploid). The present invention relates to a method for proliferating donor fish that specifically forms an egg and / or sperm derived from a donor fish.
本発明者らは、魚類において、遺伝的に改変した或いは分離した細胞を、宿主個体に移植し、これを生殖細胞系列へ分化誘導する方法について、（１）ＥＳ細胞が生殖細胞系列に分化することが知られているマウスでは、未分化な状態を維持している細胞集団が、周辺細胞からの刺激により生殖細胞へと分化していくと考えられているのに対して、魚類においては、親の卵巣内で成熟途上の卵内に蓄積されたＲＮＡやタンパク質等の母性因子が、生殖細胞系列の決定に重要な役割を果たしており、そして、これらの母性因子が受精卵中に不均一に存在するため、細胞分裂により一部の割球のみがこの因子を受け取ることとなり、その結果、母性因子を受け取った一部の細胞のみが、将来生殖細胞系列へと分化していくと考えられること、（２）このような生殖細胞系列の決定機構を考慮すると、マウスにおいては生殖細胞系列に分化可能な細胞株に求められる条件は“未分化であること”であるが、魚類の場合は“生殖細胞への分化を決定する母性因子を含むこと”であること、（３）魚類において、遺伝的に改変した或いは分離した細胞を、宿主個体に移植し、これを生殖細胞系列へ分化誘導する際に用いるべき細胞（すなわち、宿主個体に移植後、卵子又は精子に分化し、次世代個体に改変可能な細胞）は、未分化な胚細胞ではなく、将来生殖細胞に分化することが決定付けられている生殖細胞の幹細胞、すなわち始原生殖細胞であること、（４）該始原生殖細胞を、魚類の初期胚に移植することにより、始原生殖細胞を、生殖細胞系列へ分化誘導することができること、（５）魚類由来の分離始原生殖細胞を、宿主魚類の初期胚への移植、特には、初期発生段階の宿主の腹腔内腸管膜裏側への移植により、該始原生殖細胞を生殖細胞系列への分化誘導を行うことが可能であること、を報告している（例えば、特許文献１参照）。そして、このような生殖細胞への分化誘導に関する研究を応用すれば、ヒラメを宿主とし、他のヒラメやカレイの仲間を借り腹で作ることも可能である。 In the method of transplanting genetically modified or isolated cells into a host individual and inducing differentiation into germline in fish, (1) ES cells differentiate into germline. In mice that are known to be undifferentiated, cell populations that are maintained in an undifferentiated state are thought to differentiate into germ cells by stimulation from surrounding cells, whereas in fish, Maternal factors such as RNA and proteins accumulated in the maturing egg in the parent's ovary play an important role in germline determination, and these maternal factors are heterogeneously present in the fertilized egg. As a result, only some blastomeres will receive this factor due to cell division, and as a result, only some cells that have received maternal factor will be differentiated into the germline in the future. , (2 Considering the germline-determining mechanism, the requirement for cell lines that can differentiate into germline in mice is “undifferentiated”, but in the case of fish, (3) In fish, genetically modified or isolated cells should be transplanted into host individuals and used to induce differentiation into germline. Reproductive cells that have been determined to differentiate into germ cells in the future rather than undifferentiated embryonic cells (ie, cells that can be transformed into eggs or sperm after being transplanted into a host individual and can be transformed into next-generation individuals) A stem cell of a cell, that is, a primordial germ cell, (4) the primordial germ cell can be induced to differentiate into a germ cell line by transplanting the primordial germ cell into an early fish embryo, (5 Transplanting isolated primordial germ cells from fish into the early embryos of the host fish, in particular, transplantation of the primordial germ cells into the germ line of the abdominal mesentery of the host in the early developmental stage. It is reported that it can be performed (see, for example, Patent Document 1). By applying such research on differentiation induction into germ cells, it is possible to use flounder as a host and borrow other flounder and flounder friends.
他方、優良品質の魚の育種に３倍体を作製する技術が知られている。具体的には、染色体操作によって誘導した完全同型接合体の選抜によって、好ましくは通常魚をも対照群として該優良形質の個体を選び出すことによって、優良形質を遺伝的に固定し、これと、他の完全同型接合体もしくは通常魚と交配し、優良形質の品種を作出することを特徴とするヒラメ類の育種方法が知られている（例えば、特許文献２及び特許文献３参照）。また、三倍体のフナの胎盤部分を取り出し、金魚の胚に移植し、キメラを作ることにより、両者の卵を生産することも知られている。 On the other hand, techniques for producing triploids for breeding excellent quality fish are known. Specifically, by selecting a perfect homozygote induced by chromosome manipulation, preferably selecting an individual having the superior trait as a control group, including normal fish, the superior trait is genetically fixed. A breeding method of Japanese flounder is known, which is characterized by mating with a completely homozygous or normal fish and producing a variety of excellent traits (see, for example, Patent Document 2 and Patent Document 3). It is also known to produce both eggs by taking the placenta part of triploid crucian carp and transplanting it to a goldfish embryo to make a chimera.
上記特許文献１記載の分化誘導方法は極めて優れた方法であるが、ドナー魚類由来の分離始原生殖細胞を、異種のレシピエント魚類の初期胚に移植した場合、レシピエント魚類は、ドナー由来の卵子及び／又は精子を作ると共に、レシピエント由来の卵子及び／又は精子を作る。しかし、ドナー魚類の方が異種のレシピエント魚類よりも付加価値が高い場合など、ドナー由来の卵子及び／又は精子を特異的に形成させることが必要とされる場合がある。本発明の課題は、ドナー魚類由来の分離始原生殖細胞を異種のレシピエント魚類の初期胚に移植する分離始原生殖細胞の生殖細胞系列への分化誘導方法において、レシピエント由来の卵子及び／又は精子を形成させることなく、ドナー由来の卵子及び／又は精子を特異的に形成させる魚類の増殖或いは育種を効率よく行う方法を提供することにある。 Although the differentiation induction method described in Patent Document 1 is an excellent method, when the isolated primordial germ cells derived from a donor fish are transplanted into an early embryo of a different recipient fish, the recipient fish is an egg derived from the donor. And / or making sperm and making egg and / or sperm from the recipient. However, it may be necessary to specifically form donor-derived eggs and / or sperm, such as when donor fish has higher added value than heterogeneous recipient fish. An object of the present invention is to provide an egg and / or sperm derived from a recipient in a method for inducing differentiation of a separated primordial germ cell into a germ cell line by transplanting a separated primordial germ cell derived from a donor fish into an early embryo of a different recipient fish. It is an object of the present invention to provide a method for efficiently growing or breeding fish that specifically forms donor-derived ova and / or sperm without forming a sperm.
本発明者は、上記課題を解決するために鋭意研究し、ドナー魚類由来の分離始原生殖細胞を異種のレシピエント魚類の初期胚に移植する分離始原生殖細胞の生殖細胞系列への分化誘導方法において、ドナーと異なるレシピエント魚類として３倍体魚類を用いることにより、レシピエント由来の卵子及び／又は精子が殆ど形成されることなく、ドナー由来の卵子及び／又は精子、特に雌においては殆どドナー由来の卵子が特異的に形成されることを見い出し、本発明を完成するに至った。 In order to solve the above-mentioned problems, the present inventor conducted an intensive study, and in a method for inducing differentiation of a separated primordial germ cell into a germ cell line, transplanting an isolated primordial germ cell derived from a donor fish into an early embryo of a different recipient fish. By using triploid fish as recipient fish different from the donor, egg and / or sperm derived from the recipient is hardly formed, and the egg and / or sperm derived from the donor is mostly derived from the donor in females. The present invention has been completed by finding that the eggs are specifically formed.
すなわち具体的には本発明は、（１）ドナー魚類由来の分離始原生殖細胞を、孵化前後の異種のレシピエント３倍体魚類の腹腔内への移植により孵化前後の３倍体魚類に移植することを特徴とする分離始原生殖細胞の生殖細胞系列への分化誘導方法や、（２）分離始原生殖細胞が、孵化前後の胚から分離した始原生殖細胞であることを特徴とする上記（１）記載の分離始原生殖細胞の生殖細胞系列への分化誘導方法や、（３）孵化前後の異種のレシピエント３倍体魚類の腹腔内への移植が、孵化前後のレシピエント３倍体魚類の腹腔内腸管膜裏側への移植であることを特徴とする上記（１）又は（２）記載の分離始原生殖細胞の生殖細胞系列への分化誘導方法や、（４）上記（１）〜（３）のいずれか記載の分離始原生殖細胞の生殖細胞系列への分化誘導方法により、ドナー魚類由来の卵子及び／又は精子を形成せしめることを特徴とする魚類の増殖方法からなる。 Specifically, the present invention specifically (1) transplants isolated primordial germ cells derived from donor fish into triploid fish before and after hatching by transplanting into the abdominal cavity of different recipient triploid fish before and after hatching. it differentiation inducing method into germline or separation primordial germ cells, wherein, (2) separating primordial germ cells, and wherein the hatch is primordial germ cells isolated from the front and back of the embryo (1) method for inducing differentiation and to germline separation primordial germ cells as described, (3) implanted into the peritoneal cavity of the recipient triploid fish hatching before and after heterologous, abdominal cavity of the recipient triploid fish before and after hatching The method for inducing differentiation of a separated primordial germ cell into a germ cell line according to the above (1) or (2), wherein the method is a transplant to the back side of the intestinal mesentery, or (4) the above (1) to (3) The germline of the isolated primordial germ cell according to any of The method inducing differentiation, consisting of the growth process of fish, characterized in that allowed to form eggs and / or sperm from a donor fish.
本発明によると、ドナー魚類由来の始原生殖細胞を用いて、これを異種のレシピエント魚類（３倍体）の初期胚に移植することにより、レシピエント由来の卵子及び／又は精子が殆ど形成されることなく、ドナー由来の卵子及び／又は精子、特に雌においては殆どドナー由来の卵子が特異的に形成されることから、例えば、分離した始原生殖細胞を、異種系統や異種の宿主個体（３倍体）に移植して、魚類等の借り腹としての利用が可能となり、この方法により、マグロのような巨大な種の始原生殖細胞を小型の近縁種に移植することで、異種系統や異種の宿主個体由来の稚魚が殆ど混じっていないのでこれらを分離する手間が省け、マグロ特異的な稚魚生産が可能となる。また、希少種、絶滅危惧種の始原生殖細胞を凍結保存し、必要な時に飼育が容易な近縁種に移植することにより、近縁種由来の卵子や精子が混じっていない希少種、絶滅危惧種特異的な遺伝子資源の保存への利用が可能となる。 According to the present invention, primordial germ cells derived from donor fish are used and transplanted into early embryos of different recipient fish (triploid), whereby recipient-derived eggs and / or sperm are almost formed. Without being formed, donor-derived ova and / or sperm, especially in females, most donor-derived ova are specifically formed. For example, isolated primordial germ cells are transformed into heterologous strains or heterologous host individuals (3 It is possible to use it as a litter for fish and the like, and by this method, transplanting primordial germ cells of huge species such as tuna into small related species, Since almost no fry from different host individuals are mixed, the labor of separating these can be saved, and tuna-specific fry production is possible. In addition, by storing cryogenically preserved rare and endangered primordial germ cells and transplanting them to related species that are easy to breed when needed, rare species that are not mixed with eggs or sperm from related species are endangered. It can be used to preserve species-specific genetic resources.
本発明の分離始原生殖細胞の生殖細胞系列への分化誘導方法としては、ドナー魚類由来の分離始原生殖細胞を、異種のレシピエント魚類（３倍体）の初期胚に移植する方法であれば特に制限されるものではなく、本発明の魚類の増殖方法としては、上記本発明の分化誘導方法により、ドナー魚類由来の卵子及び／又は精子を形成せしめる方法であれば特に制限されるものではなく、魚類としては海水魚、淡水魚等特に制限されず、異種の魚類としては同属異種や異属の魚類を挙げることができ、例えば、ドナーがニジマスの場合、同属異種としてヤマメ、異属としてブラウントラウト、イワナを具体的に例示することができる。 The method for inducing differentiation of isolated primordial germ cells into germ line of the present invention is particularly a method for transplanting isolated primordial germ cells derived from donor fish to the early embryos of different recipient fish (triploid). The method for proliferating the fish of the present invention is not particularly limited as long as it is a method for forming eggs and / or sperm derived from donor fish by the differentiation induction method of the present invention, The fish is not particularly limited, such as saltwater fish, freshwater fish, etc., and the heterogeneous fish can include the same genus different species and different genus fish, for example, when the donor is a rainbow trout, the same genus different species yamame, the different genus brown trout, A char can be illustrated specifically.
また、ドナー分離始原生殖細胞のレシピエントの初期胚への移植は、初期発生段階の宿主レシピエントの腹腔内腸管膜裏側への移植によって行うことができる。ドナー分離始原生殖細胞を、レシピエント（３倍体）の孵化胚の腹腔内に、マイクロインジェクション法のような方法で移植すると、移植した始原生殖細胞は、自発的に宿主生殖腺内へと移動し、そこで増殖、分化を誘導することができる。魚類においては、移植に用いる始原生殖細胞は、孵化前後の胚から分離した始原生殖細胞を用いることができ、又、宿主の初期胚への移植は、孵化前後の発生段階にある宿主レシピエント（３倍体）を用いることができる。 In addition, transplantation of donor isolated primordial germ cells into the recipient's early embryo can be performed by transplanting the host recipient at the early developmental stage into the peritoneal cavity of the peritoneal mesentery. When donor isolated primordial germ cells are transplanted into the peritoneal cavity of a recipient (triploid) embryo by a method such as microinjection, the transplanted primordial germ cells spontaneously move into the host gonad. Therefore, proliferation and differentiation can be induced. In fish, primordial germ cells used for transplantation can be primordial germ cells isolated from embryos before and after hatching, and transplantation into the early embryo of a host can be performed by a host recipient ( Triploid) can be used.
上記ドナーの始原生殖細胞を移植に用いるに際しては、該細胞の分離、精製を行い、取得することが好ましい。魚類の始原生殖細胞は、初期発生の限られた段階、例えば孵化前後の限られた初期発生の段階でのみ出現する細胞集団であることから、その取得のためには、その可視化を行い、分離、精製を可能として、取得を行うことが好ましい。始原生殖細胞の可視化には、本発明者らが報告した方法（Int. J. Dev. Biol.44: 323-326, 2000）を用いることができる。即ち、例えば、魚類の始原生殖細胞の可視化には、まず、魚類の生殖細胞で特異的に発現している遺伝子を取得し（Mol. Reprod. Develop. 55: 364-371, 2000）、その調節領域を利用する。魚類においては、ｖａｓａ遺伝子を利用することができる。ｖａｓａとは、ショウジョウバエでＦ１世代が不妊になる（Ｆ２世代が取れない）突然変異の原因遺伝子として単離され、生殖細胞におけるｍＲＮＡの翻訳に関与するＲＮＡヘリカーゼ活性を有するといわれている遺伝子で（蛋白質核酸酵素，43，356-363，1998）、ニジマスのような魚類においても、始原生殖細胞において特異的に発現しており、この遺伝子の発現を、始原生殖細胞の可視化に利用することができる。 When the primordial germ cells of the donor are used for transplantation, it is preferable to obtain them by separating and purifying the cells. Fish primordial germ cells are a cell population that appears only at a limited stage of early development, for example, at a limited stage of early development before and after hatching. It is preferable to perform the acquisition to enable purification. For visualization of primordial germ cells, the method reported by the present inventors (Int. J. Dev. Biol. 44: 323-326, 2000) can be used. That is, for example, in order to visualize the primordial germ cells of fish, first, a gene specifically expressed in the germ cells of fish is obtained (Mol. Reprod. Develop. 55: 364-371, 2000) and its regulation is performed. Use area. In fish, the vasa gene can be used. Vasa is a gene that has been isolated as a causative gene of a mutation that causes infertility in the F1 generation in Drosophila (the F2 generation cannot be removed) and is said to have RNA helicase activity involved in translation of mRNA in germ cells ( Protein nucleic acid enzyme, 43, 356-363, 1998), fish such as rainbow trout are also specifically expressed in primordial germ cells, and the expression of this gene can be used for visualization of primordial germ cells .
始原生殖細胞の可視化に際して、始原生殖細胞において特異的に発現している遺伝子の発現を用いて、生体中で始原生殖細胞を標識して分離するためには、ｖａｓａ遺伝子のように生殖細胞で特異的に発現している遺伝子の調節領域に、例えば、生体中で始原生殖細胞を可視化しうるタンパク質等をコードするマーカー遺伝子を組込んだプラスミドを、魚類の受精卵の細胞質内に導入することによって行うことができる。該マーカー遺伝子としては、蛍光タンパク質ＦＰをコードする遺伝子、例えばオンワンクラゲ由来の緑色蛍光タンパク質ＧＦＰ（green fluorescent protein）やＥＧＦＰ（Enhanced Green Fluorecent Protein）のようなマーカー遺伝子を用いることができる。 When visualizing primordial germ cells, using the expression of genes that are specifically expressed in primordial germ cells to label and isolate primordial germ cells in the body, it is specific in germ cells like the vasa gene By introducing into the cytoplasm of fertilized eggs of fish, for example, a plasmid incorporating a marker gene encoding a protein that can visualize primordial germ cells in the living body into the regulatory region of the gene that is expressed It can be carried out. As the marker gene, a gene encoding a fluorescent protein FP, for example, a marker gene such as green fluorescent protein (GFP) derived from Onwan jellyfish or Enhanced Green Fluorecent Protein (EGFP) can be used.
可視化した始原生殖細胞を、生殖細胞組織から分離するには、適宜公知の方法を用いることができる。例えば、孵化胚から始原生殖細胞を含む生殖隆起を回収し、これをタンパク質分解酵素でバラバラになるまで解離し、更にこれをセルソーターに掛けて、分取することができる。始原生殖細胞は、マーカー遺伝子の発現により、蛍光陽性に標識されているから、蛍光を発している生殖細胞と蛍光を発していない他の体細胞とを、セルソーターにより、容易に分離することができる。 In order to separate the visualized primordial germ cells from germ cell tissues, known methods can be used as appropriate. For example, a genital ridge containing primordial germ cells can be recovered from a hatched embryo, dissociated with a proteolytic enzyme until separated, and further applied to a cell sorter for separation. Since primordial germ cells are fluorescently labeled by the expression of the marker gene, germ cells that emit fluorescence and other somatic cells that do not emit fluorescence can be easily separated using a cell sorter. .
取得した始原生殖細胞は、例えば、マイクロインジェクション等の適宜の方法により、初期発生段階のレシピエント（３倍体）の腹腔内腸管膜裏側への移植を行うことによって、始原生殖細胞は、自発的に宿主生殖腺内へと移動し、卵母細胞或いは精原細胞への分化誘導、更には卵子或いは精子への分化誘導を行うことができる。本発明の分離始原生殖細胞の生殖細胞系列への分化誘導方法は、これを、新規個体の作製に用いることができ、ドナー魚類の増殖方法に用いることができる。 The obtained primordial germ cells are spontaneously transplanted to the back side of the intraperitoneal mesentery of the recipient (triploid) in the early development stage by an appropriate method such as microinjection. It can move into the host gonad and induce differentiation into an oocyte or spermatogonia, and further induce differentiation into an egg or sperm. The method for inducing differentiation of isolated primordial germ cells into germ line of the present invention can be used for production of a new individual, and can be used for a method for growing donor fish.
ドナー魚類由来の分離始原生殖細胞を移植する３倍体のレシピエント魚類の作製方法としては特に制限されないが、実施例に記載されているように、通常２倍体魚類の雌親からの卵と、通常２倍体魚類の雄親からの精子を用いて、通常の方法で人工授精を施した後、１０℃の流水下で発生を開始させ、受精後１５分経過した後に２８℃の温水に浸漬し、その後１０℃の流水で通常卵となると同様に発生させる方法の他、通常２倍体魚類の雌親からの卵と、通常２倍体魚類の雄親からの精子を用いて、通常の方法で人工授精を施した後、圧力処理によって第二極体の放出を阻止することにより卵の染色体を三倍体化した受精卵を経て、３倍体稚魚を作出する方法や、紫外線やγ線によって不活性化した精子を卵にかけ、発生させることで雌性発生を行わせ、第二極体放出阻止法（発生の過程で、圧力刺激や温度刺激によって第二極体の放出を阻止して２倍体にする方法）又は第一卵割阻止法（発生の段階で第二極体を放出して雌性前核のみで発生を開始したところに、物理化学的な刺激を与え、第１回の卵割を阻止して２倍体とする方法）により染色体を倍加する方法や、雌魚を雄性ホルモン（テストステロン）で処理して、性転換を起こさせ雄化させ、この偽雄と普通の雌を交配する方法等を挙げることができる。３倍体のレシピエントとしては、殆どドナー由来の卵子が特異的に形成される雌が好ましい。 Although it does not restrict | limit especially as a production method of the triploid recipient fish which transplants the isolation | separation primordial germ cell derived from a donor fish, As described in an Example, Usually, the egg from the diploid fish female parent and Usually, spermatozoa from diploid fish male parents are used, and artificial insemination is performed in the usual manner. Then, development is started under flowing water at 10 ° C, and after 15 minutes from fertilization, the water is heated to 28 ° C. In addition to the method of immersing and then generating a normal egg in flowing water at 10 ° C., usually using an egg from a diploid fish female parent and a sperm from a normal diploid fish male parent, After the artificial insemination by the method of, after the fertilized egg that triploidized the chromosome of the egg by blocking the release of the second polar body by pressure treatment, the method of producing a triploid juvenile, By applying sperm inactivated by gamma rays to eggs and generating them, The second polar body release prevention method (a method of preventing the second polar body from being released by pressure stimulation or temperature stimulation in the course of generation to make a diploid) or the first cleavage prevention method (generation of At this stage, the second polar body is released and development begins only in the female pronucleus, and then a physicochemical stimulus is applied to prevent the first cleavage and make a diploid. Examples include a method of doubling, a method in which a female fish is treated with a male hormone (testosterone) to cause sex change and maleize, and this pseudo male and a normal female are mated. As a triploid recipient, a female in which an egg derived from a donor is almost specifically formed is preferable.
本発明の分離始原生殖細胞の生殖細胞系列への分化誘導方法の魚類の増殖方法への利用としては、該分化誘導方法を魚類の異種個体の卵子及び／又は精子の形成に用い、これを用いて魚類等の大量の増殖に利用することができる。本発明の増殖方法の特徴として、レシピエントとして３倍体宿主を用いるため殆ど始原生殖細胞を保持していない。ここにドナー由来の始原生殖細胞を移植することで、レシピエント（３倍体）宿主生殖腺内で、ドナー始原生殖細胞に由来する卵・精子、が殆どを占めることになる。 As the utilization of the method for inducing differentiation of isolated primordial germ cells of the present invention into a germ line, the method for inducing differentiation is used for the formation of eggs and / or sperm of different species of fish. Can be used for large-scale growth of fish. As a feature of the growth method of the present invention, since a triploid host is used as a recipient, almost no primordial germ cells are retained. By transplanting donor-derived primordial germ cells here, most of the eggs and sperm derived from donor primordial germ cells occupy within the recipient (triploid) host gonad.
また、ドナー始原生殖細胞は孵化前後の極めて若い胚に由来するため、元来その増殖能力が極めて高く、移植後の異種の３倍体宿主個体内であっても、その増殖が極めて早いことが確認されている。さらに精原細胞の移植では精子しか作れないが、始原生殖細胞は性が分化する以前の細胞であるため、卵・精子の両者の形成が可能である。ちなみに魚類では卵の凍結保存技術が完成していないため、本始原生殖細胞を凍結保存後、移植により卵・精子を作れば遺伝子始原の保存と技術となる。 In addition, since the donor primordial germ cells are derived from very young embryos before and after hatching, their ability to grow is inherently extremely high, and even within heterologous triploid host individuals after transplantation, the growth is extremely fast. It has been confirmed. Moreover, only sperm can be produced by spermatogonia transplantation, but since primordial germ cells are cells before sex differentiation, both eggs and sperm can be formed. By the way, since the cryopreservation technique for eggs has not been completed in fish, the preservation and technique of gene primitives can be achieved by making eggs and sperm by transplantation after cryopreserving primitive germ cells.
（実施例）
以下、実施例により本発明をより具体的に説明するが、本発明の技術的範囲はこれらの例示に限定されるものではない。本発明の実施例として、ニジマスを用いた例を示し、本発明を説明する。
(Example)
EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations. As an example of the present invention, an example using rainbow trout will be shown to explain the present invention.
（実施例１）
［ヤマメ（３倍体）の調製］
宿主となるヤマメ（３倍体）の調製は、通常２倍体ヤマメの雌親からの卵と、通常２倍体ヤマメの雄親からの精子を用いて、通常の方法で人工授精を施した後、１０℃の流水下で発生を開始させ、受精後１５分経過した後に２８℃の温水に浸漬し、その後１０℃の流水で通常卵となると同様に発生させ、受精後３５日に達した個体を宿主ヤマメ（３倍体）として実験に供した。なお、受精後３５日に達した段階の幼魚の３倍体化率を、ＤＮＡ量の顕微測光法で調べたところ、９９．８％であり、殆どの幼魚が３倍体となっていた。
Example 1
[Preparation of yamame trout (triploid)]
For the preparation of the yamame trout (triploid) as the host, artificial insemination was performed in the usual manner using eggs from the parent of the diploid yamame and sperm from the male parent of the diploid yamame. Thereafter, the development was started under running water at 10 ° C, and after 15 minutes from fertilization, it was immersed in warm water at 28 ° C. After that, when it became a normal egg with running water at 10 ° C, it was generated in the same manner and reached 35 days after fertilization. The individual was subjected to the experiment as a host yamame (triploid). In addition, when the triploidization rate of the young fish at the stage of reaching 35 days after fertilization was examined by microphotometry of the amount of DNA, it was 99.8%, and most of the young fish were triploid.
（実施例２）
［分離した始原生殖細胞のヤマメ（３倍体）初期胚での分化誘導］
（分離始原生殖細胞のヤマメ（３倍体）初期胚への導入）
特開２００３−２３５５５８号公報記載の方法により、ニジマスのｖａｓａ遺伝子の発現調節領域にオワンクラゲ由来のＧＦＰ遺伝子を接続し、可視化したニジマスの始原生殖細胞（２倍体）を、異種個体である宿主ヤマメ（３倍体）の初期胚に導入した。このＧＦＰ遺伝子を導入した始原生殖細胞は特異的に緑色蛍光を発するため、生きたヤマメ（３倍体）個体内で可視化することができる。ヤマメ（３倍体）の初期胚への始原生殖細胞の導入は、ニジマスの孵化前後の胚（水温１０℃で飼育した場合、受精後３０−４０日の胚、孵化は受精後３２日）から、前記の方法で単離した始原生殖細胞１０個程度を、マイクロインジェクターに装着したガラス製のマイクロピペットで吸引し、同じ初期発生段階のヤマメ（３倍体）個体の腹腔内腸管膜裏側に移植した。
(Example 2)
[Induction of differentiation of isolated primordial germ cells in early embryo of yamame (triploid)]
(Introduction of isolated primordial germ cells into early embryo of yamame (triploid))
A rainbow trout primordial germ cell (diploid) visualized by connecting a GFP gene derived from Aequorea jellyfish to a rainbow trout vasa gene expression regulatory region by the method described in JP-A-2003-235558, It was introduced into (triploid) early embryos. Since the primordial germ cell into which this GFP gene has been introduced emits green fluorescence specifically, it can be visualized in a living yamame (triploid) individual. The introduction of primordial germ cells into the early embryo of yamame trout (triploid) is from embryos before and after hatching of rainbow trout (embryos at 30 to 40 days after fertilization, hatching is 32 days after fertilization) About 10 primordial germ cells isolated by the above method are sucked with a glass micropipette attached to a microinjector and transplanted to the back side of the intraperitoneal mesentery of a yamame trout (triploid) individual at the same initial development stage. did.
（移植した始原生殖細胞の生殖細胞系列への分化誘導）
初期発生段階のヤマメ（３倍体）個体の腹腔内腸管膜裏側に移植した始原生殖細胞は、自発的に未熟生殖腺に向かって移動を開始し、そこで生殖腺内に取り込まれる。導入効率の確認は、移植後に経時的に宿主生殖線を蛍光観察し、ドナー由来の生殖細胞の割合を調査した。移植後、６０日目において、移植した始原生殖細胞が生殖腺中の生殖隆起へ移動し、生殖隆起内に取り込まれて、更に増殖しているのが観察された。その後、移植した始原生殖細胞は、宿主レシピエント生殖腺内で効率よく増殖し、精巣において精原細胞に、卵巣において卵原細胞・卵母細胞に分化誘導しているのが観察された。これらの卵母細胞や精原細胞は、ヤマメ（３倍体）個体内で、卵子又は精子に分化するので、これらの生殖細胞を用いることにより、新しいニジマス個体を作製することができる。
(Induction of differentiation of transplanted primordial germ cells into germ line)
The primordial germ cells transplanted to the back side of the intraperitoneal mesentery of an early developmental yamame (triploid) individual spontaneously begin to move toward the immature gonad, where it is taken into the gonad. To confirm the introduction efficiency, the host germ line was observed with fluorescence over time after transplantation, and the proportion of germ cells derived from donors was investigated. On the 60th day after transplantation, it was observed that the transplanted primordial germ cells migrated to the genital ridge in the gonad, taken into the genital ridge, and further proliferated. Thereafter, it was observed that the transplanted primordial germ cells efficiently proliferated in the host recipient gonad, and induced to differentiate into spermatogonia in the testis and into oocytes and oocytes in the ovary. Since these oocytes and spermatogonia differentiate into ova or sperm in a yamame (triploid) individual, a new rainbow trout individual can be produced by using these germ cells.
（実施例３）
［精子・卵子の形成］
ニジマスの始原生殖細胞（２倍体）を移植したヤマメ（３倍体）レシピエント（recipient）を、移植１８ヶ月後に精巣と卵巣の蛍光顕微鏡による解剖観察を行った。対照としては、移植したヤマメ（３倍体）レシピエント（recipient）と同ステージの移植をしなかった３倍体ヤマメ（3n control）、及びニジマスの始原生殖細胞（２倍体）を移植した移植１８ヶ月後ヤマメ（２倍体）レシピエント（2n control）を用いた。精巣における解剖結果を図１（参考写真１）に、卵巣における解剖結果を図２（参考写真２）に示す。図１からわかるように、ヤマメ（３倍体）レシピエント（recipient）精巣内に大きなコロナイズが認められた。また、図２からわかるように、ヤマメ（２倍体）レシピエント（2n control）と同程度に発達したドナー由来の卵がヤマメ（３倍体）レシピエント（3n recipient）の卵巣を満たしていた。
(Example 3)
[Formation of sperm and egg]
A yamame trout (triploid) recipient transplanted with rainbow trout primordial germ cells (diploid) was subjected to anatomical observation of the testis and ovary 18 months after transplantation. Controls were transplanted with transplanted yamame trout (triploid) recipients and triploid yamame trout that did not transplant at the same stage (3n control) and rainbow trout primordial germ cells (diploid) After 18 months, a yamame trout (diploid) recipient (2n control) was used. The anatomical result in the testis is shown in FIG. 1 (Reference Photo 1), and the anatomical result in the ovary is shown in FIG. 2 (Reference Photo 2). As can be seen from FIG. 1, large colonization was observed in the yamame (triploid) recipient testis. Also, as can be seen from FIG. 2, the donor-derived egg developed to the same extent as the yamame (diploid) recipient (2n control) filled the ovary of the yamame (triploid) recipient (3n recipient). .
Claims (4)
A method for proliferating fish, characterized in that an egg and / or sperm derived from a donor fish is formed by the method for inducing differentiation of a separated primordial germ cell into a germ cell line according to any one of claims 1 to 3.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2004296852A JP4581083B2 (en) | 2004-10-08 | 2004-10-08 | Method for breeding fish by germ cell transplantation using triploid recipients |
| PCT/JP2005/017801 WO2006040926A1 (en) | 2004-10-08 | 2005-09-28 | Method of growing fish by transplanting genital cell with the use of triploid recipient |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2004296852A JP4581083B2 (en) | 2004-10-08 | 2004-10-08 | Method for breeding fish by germ cell transplantation using triploid recipients |
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| JP2006101845A JP2006101845A (en) | 2006-04-20 |
| JP4581083B2 true JP4581083B2 (en) | 2010-11-17 |
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| JP2004296852A Expired - Lifetime JP4581083B2 (en) | 2004-10-08 | 2004-10-08 | Method for breeding fish by germ cell transplantation using triploid recipients |
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| Country | Link |
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| JP (1) | JP4581083B2 (en) |
| WO (1) | WO2006040926A1 (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009118778A1 (en) * | 2008-03-25 | 2009-10-01 | 国立大学法人東京海洋大学 | Method of separating fish immature germ cell using fish immature germ cell surface-specific protein |
| US8921643B2 (en) | 2010-02-09 | 2014-12-30 | National University Corporation Hokkaido University | Method for acquiring genetically identical gamete from lethal fish haploid-derived germ cell via germ line chimera |
| WO2013145703A1 (en) * | 2012-03-27 | 2013-10-03 | 国立大学法人東京海洋大学 | Improvement to survival rate of separated germ cells implanted into host fish gonads |
| JP6536961B2 (en) * | 2014-03-26 | 2019-07-03 | 国立大学法人東京海洋大学 | Method for producing fish gametes for cultured fish production using surrogate parent fish, which is applicable to parent species of another genus fish species |
| JP6999072B2 (en) * | 2015-03-26 | 2022-02-04 | 国立大学法人東京海洋大学 | How to make transplanted fish, transplanted fish, how to make hybrid fish species and hybrid fish species |
| CN105475202B (en) * | 2015-12-25 | 2018-07-24 | 武汉百瑞生物技术有限公司 | The method that a generation is bred as complete female Pelteobagrus fulvidraco |
| CN105494205B (en) * | 2015-12-25 | 2018-08-21 | 武汉百瑞生物技术有限公司 | A method of shortening fish sexual maturation cycle |
| WO2022211138A1 (en) * | 2021-03-29 | 2022-10-06 | 주식회사 노아바이오텍 | Method for decreasing mortality rate during seawater acclimation of trout |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH10327706A (en) * | 1997-03-29 | 1998-12-15 | Tottori Pref Gov | Breeding of flatfish using complete homozygote, produced flatfish and proliferation of flatfish |
| JP2000135038A (en) * | 1997-03-29 | 2000-05-16 | Tottori Prefecture | Breeding of flatfishes by utilization of full homogenote, created flatfishes and increased culture of flatfishes |
| JP2003235558A (en) * | 2002-02-14 | 2003-08-26 | Japan Science & Technology Corp | Method for differentiation and induction to germ line by transplantation of separated primordial germ cell |
-
2004
- 2004-10-08 JP JP2004296852A patent/JP4581083B2/en not_active Expired - Lifetime
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2005
- 2005-09-28 WO PCT/JP2005/017801 patent/WO2006040926A1/en active Application Filing
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH10327706A (en) * | 1997-03-29 | 1998-12-15 | Tottori Pref Gov | Breeding of flatfish using complete homozygote, produced flatfish and proliferation of flatfish |
| JP2000135038A (en) * | 1997-03-29 | 2000-05-16 | Tottori Prefecture | Breeding of flatfishes by utilization of full homogenote, created flatfishes and increased culture of flatfishes |
| JP2003235558A (en) * | 2002-02-14 | 2003-08-26 | Japan Science & Technology Corp | Method for differentiation and induction to germ line by transplantation of separated primordial germ cell |
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| JP2006101845A (en) | 2006-04-20 |
| WO2006040926A1 (en) | 2006-04-20 |
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