WO2015146184A1

WO2015146184A1 - Method for producing fish gametes for farmed fish production using surrogate parent fish, applicable to surrogate parent fish of different species

Info

Publication number: WO2015146184A1
Application number: PCT/JP2015/001747
Authority: WO
Inventors: 吉崎　悟朗; 承起李; 幸典嶋田; 良輔矢澤; 竹内　裕; 森田　哲朗; 輝森島
Original assignee: 国立大学法人東京海洋大学; 日本水産株式会社
Priority date: 2014-03-26
Filing date: 2015-03-26
Publication date: 2015-10-01
Also published as: JPWO2015146184A1; JP6536961B2

Abstract

The present invention addresses the problem of providing a method for producing fish gametes in techniques of farmed fish production using a surrogate parent fish, said method being applicable to a surrogate parent fish of a different species and thus broadening the scope of fish species usable as the surrogate parent fish. To solve this problem, provided is a method for producing fish gametes using a surrogate parent fish, said method comprising: hybridizing a first fish, which can serve as a surrogate parent fish for a donor fish, with a second fish, which cannot serve as a surrogate parent fish for the donor fish, to construct a hybrid fish capable of serving as a surrogate parent fish for the donor fish; and, using the hybrid fish thus constructed as a recipient, transplanting separated germ cells of the donor fish thereinto and thus inducing differentiation to a germ cell line.

Description

Method for producing fish gametes for production of cultured fish using surrogate parent fish that can be applied to surrogate parent fish of other species

The present invention relates to a method for producing fish gametes (sperm and eggs) for producing cultured fish using surrogate parent fish, which can be applied to surrogate parent fish of other genus species. In the fish gamete production method, a donor fish species that cannot be a surrogate parent of the donor fish species is supplied to a donor fish species that supplies isolated germ cells for production of the target fish gamete. By using a hybrid with a fish species that can be a surrogate parent, it can be applied as a surrogate parent fish to other species that cannot originally be a surrogate parent of a donor fish species. By expanding the application range of the species, it is possible to select surrogate parent fish having practically advantageous characteristics suitable for breeding, etc., and fish that can be put to practical use as a fish gamete production method for farmed fish production Provide gamet production methods It on.

In recent years, the catch of natural fishery resources has been on a gradual decreasing trend, and the proportion of aquaculture production in the total catch has greatly increased. On the other hand, in the cultivation of marine fish, natural seedlings are still often cultivated to the product size. For this reason, with the increasing importance of resource management, the introduction of complete aquaculture is strongly demanded.

Complete aquaculture means artificially producing seeds and seeds from fertilized eggs of the target fish species, distributing the obtained artificial seeds as products, and cultivating some individuals of the obtained artificial seeds to parent fish, It refers to a culture style that is completed without depending on natural resources, producing gametes (sperms and eggs) and using the resulting gametes for aquaculture. In order to realize complete aquaculture, first of all, it is necessary to obtain gametes that are sufficient for seed production in terms of quality and quantity from the parent fish of the target fish. In order to obtain gametes sufficient for seedling production, a breeding facility sufficient for the maintenance of parent fish, breeding with appropriate feed and spawning induction are essential, and a lot of space and labor are required. Therefore, it can be said that there are few fish species that have been established for seed production so that at least many farmers can easily introduce them.

As a technique that can solve this problem, fish gamete production techniques necessary for seedling production using surrogate parent fish, that is, surrogate parent fish techniques, are attracting attention. This technique is exemplified for donor primordial germ cells, spermatogonia, or ovary cells, with the target fish species to be gameted as the donor and the fish species that are to produce gametes as the recipient. In this method, undifferentiated germ cells are transplanted into a recipient, and the recipient is used as a surrogate parent fish. In other words, the surrogate parent fish technique is a technique for producing donor gametes and producing a next generation individual population of donors by growing or differentiating donor germ cells in the recipient gonad. In the case of fish, male gametes have motility and are called sperm, whereas female gametes have no motility and are called eggs. In surrogate parenting techniques, if one wants to obtain sperm, transplant the donor germ cells into a male recipient, while if one wants to obtain an egg, give the donor germ cells to a female recipient. Transplant.

The surrogate parent fish technique has previously been performed by the present inventors to transfer a donor isolated germ cell, which is a germ cell derived from a different strain or a different species of fish from the host (recipient) fish, into the peritoneal cavity of the host fish before and after hatching. By transplanting into an individual host fish, it is possible to induce differentiation of germ cells into the germline, that is, isolated germ cells derived from vertebrates such as fish before and after hatching of the host vertebrates. It has been found that it is possible to induce differentiation of the germ cells into a germ line by transplanting to a fish individual, and a method for inducing differentiation of a separated germ cell into a germ line by a different lineage or a heterogeneous host fish It is constructed and disclosed as (Japanese Patent No. 4300287). The non-patent literature Fisheries for Global Welfare and Environment, 5th World Fisheries Congress 2008 (2008) p209-219 includes the spermatogonia of the donor yamame trout (masu salmon, Oncorhynchus masaou) A method has been reported in which a rainbow trout (rainbouw traut, Oncorhynchus mykiss) is transplanted to obtain a yamame trout egg and sperm and a next-generation yama trout is used as a surrogate parent fish.

In the method disclosed above, the host (recipient) fish produces a gamete (sperm and / or egg) from the donor and also a gamete (sperm and / or egg) from the host (recipient). For this reason, in order to specifically form and separate donor-derived gametes (sperm and / or eggs), an operation for sorting them was necessary, which was a problem in practice. For example, if you want to induce differentiation into the germline, using a rainbow trout spermatogonia or oocyte as a donor fish species and a rainbow trout as a recipient, select a yamame sperm from among many rainbow trout sperm. In addition, it is difficult to select a yamame trout egg from among many rainbow trout eggs, and there remains a problem as an industrial application technique that requires mass production of only donor fish species.

Therefore, in order to solve this problem, the present inventors produce a triploid fish species of a host fish species, sterilize the fish species, and use the triploid fish species as a substitute parent fish technique. A method of suppressing the formation of germ cells of the host (recipient) itself by constructing it as a host (recipient) has been constructed and disclosed (Japanese Patent No. 4581083). Non-patent document Science Vol.317 (2007) p1517 produces a triploid rainbow trout to obtain an infertile surrogate parent fish that does not produce rainbow trout-derived sperm and eggs. A method has been reported for efficiently obtaining spermatozoa and eggs of yamame trout by transplanting and producing the next generation of yamame trout.

However, with the surrogate parent fish technology so far, the fish species that can be used as the surrogate parent fish are limited to those that are closely related to the donor, and the fish species to which the surrogate parent fish technology can be applied are limited. For example, in the non-patent document Fishories Science Vol.317 (2011) p.60-77, when a yellowtail spermatogonia was transplanted into a nibe, it was taken into the nibe gonad but did not produce gametes. It is described. Non-patent literature Biology of Reproduction Vol. 82 (2010) p.896-904 shows that when the spermatogonial cells of nibe are transplanted into the chub mackerel, the gonad germ cells are taken up and proliferated. However, it was described that it did not lead to gamete production.

In the formation of donor-derived gametes (sperm and / or eggs) by surrogate parent fish technology, for example, from a practical point of view, large fish gametes (sperm and / or eggs), small fish that are easy to breed, etc. Need to be formed as surrogate parent fish, and in many cases, selection of surrogate parent fish beyond the genus to which the fish species belongs is required. However, in many cases, surrogate parental fish for a donor fish species become surrogate parent fish because, for example, the isolated germ cells of the donor fish species do not survive the germline of the surrogate parent fish. There is a problem that it is not possible. Therefore, the development of surrogate parent fish technology that enables the use of surrogate parent fish beyond the scope of the genus, etc. is a challenge for donor fish species in order to respond to practical demands for the use of surrogate parent fish technology. .

Japanese Patent No. 4300287 Japanese Patent No. 4581083

An object of the present invention is to enable application to surrogate parent fish such as other species of fish in the technology of fish gamete (sperm and egg) production method for production of cultured fish using surrogate parent fish. It is possible to use a fish species that cannot be a surrogate parent of a species as a surrogate parent of a donor fish species, expand the range of fish species that can be a surrogate parent, and for the practical application of this technology, Therefore, it is intended to provide a method for producing fish gametes (sperm and eggs) for producing cultured fish using surrogate parent fish, which can select and apply surrogate parents with good applicability.

In order to solve the above-mentioned problems, the present invention provides an isolated germ cell donor for a fish species used as a surrogate parent fish in a fish gamete (sperm and egg) production method for producing cultured fish using the surrogate parent fish. A surrogate that has both the ability to be a surrogate parent for fish species (fish species that can engraft undifferentiated germ cells) and the ability to be a breeding species that is easy to breed as a surrogate parent fish In the intensive study on the selection of the parent, with respect to the donor fish species such as other species, the fish species that cannot be the surrogate parent of the donor fish species and the fish species that can be the surrogate parent of the donor fish species It has been found that by using a hybrid, the ability to be a surrogate parent fish can be imparted to other genus fish species that cannot be a surrogate parent of a donor fish species. By forming the hybrid, the range of selection of the surrogate parent fish can be expanded to other species of fish that cannot originally be a surrogate parent fish of the donor fish species, and the surrogate parent fish of the other genera of species that are easy to rear. It has been found that it can be used, and the present invention has been made. According to the method of the present invention, for example, it is possible to select a small species of other genera that is easy to breed as a surrogate parent of a donor species of a large fish such as tuna, for the production of cultured fish using the surrogate parent fish. It came to provide the practical use method as a production method of the fish gamete (sperm and egg).

That is, the present invention uses a surrogate parent fish that uses a recipient fish and transplants a separated germ cell of a fish of a different or different species from the recipient fish into the recipient fish to induce differentiation into a germ line. In the production method of fish gametes
(A) selecting a donor fish species that supplies isolated germ cells;
(B) selecting a fish of a first species that can be a surrogate parent for the donor species;
(C) selecting a fish of a second species that cannot be a surrogate parent for the donor species;
(D) The fish of the first species selected in (B) and the fish of the second species selected in (C) are hybridized to become a surrogate parent for the donor species. Creating hybrid fish species with the ability to gain,
(E) Using the hybrid fish species produced in (D) as a recipient, transplanting the isolated germ cells of the donor fish species to induce differentiation into the germ line,
A method for producing fish gametes using surrogate parent fish containing

In the method for producing fish gametes using the surrogate parent fish of the present invention, it is possible to use, as a surrogate parent of a donor fish species, a fish species that cannot originally be a surrogate parent of a donor fish species, such as other species. By expanding the application range of fish species, the selection range of easy substitute parent fish such as breeding can be expanded as a substitute parent fish, and it is suitable for practical use as a fish gamete production method for farmed fish production It becomes possible to provide a method for producing fish gametes.

In the method for producing fish gametes using the surrogate parent fish of the present invention, examples of the fish gametes include sperm or eggs of donor fish species. In the method for producing fish gametes using the surrogate parent fish of the present invention, the first fish species that can be the surrogate parent fish for the donor fish species is the gonad of the isolated germ cell of the transplanted donor fish species. The second species that cannot be a surrogate parent for the donor species is the gonad of the isolated germ cell of the transplanted donor species. The fish of the kind which does not have the engraftment ability can be mentioned.

In the fish gamete production method using the surrogate parent fish of the present invention, the first fish species that can be the surrogate parent fish for the donor fish species is a fish of the same species as the donor fish species. The fish of the second fish species that can be mentioned and cannot be a substitute parent fish for the donor fish species include fish of a fish species that is different from the donor fish species. In the method for producing gametes of fish using the surrogate parent fish of the present invention, examples of the genus of the donor fish species include the genus Ivana, the yellowtail or the tuna genus.

In the method for producing fish gametes using the surrogate parent fish of the present invention, a hybrid fish species capable of becoming a surrogate parent fish for a donor fish species is a first that can be a surrogate parent fish for a donor fish species. It is preferable to have the genome of the fish species at half or less per cell, and more preferably, it has the genome of the first fish species that can be a surrogate parent fish for the donor fish species at 1/3 or less per cell. By making the genome of the first fish species that can be a surrogate parent for the donor fish species within the above range per cell, the surrogate parent fish for the donor fish species while preserving the breeding characteristics of the fish of the second fish species The ability to become a surrogate parent of the first fish species that can become a surrogate can be introduced to give the donor fish species the ability to become a surrogate parent.

In the method for producing fish gametes using the surrogate parent fish of the present invention, a hybrid fish species capable of becoming a surrogate parent fish with respect to the donor fish species is produced as a heteroploid and a gamete derived from the hybrid fish species is produced. It can be produced as an infertile hybrid fish species. As an allopolyploid of a hybrid fish species capable of becoming a surrogate parent fish for the donor fish species, an infertile hybrid fish species in which sperm and eggs derived from the hybrid fish species are not produced is a triploid hybrid fish species. Can be mentioned.

The present invention relates to a method for producing a hybrid fish as a recipient,
(A) selecting a donor fish species that supplies isolated germ cells;
(B) selecting a fish of a first species that can be a surrogate parent for the donor species;
(C) selecting a fish of a second species that cannot be a surrogate parent for the donor species;
(D) The fish of the first species selected in (B) and the fish of the second species selected in (C) are hybridized to become a surrogate parent for the donor species. Creating hybrid fish species with the ability to gain,
The production method of the hybrid fish containing is included.

The present invention also uses a recipient fish, transplants a separated germ cell of a donor fish species of a different lineage or heterogeneous fish from the recipient fish, and induces differentiation into a germline. In the method of inducing differentiation of a separated germ cell of a fish using a parent fish into a germ line, using the hybrid fish produced above as a recipient fish, transplanting the isolated germ cell of a donor fish species and inducing differentiation. The invention includes an invention of a method for inducing differentiation of a separated germ cell into a germ line using the characteristic surrogate parent fish.

That is, the present invention is as follows.
[1] Fish using surrogate parent fish using recipient fish and transplanting isolated germ cells of a fish of a different or different species from recipient fish to recipient fish to induce differentiation into germline In the gamete production method of
(A) selecting a donor fish species that supplies isolated germ cells;
(B) selecting a fish of a first species that can be a surrogate parent for the donor species;
(C) selecting a fish of a second species that cannot be a surrogate parent for the donor species;
(D) The fish of the first species selected in (B) and the fish of the second species selected in (C) are hybridized to become a surrogate parent for the donor species. Creating hybrid fish species with the ability to gain,
(E) Using the hybrid fish species produced in (D) as a recipient, transplanting the isolated germ cells of the donor fish species to induce differentiation into the germ line,
Method for producing fish gametes using surrogate parent fish including
[2] The fish gamete production method using the surrogate parent fish according to [1] above, wherein the fish gamete is a sperm or egg of a donor fish species.
[3] The fish of the first fish species that can be a surrogate parent for the donor fish species is a fish of the species that has the ability to engraft the isolated germ cells of the transplanted donor fish species, and the donor fish The fish of the second species that cannot be a surrogate parent for the species is a fish of a species that does not have the ability to engraft the isolated germ cells of the transplanted donor species A method for producing a fish gamete using the surrogate parent fish according to [1] or [2].
[4] The fish according to any one of [1] to [3] above, wherein the fish of the first fish species that can be a surrogate parent fish for the donor fish species is the same genera as the donor fish species Production of fish gametes using surrogate parent fish.
[5] Any one of the above [1] to [4], wherein the fish of the second fish species that cannot be a surrogate parent fish for the donor fish species is different from the donor fish species Production method of fish gametes using the surrogate parent fish described.
[6] The method for producing fish gametes using the surrogate parent fish according to any one of [1] to [5] above, wherein the donor fish species is genus Ivana, Yellowtail or Tuna.
[7] A hybrid fish species capable of being a surrogate parent for a donor fish species has a genome of a first fish species that can be a surrogate parent for a donor fish species in less than half of the cells. A method for producing fish gametes using the surrogate parent fish according to any one of [1] to [6] above.
[8] The hybrid fish species capable of becoming a surrogate parent fish for the donor fish species has the genome of the first fish species that can be the surrogate parent fish for the donor fish species at 1/3 or less per cell. A method for producing a fish gamete using the surrogate parent fish according to any one of [1] to [7] above.
[9] The hybrid fish species having the ability to be a surrogate parent fish for the donor fish species are heteroploid, and are infertile hybrid fish species that do not produce gametes derived from the hybrid fish species. A method for producing fish gametes using the surrogate parent fish according to any one of [1] to [8] above.
[10] A hybrid fish species capable of becoming a surrogate parent for a donor fish species is an allopolyploid, and an infertile hybrid fish species in which sperm and eggs derived from the hybrid fish species are not produced is a triploid. A method for producing a gamete of a fish using the surrogate parent fish according to any one of [1] to [9] above.
[11] In a method for producing hybrid fish as a recipient,
(A) selecting a donor fish species that supplies isolated germ cells;
(B) selecting a fish of a first species that can be a surrogate parent for the donor species;
(C) selecting a fish of a second species that cannot be a surrogate parent for the donor species;
(D) The fish of the first species selected in (B) and the fish of the second species selected in (C) are hybridized to become a surrogate parent for the donor species. Creating hybrid fish species with the ability to gain,
Of hybrid fish including
[12] The fish of the first fish species that can be a surrogate parent for the donor fish species is a fish of the fish species that has the ability to engraft the isolated germ cells of the transplanted donor fish species, and the donor fish The fish of the second species that cannot be a surrogate parent for the species is a fish of a species that does not have the ability to engraft the isolated germ cells of the transplanted donor species The method for producing hybrid fish according to [11] above.
[13] The hybrid fish according to [11] or [12] above, wherein the fish of the first fish species that can be a surrogate parent for the donor fish species is the same genera as the donor fish species Production method.
[14] Any one of the above [11] to [13], wherein the fish of the second fish species that cannot be a surrogate parent for the donor fish species is different from the donor fish species A method for producing the described hybrid fish.
[15] The method for producing a hybrid fish according to any one of the above [11] to [14], wherein the donor fish species is the genus Ivana, Yellowtail or Tuna.
[16] The hybrid fish species capable of becoming a surrogate parent for a donor fish species has a genome of a first fish species that can be a surrogate parent for a donor fish species in less than half of the cells. The method for producing hybrid fish according to any one of [11] to [15] above.
[17] A hybrid fish species capable of becoming a surrogate parent fish for a donor fish species has a genome of a first fish species that can be a surrogate parent fish for a donor fish species at 1/3 or less per cell. The method for producing hybrid fish according to any one of [11] to [16] above, wherein
[18] The hybrid fish species capable of becoming a surrogate parent for the donor species is an allogeneic polyploid and is an infertile hybrid species that does not produce gametes derived from the hybrid species. A method for producing hybrid fish using the surrogate parent fish according to any one of [11] to [17] above.
[19] A hybrid fish species capable of becoming a surrogate parent for a donor fish species is an allopolyploid, and an infertile hybrid fish species that does not produce sperm and eggs derived from a hybrid fish species is a triploid. The method for producing a hybrid fish according to any one of the above [11] to [18], characterized in that it exists.
[20] Using a surrogate parent fish that uses a recipient fish and transplants a separated germ cell of a donor fish species of a different or different species from the recipient fish into the recipient fish to induce differentiation into a germ cell line. In the method for inducing differentiation of isolated fish germ cells into germ line, the hybrid fish species produced in any one of [11] to [19] above are used as recipient fish, and the isolated germ cells of the donor fish species A method for inducing differentiation of a separated germ cell of a fish into a germ line using a surrogate parent fish, characterized by transplanting and differentiation.
[21] A hybrid fish produced by the method according to any one of [11] to [19] above.
[22] An egg or sperm of a donor fish species produced by the fish gamete production method using the surrogate parent fish according to any one of [1] to [10] above.
[23] A seedling for aquaculture of a donor fish species produced from an egg or sperm of the donor fish species according to [22] above.
[24] An adult donor fish species obtained by culturing the seedling for aquaculture of the donor fish species according to [23].
[25] Fish using a surrogate parent fish that includes using a recipient fish and transplanting isolated germ cells of a fish of a different or different species from the recipient fish into the recipient fish to induce differentiation into a germ line In the method for producing transplanted fish of
(A) selecting a donor fish species that supplies isolated germ cells;
(B) selecting a fish of a first species that can be a surrogate parent for the donor species;
(C) selecting a fish of a second species that cannot be a surrogate parent for the donor species;
(D) The fish of the first species selected in (B) and the fish of the second species selected in (C) are hybridized to become a surrogate parent for the donor species. Creating hybrid fish species with the ability to gain,
(E) transplanting the isolated germ cells of the donor fish species with the hybrid fish species produced in (D) as recipients;
Of transplanted fish using surrogate parent fish including
[26] The fish of the first fish species that can be a surrogate parent for the donor fish species is a fish of the fish species that has the ability to engraft the isolated germ cells of the transplanted donor fish species, and the donor fish The fish of the second species that cannot be a surrogate parent for the species is a fish of a species that does not have the ability to engraft the isolated germ cells of the transplanted donor species A method for producing transplanted fish using the surrogate parent fish according to [25] above.
[27] The surrogate parent fish according to [25] or [26] above, wherein the fish of the first fish species that can be a surrogate parent fish for the donor fish species is the same genera as the donor fish species The transplanted fish production method used.
[28] The fish according to any one of [25] to [27] above, wherein the fish of the second fish species that cannot be a surrogate parent fish for the donor fish species is different from the donor fish species A method for producing transplanted fish using the surrogate parent fish described.
[29] The method for producing transplanted fish using the surrogate parent fish according to any one of the above [25] to [28], wherein the donor fish species is Ivana, Yellowtail or Tuna.
[30] The hybrid fish species capable of becoming a surrogate parent fish for the donor fish species has the genome of the first fish species that can be the surrogate parent fish for the donor fish species in less than half of the cells. A method for producing transplanted fish using the surrogate parent fish according to any one of [25] to [29] above.
[31] A hybrid fish species capable of becoming a surrogate parent fish for a donor fish species has a genome of the first fish species that can be a surrogate parent fish for a donor fish species at 1/3 or less per cell. A method for producing a transplanted fish using the surrogate parent fish according to any one of [25] to [30] above.
[32] The hybrid fish species capable of becoming a surrogate parent fish for the donor fish species are heteroploid, and are infertile hybrid fish species that do not produce gametes derived from the hybrid fish species. A method for producing transplanted fish using the surrogate parent fish according to any one of [25] to [31] above.
[33] A hybrid fish species capable of becoming a surrogate parent for a donor fish species is an allopolyploid, and an infertile hybrid fish species that does not produce sperm and eggs derived from a hybrid fish species is a triploid. A method for producing transplanted fish using the surrogate parent fish according to any one of the above [25] to [32], wherein
[34] A transplanted fish obtained by the method according to any one of [25] to [33] above.
[35] A gamete obtained from a transplanted fish obtained by the method according to any one of [25] to [33] above.
[36] A seedling for aquaculture produced from the gamete described in [35] above.
[37] An adult donor fish species obtained by culturing the aquaculture seedling according to [36].

Until now, in the surrogate parent fish technology, the fish species that can be used as the surrogate parent fish are limited to those that are closely related to the donor, and the fish species to which the surrogate parent fish technology can be applied are limited. Although it is necessary to use surrogate parent fish such as other genus fish species that are easy to breed, it has been difficult to use surrogate parent fish such as other genus fish species. The present invention enables use for surrogate parent fish such as other genus fish species, and allows easy use of fish species such as other genus breeding as recipients in surrogate parent fish technology. It is possible to select a fish species that is suitable as an ent, and it is possible to provide technology for practical use as a production method of fish gametes (sperm and eggs) for the production of cultured fish using surrogate parent fish did.

The present invention uses a recipient fish that uses a recipient fish and includes transplanting germ cells of a different lineage or a heterogeneous fish from the recipient fish into the recipient fish to induce differentiation into a germline. In the production of fish gametes,
(A) selecting a donor fish species that supplies isolated germ cells;
(B) selecting a fish of a first species that can be a surrogate parent for the donor species;
(C) selecting a fish of a second species that cannot be a surrogate parent for the donor species;
(D) The fish of the first species selected in (B) and the fish of the second species selected in (C) are hybridized to become a surrogate parent for the donor species. Creating hybrid fish species with the ability to gain,
(E) The mating of the fish using the surrogate parent fish including transplanting the isolated germ cells of the donor fish species and inducing differentiation into the germ line using the hybrid fish species produced in (D) as the recipient Includes child production methods.

The surrogate parent fish technique in the present invention uses a fish species intended to obtain gametes (sperm and eggs) as a donor, and a fish species intended to produce gametes as a recipient. Transplant undifferentiated germ cells exemplified by protoblasts or oocyte cells to a recipient to produce a transplanted fish transplanted with the undifferentiated germ cells, and within the gonad of the transplanted fish (recipient) This includes engrafting, proliferating and differentiating the undifferentiated germ cells of the donor to obtain gametes derived from the donor in the body of the transplanted fish (recipient), and obtaining seedlings that become the offspring of the donor.

In the method for producing fish gametes using the surrogate parent fish of the present invention, sperm or eggs are produced as gametes. When trying to obtain sperm, a male germ cell is transplanted with a donor germ cell, and when trying to obtain an egg, a female germ cell is transplanted with a donor germ cell. As the gonad, the testis can be used to obtain sperm, and the ovary can be used to obtain eggs. Donor germ cells transplanted into a male recipient can grow and differentiate within the recipient's testis to produce donor sperm. Donor germ cells transplanted into a female recipient can grow and differentiate in the recipient's ovaries to produce donor eggs. The sperm or egg thus obtained can be fertilized eggs by crossing each other or by multiplying separately obtained eggs and sperm. By generating the obtained fertilized eggs, seedlings of the target fish species can be obtained.

As a donor, fish for the purpose of aquaculture can be selected as appropriate. For the purpose of aquaculture, the donor can select various fish species for aquaculture, such as edible fish species, material collecting fish species, ornamental fish species, and the like. Examples of edible fish species for aquaculture include salmonids such as char, genera such as yellowtail, mackerel such as tuna, etc. Or the food fish of the genus Tuna can be mentioned.

In the method of producing fish gametes using the surrogate parent fish of the present invention, the undifferentiated germ cells of the donor used in the surrogate parent fish technology include primordial germ cells obtained from the gonad before sex differentiation, and sperm obtained from the testis. Progenitor cells, oocytes obtained from ovaries and the like are included. These undifferentiated germ cells can be used in combination with cells at different stages of differentiation, or cells at the same stage of differentiation alone.

In order to obtain undifferentiated germ cells from a donor, it can be collected from the donor tissue according to the differentiation stage of the target undifferentiated germ cells by a usual method. For example, by removing gonads before sex differentiation from a donor, or tissues after sex differentiation, such as testis or ovaries, and dispersing them from the tissues to individual cells by physical detachment or treatment with proteolytic enzymes, etc. Undifferentiated germ cells can be obtained. The dispersed individual cells can be isolated using, for example, an antibody that serves as a marker, or using a cell sorter.

Undifferentiated germ cells can be obtained from frozen bodies or living individuals. In order to increase the success rate of surrogate parent fish technology, it is preferable to obtain undifferentiated germ cells from live individuals. As the frozen body, those obtained by freezing any unit of an individual, an organ, and a cell can be used. When freezing cells, it is preferable to use an appropriate cryoprotectant. Suitable cryoprotectants include 0.1 wt% to 10 wt% glucose or trehalose, or 0.1 mol / liter to 20 mol / liter dimethyl sulfoxide (DMSO), ethylene glycol or propylene glycol. These cryoprotectants can be used alone or in combination of two or more.

The step of collecting primordial germ cells preferably includes confirming that the obtained cells are primordial germ cells using a marker of primordial germ cells. Markers applicable to confirming that they are primordial germ cells include Vasa gene, CD205 gene and the like. Confirmation can be performed by confirming the expression, modification, localization, or a combination of two or more of the genes or gene products.

In order to confirm gene expression, it is convenient to confirm mRNA transcribed from a specific gene by PCR (polymerase chain reaction). It is easy to confirm the expression and localization of a gene using RNA transcribed from a specific gene using “in situ hybridization”. Expression, modification and expression of a gene product can be easily confirmed by using an antibody specific for the gene product expressed from a specific gene and translated.

When the donor cells are introduced into the recipient, it is preferably introduced into an individual at the embryo or larva stage before the recipient's immune system works sufficiently. The introduction can be performed using a manipulator such as a micromanipulator, an electric knife, or a laser knife. The introduction may be performed in any tissue or site of the recipient, and examples of the tissue or site to be introduced include the epidermis or the abdominal cavity. There is no restriction | limiting in particular as a cell number at the time of introduce | transducing into the individual | organism | solid of an embryo or a larva stage, For example, 100,000 cells can be introduce | transduced from 1 cell.

Confirmation of engraftment of donor germ cells in the recipient gonad can be performed using an index that can distinguish donor cells from recipient cells. Examples of usable indicators include genes, antibodies, fluorescent dyes and the like, and these indicators can be used alone or in combination of two or more. Examples of the gene serving as an index include genes that are expressed in undifferentiated germ cells, such as vasa gene and dead-end gene. Examples of the fluorescent dye serving as an index include PKH26 and CFSE.

When using a gene, PCR is performed by designing a primer using a site where the sequence is different between the donor and the recipient, or a site where the sequence is different between the donor and the recipient. It is easy to confirm using In situ hybridization. By labeling donor germ cells with a fluorescent dye prior to introduction, engraftment can be confirmed under a fluorescence microscope. Confirmation of engraftment can be carried out immediately after introduction, and preferably, it is carried out 7 days or more after introduction in order to confirm whether or not the recipient has engrafted and functions as a cell. it can. It is expected that donor sperm or eggs will be obtained from individuals whose engraftment was observed in the recipient's gonads 7 days after introduction.

In the present invention, a hybrid means an individual having genomes derived from different varieties, strains, or biological species in a cell, and the hybrid is produced by crossing, cell fusion or gene transfer, or a combination of two or more thereof. Including individuals In fish, different reproductive populations biologically isolated by habitats, etc. are recognized as different varieties, strains, or species. May occur. However, it is not well understood when the hybridization occurs in the natural environment and the hybrid is obtained, and the hybrid is exclusively produced artificially. Similarly, when artificially producing and maintaining varieties and lines, hybrids can be artificially produced.

When producing individuals as hybrids by crossing, hybrids can be obtained by crossing individuals of different varieties, strains or species.

In particular, when a hybrid is produced by cross-breeding between different species, a fertilized egg is produced by contacting the sperm of the other species with the egg of one species obtained by artificial breeding or natural collection, A heterogeneous hybrid can be produced by hatching the obtained fertilized egg. At this time, if the egg to be fertilized is artificially doubled and brought into contact with the sperm of the other species, the heterogeneous hybrid obtained becomes a heteroploid. Heterogeneous polyploid individuals are particularly preferred for use as recipients because they are less likely to produce their own eggs or sperm. Examples of polyploids include diploids, triploids, tetraploids, and the like. Any of these polyploids can be used.

As a method of artificially doubling fertilized eggs, polar body release is suppressed by using a cell division inhibitor, electrical stimulation, light stimulation or temperature stimulation, pressure stimulation, or a combination of two or more thereof at the time of fertilization. The method can be used. For example, colchicine can be used as the cell division inhibitor. Also, a tetraploid can be created as a polyploid, and a triploid can be created by multiplying this with a diploid individual.

When producing hybrids by cell fusion, a method of fusing different types of eggs or isolated embryo cells can be used. For cell fusion, a method of directly fusing cells using a cell membrane fusion agent, electrical stimulation, light stimulation or thermal stimulation, or a combination thereof can be used. For example, polyethylene glycol can be used as the cell membrane fusion agent. Hybrid cells obtained by cell fusion may retain all chromosomes or only some chromosomes. When used in the present invention, it is sufficient that the genome necessary for functioning the germ cells after transplantation as an egg or sperm when the undifferentiated germ cells of the donor are transplanted.

When an individual as a hybrid is produced by gene introduction, it can be produced by introducing the chromosome or gene of the other species into the egg of one species. The amount of chromosomes to be introduced can be any amount necessary for the transplanted germ cells to function as eggs or sperm when the donor undifferentiated germ cells are transplanted into the recipient hybrid. May be. For example, one or more chromosomes or a partial fragment thereof may be used as the chromosome to be introduced. Different chromosome combinations can also be introduced to find appropriate chromosomes that function after transplantation.

The gene to be introduced can be any gene as long as it is necessary for the recipient germ cell to function as an egg or sperm when the donor's primordial germ cell is transplanted into the recipient hybrid. Of course, one or more genes may be introduced. An example of such a gene is the gsdf gene.

In the present invention, the hybrid used for the recipient is preferably a cultivar, strain or organism having properties suitable for the recipient. Properties suitable for recipients include high survival rates in artificial breeding environments, small breeding equipment required, cost of equipment for temperature control needed, food cost, eggs or This is exemplified by the ease of collecting sperm. In the present invention, the recipient to be used preferably has inherited properties suitable for the recipient. In order for the created hybrid to inherit properties suitable for recipients, it must be obtained by including more of the genomes of varieties, strains or species with properties suitable for recipients in the genome of the hybrid. Can do. In order to include more genomes of varieties, strains or species that have properties suitable as recipients, artificially double eggs obtained from varieties, strains or species that have properties suitable as recipients. By doing so, you can have more genomes.

The hybrid preferably has a genome of a fish species that can be a surrogate parent fish for the donor (first) at less than half per cell in that it has less disadvantageous properties as a surrogate parent fish. More preferably, the genome of the (first) fish species that can be a surrogate parent fish for the donor is preferably 1/3 or less per cell. The amount of genome in the hybrid can be an amount calculated by DNA flow cytometry using somatic cells or blood cells selected as cells and using cells collected by a conventional method. For example, in the case of a hybrid cell having one set of genomes of fish species that can be surrogate parent fish (1) and one species of fish species that can be surrogate parent fish (1) fish species, hybrid cells The amount of genomes of fish species that can be surrogate parent fish in (1) is halved. At this time, the chromosome of the species of the fish species that can be a surrogate parent fish (first) if the chromosome of the fish species is missing or the DNA is deficient or can be the surrogate parent fish (first) In the case of a hybrid cell prepared by introducing into the genome of a fish species other than the fish species that can be a surrogate parent fish (first), the amount is less than half the amount of the donor genome. In the case of a hybrid cell with one set each of the genome of a fish species that can be a surrogate parent fish (1) and a doubled genome of a fish species other than the fish species that can be a surrogate parent fish (1) The amount of the genome of the fish species that can be a surrogate parent in this cell is 1/3. At this time, if the donor-derived chromosome is missing or if the donor-derived DNA is missing or can be a surrogate parental fish (first) a part of the chromosome or DNA of the fish species genome can be a surrogate parental fish (first 1) In the case of a hybrid cell prepared by introducing into the genome of a fish species other than the fish species, it can be confirmed that it is 1/3 or less of the amount of the genome of the fish species that can be a surrogate parent fish (first).

In the present invention, the first fish selected for obtaining the hybrid is a fish species that can be a surrogate parent fish for the donor. A fish species that can serve as a surrogate parent for a donor means that when a donor's germ cells are transplanted using a fish belonging to the fish species as a recipient, the donor's germ cells are engrafted and a donor gamete is obtained. A fish species that can The fish species that can be a surrogate parent fish for the first donor is preferably a donor or a fish species closely related to the donor. When a donor or a fish species closely related to the donor is transplanted with a donor germ cell, the donor germ cell is likely to be established and a donor gamete is easily obtained. In a hybrid, one or more genes of a donor or a fish species closely related to the donor help the donor's germ cells transplanted to the recipient to obtain donor-derived eggs or sperm It is considered possible. One or more genes that assist the donor's germ cells in the recipient transplant are considered to be on one or more chromosomes in the genome of the fish species closely related to the donor or donor. ing.

The fish species closely related to the donor are preferably selected from the same genera of the donor, and more preferably selected from the same genera of the donor. Specifically, when the donor is a genus char, the first fish species includes the same or a different species of fish as the donor, for example, a trout of the genus char, ashokokoma, Miyabeiwana, Brook trout, char, etc. Can be mentioned. When the donor is a species of yellowtail, examples of the first fish species include the same or different species of fish as the donor, and examples include the yellowtail yellowtail, kingfish, amberjack, and the like. When the donor is a genus Tuna, examples of the first fish species include the same or different species of fish as the donor, such as yellowfin tuna, albacore, southern bluefin tuna, Atlantic bluefin tuna, coshinaga and the like.

In the present invention, a (second) fish species that cannot be a surrogate parent fish for a donor means that the donor's germ cells are generated when the donor's germ cells are transplanted using the fish belonging to the fish species as a recipient. A fish species that is difficult to dress and obtain a donor gamete. The second fish species that cannot be a surrogate parent fish for the donor can include donor and heterogenous fish species. Recipients in surrogate parenting techniques are generally selected as closely related fish species that have suitable properties, but should be raised in the case of donors such as large fish. Due to easiness and the like, there are cases where it is not possible to select a breed, a line or a biological species having properties suitable as a recipient from a donor or a fish species belonging to the donor. That is, low survival rate in the artificial breeding environment, the size of the necessary breeding equipment, the cost of the equipment for the required temperature control, the cost of food, the difficulty of collecting eggs or sperm, For these reasons, it may not be possible to select varieties, strains or species having properties suitable as recipients. In such cases, a need arises to select donor and heterogenous fish species as recipients. However, when a donor's germ cells are transplanted, donor fish and heterogenous fish species are difficult to establish donor germ cells and to obtain donor gametes.

In the present invention, when such a second fish species is selected and used as a recipient, a hybrid of the second fish species and the first fish species is produced to produce the second fish species. Giving the fish species the ability to be a surrogate parent fish for the donor, allowing it to be used as a recipient, and using the breeding characteristics of the second fish species, etc., to efficiently obtain the gametes of the donor it can. The method for producing a donor gamete using the surrogate parent fish of the present invention is particularly effective as a method for artificially producing a hybrid in marine fish that does not often form a hybrid in nature. In particular, in the field of cultured fish, it has economic utility value in securing the supply of necessary seeds for fish.

In the case of exceeding the genus range, for example, if the donor is the genus Iwana, the second fish species can include salmon salmon genus salmon, rainbow trout, chinook salmon, etc. If the donor is a genus Buri, the second species of fish is the genus Maji of the genus Majiaceae, the maji of the genus Majiae, the Shimaji of the genus Ajiida, And the like, and the species of the family of the family H. When the donor is a tuna genus, the second fish species include mackerel of the family Sabaaceae Suma, yam, etc., mackerel Saba genus mackerel, sesame mackerel, etc., mackerel Ganoderma spp. , Species of the same genera with the donor, and fish species of the same family with the donor.

Furthermore, as a specific example, in the case of the salmonid genus Ivana, the survival rate is low due to poor feeding of the feed under artificial breeding, and a method for collecting eggs or sperm is well established. Because it is not, it is not suitable as a surrogate parent fish. Oshorokoma, which belongs to the same genus as char, has not been bred. The brook trout belonging to the same genera is an alien species, although its breeding method has been found, it is difficult to obtain it quickly. However, the same salmonid as char, like rainbow trout and yamame trout, is easy to collect eggs and spermatozoa, and there are fish species that have been well studied for surrogate parent fish technology. Are known. Therefore, in order to produce charcoal eggs or spermatozoa using surrogate parental fish technology, a hybrid that combines charcoal and horcoma genomes or genes of rainbow trout and yamame trout with either charcoal or charcoal genomes or genes closely related to char or char. Preferably, the hybrid is used as a recipient.

In addition, for example, in the case of the genus Yellowtail, a large facility is required for breeding, and the temperature control for collecting eggs or sperm is often expensive, so it is suitable as a surrogate parent fish. Not. For the same reason, amberjack and hiramasa that belong to the same genus are not suitable as surrogate parent fish. However, in the same family as the yellowtail, there are some fish species that are remarkably small in breeding facilities compared to yellowtails such as maji, blue mackerel, and yellowtail, and are suitable for use as surrogate parent fish. . Therefore, in order to obtain a gamelet of a yellowtail using a surrogate parent fish, either a yellowtail or a genome or gene of amberjack and whitefish closely related to the yellowtail, and a small size exemplified by horse mackerel, blue horse mackerel, and sea mackerel It is preferable to prepare a hybrid having both genomes or genes of a family of phlogopaceae and use the hybrid as a recipient. The hybrid obtained in this way can also be used as a surrogate parent fish of other fish species of the genus Buri, such as amberjack and kingfish.

For example, the bluefin tuna genus Tuna is not suitable as a surrogate parent fish because it requires extensive equipment for breeding and requires at least 3 years of breeding to collect eggs or sperm. In addition, the tuna species that are closely related to bluefin tuna, SBT, bigeye, albacore, cocinaga, yellowfin, etc. require extensive facilities for breeding, and no method for collecting eggs or sperm has been established. . However, in the same mackerel family as bluefin tuna, there are fish species that are small in breeding facilities and easy to collect eggs and sperm, such as cassava, sesame mackerel, suma, hirasoda and marsoda, and have characteristics suitable as surrogate parent fish. . Therefore, in order to obtain bluefin tuna eggs or sperm using surrogate parent fish technology, any of the bluefin tuna or bluefin bluefin tuna, bigeye, albacore, cocinaga, and yellowfin genomes or genes; It is preferable to prepare a hybrid having both the genome or gene of the mouse and the smartphone and use the hybrid as a recipient. The hybrid thus obtained can be used as a recipient of other fish species of the genus Tuna, such as southern bluefin tuna, bigeye, albacore, cocinaga and yellowfin.

In order to produce eggs or sperm using the surrogate parent fish technology of the present invention, a hybrid is produced, and the produced hybrid is used as a recipient to transplant a primordial germ cell of a donor and produce a transplanted fish. be able to. The transplanted donor primordial germ cells enter the recipient's gonads and produce donor-derived eggs or sperm. Accordingly, in the present invention, the production of a transplanted fish transplanted with a donor's primordial germ cells includes obtaining a donor-derived egg or sperm by the transplanted fish.

In order to obtain an egg or sperm derived from a donor from a transplanted fish, it can be obtained by inducing maturation and collecting by a conventional method according to the fish species (Japanese Patent No. 4300287). In particular, by preparing the hybrid as a heteroploid (Japanese Patent No. 4581083), a hybrid recipient that is a heteroploid can be used. In such a case, a recipient-derived egg or sperm is produced. Therefore, donor-derived eggs or sperm in the transplanted fish can be easily obtained.

In the present invention, the seedlings of fish used for aquaculture are produced from the transplanted fish obtained by breeding the produced hybrid and transplanting the donor's primordial germ cells using the hybrid as a recipient. A fertilized egg is obtained by crossing with a sperm or egg obtained from an individual, and the fertilized egg can be grown. Fish seeds and seedlings used for aquaculture can also be obtained by breeding the produced hybrid and crossing the donor's egg or sperm with the donor's egg or sperm obtained by transplanting the donor's primordial germ cells using the hybrid as a recipient. Obtainable. It can also be obtained by breeding the produced hybrid and crossing with eggs or sperm obtained by transplanting the donor's primordial germ cells using the hybrid as a recipient. The seedlings can be further grown by aquaculture to obtain adult fish.

The method for producing fish gametes (sperm and eggs) for the production of cultured fish using the surrogate parent fish of the present invention makes it possible to secure seedlings for aquaculture of donor fish species for which seedling production has been difficult until now. As a result, it becomes easy to cultivate the target donor fish species. The obtained adult fish can be widely used as cultured fish. Thus, the hybrid fish and surrogate parent fish technique hybrid in the present invention described above, sperm or egg obtained from the transplanted fish, seedlings obtained from the transplanted fish, adult fish obtained by growing the seedlings obtained from the transplanted fish, The present invention can be applied to artificial culture of various fishes, and can contribute to the construction of practical techniques for artificial culture of the fishes.

Hereinafter, the present invention will be described in detail with reference to examples. However, the present invention is not limited to them. In the following examples, “%” means “% by weight” unless otherwise specified.

[Example 1]
<(1) Transplantation of char spermatogonia into allotriploid host and creation of next generation>
A 13-month-old immature male char (weight approximately 43 g, body length approximately 15 cm) was used as a donor. The testis was removed from the char as donor, and cryoprotective solution (132 mM sodium chloride, 2.56 mM potassium chloride, 8.13 mM potassium dihydrogen phosphate, 1.34 mM disodium hydrogen phosphate, 0.9 mM calcium chloride, magnesium chloride) 0.5 mM, sodium pyruvate 1.28 mM, Hepes 19.5 mM, dimethyl sulfoxide 1.3 M, chicken egg yolk 10%, trehalose 0.1 M) and frozen in liquid nitrogen. Freezing was slowly frozen to -80 ° C at a rate of -1 ° C / min. One week after frozen storage, thawing was performed. The thawed char testis was dispersed by reacting in PBS containing trypsin (0.9 U / ml), fetal calf serum (5%), DNase (100 U / ml) to obtain a cell suspension. The obtained cell suspension was transplanted into the peritoneal cavity of the host by 5000 cells using an injector.

The recipients used were triploid brook trout embryos that had passed 43 days after fertilization, allotriploid rainbow trout-river hybrid embryos 38 days after fertilization, and triploid rainbow trout embryos 30 days after fertilization. Transplantation was performed on 58 triploid brook trout embryos, 60 allogeneous triploid rainbow trout-river hybrid embryos and 61 triploid rainbow trout embryos. The rainbow trout and brook trout hybrid embryos were obtained by artificial insemination of unfertilized eggs and brook trout semen extracted from ovulated female rainbow trout. The brook trout, rainbow trout-river trout hybrid, and rainbow trout eggs were doubled by treating each egg for 5 minutes at 38 ° C. 5 minutes after fertilization.

One year after transplantation, 52 hosts (89.7% of the total number of transplants) were obtained for each host of triploid brook trout, allotriploid rainbow trout-river trout hybrid, and triploid rainbow trout transplanted with testicular cells of donor char. 53 (80.0%) and 55 (90.2%) survived, 5 (19.2% of the number of surviving), 4 (16.0%), 2 ( 6.5%) matured and produced gametes. All of the mature individuals were male, and as a result of fertilizing the obtained sperm with wild-type char char eggs, the char char next generation individual was obtained. The obtained next generation was confirmed to be char by the PCR method for detecting wrinkle patterns, body patterns, vertebral number, and restriction fragment length polymorphism. The results are shown in Table 1.

Two years after transplantation, the host of triploid brook trout, allogeneic triploid rainbow trout-river trout hybrid, triploid rainbow trout hosts were 48 (82.7% in the number of transplants) and 45 (75.0%), respectively. ), 48 (78.7%) survived, 14 (29.2% of surviving), 9 (20.0%), and 3 (6.3%) mature did. The breakdown of mature males and females (number of females: males) was 5: 9 for triploid brook trout hosts and 3: 6 for allotriploid rainbow trout-river hybrids. Adults of triploid rainbow trout were males. As a result of artificial insemination using sperm and eggs obtained from each host, the next generation individual of char was derived from donor char. The results are shown in Table 1.

Three years after transplantation, each host of triploid brook trout, allotriploid rainbow trout-river trout hybrid, triploid rainbow trout was 45 (77.6% in the number of transplants) and 39 (65.0%), respectively. ), 41 (67.2%) survived, 19 (42.2% of surviving), 9 (23.1%), 4 (9.8%) mature did. The breakdown of mature males and females (number of females: males) was 7:12 for the triploid brook trout host and 3: 6 for the allotriploid rainbow trout-river hybrid host. Adults of triploid rainbow trout were males. As a result of artificial insemination using sperm and eggs obtained from each host, it was found that the transplanted fish can survive for more than 3 years, and the next generation individual of char char derived from donor char is obtained. The results are shown in Table 1.

From the above results, when char was used as a donor, gametes derived from the donor were most efficiently produced when triploid brook trout was used as a host. In addition, if the host is a distantly related rainbow trout, it will produce char spermatozoa, but will not produce char, so it would be advantageous to use a more closely related host to produce donor-derived eggs. It was shown that. On the other hand, donor-derived eggs have been produced by using a hybrid of a distantly related rainbow trout and a closely related brook trout as a host. From this, it was considered that the production efficiency of donor-derived gametes can be remarkably increased by using a recipient having a closely related gene.

<(2) Properties of gametes of triploid recipients>
Of the triploid brook trout, triploid rainbow trout, and heterogeneous triploid rainbow trout-river trout hybrids obtained above, eggs from each female (2 and 3 years old) were collected by squeezing and I counted. The results are shown in Table 2.

Of the triploid brook trout, triploid rainbow trout, and heterogeneous triploid rainbow trout-river trout hybrids obtained above, sperm at each male (2 and 3 years of age) was collected by the extraction method to obtain blood cells. The number was confirmed using a calculation panel. The results are shown in Table 3. For comparison, the number of eggs in diploid rainbow trout, diploid brook trout, diploid char, 2 and 3 year old females, and the number of spermatozoa in males, which were not subjected to triploidization treatment, were similarly determined. Confirmed and shown in Table 3.

The function of the gametes obtained from each recipient was confirmed. The function of the gametes obtained by fertilizing the eggs and sperm obtained above by the dry induction method was confirmed by the eye rate and hatching rate. The eye development rate was evaluated by counting the number of eggs with black pigment deposited on the retina 3 weeks after fertilization. The hatching rate was evaluated by counting individuals that had completed hatching 35 days after fertilization. The results are shown in Table 4. * Indicates that all triploid female individuals were infertile.

As shown in Table 3, it was found that any triploid host gametes transplanted with frozen testis cells can produce donor-derived functional gametes. Donor sardines and brook trout have the same genus. Since triploid brook trout and triploid rainbow trout-river trout hybrids can produce both eggs and sperm, introducing a single set of donor and closely related genomes into a host will increase the gametogenic efficiency of infertile hosts. It was shown to be.

[Example 2]
<Transplantation of yellow spermatogonia into heterologous triploid hosts>
A 10-month-old immature male yellowtail (weight: about 1 kg, body length: about 40 cm) was used as a donor. The testis was extracted from the donor yellowtail, and the cell suspension obtained by dispersing by the enzyme treatment was transplanted into the peritoneal cavity of the host 20,000 cells. As a host, 92 alien haploid Blima maji hybrid larvae 10 days after hatching were used. Heterotriploid yellowtail horse mackerel hybrid larvae were obtained by artificially inseminating a horse mackerel unfertilized egg and a yellowtail sperm and pressurizing to 650 kg / cm ² with a French press for 5 minutes after fertilization. One month after transplantation, 18 fish (19.5% of the number of transplanted fish) survived. The resulting allotriploid burma-maji hybrid is suitable as a recipient for the germ cell of the genus Buri.

[Example 3]
<Creation of allotriploid fish for tuna germ cell transplant host>
Suma eggs and bluefin tuna spermatozoa, which are relatively small fishes from the mackerel family, were artificially crossed (population cross) to produce a Suma-Tuna hybrid. By applying the bluefin tuna sperm to the egg of the suma, a fertilized egg of the suma-bluefin tuna hybrid was obtained. The fertilized egg of the Suma-bluefin tuna hybrid hatched, but the larvae died without feeding. Therefore, the fertilized egg of the Suma-bluefin tuna hybrid was subjected to a triploidization treatment. The triploidization treatment was performed by immersing a fertilized egg of a Suma-bluefin tuna hybrid in seawater cooled to 4 ° C. for 5 minutes after fertilization. The hybrid triploid fertilized egg thus obtained possesses two sets of suma chromosomes and one set of bluefin tuna chromosomes.

The hatched suma-bluefin tuna hybrid larvae are composed of primer sets (TGC サイズ AGA ACG AAC AGG ATG AG and CCC ATT GAG GAG ATT GGA GA) and primers that are amplified only in suma and bluefin tuna, each species. The PCR method using the sets (ACA-TGG-TCC-ATC-CAT-CCA-TT and TGG-CTT-AGC-TCT-ACC-CCA-AA) was shown to possess each genetic information. Moreover, it was confirmed by DNA flow cytometry using somatic cells that the triploid has 3 sets of chromosomes. Thus, it was shown that a hetero-triploid of a suma-bluefin tuna hybrid could be produced. This allotriploid survived until 5 days after hatching, and feeding was also observed. The obtained Suma-Tuna hybrid allotriploid is suitable as a recipient of a germ cell of a Tuna donor.

The entire disclosure of Japanese Patent Application No. 2014-064639 filed on March 26, 2014 is incorporated herein by reference. All documents, patent applications, and technical standards mentioned in this specification are to the same extent as if each individual document, patent application, and technical standard were specifically and individually stated to be incorporated by reference, Incorporated herein by reference.

The present invention enables use for surrogate parent fish such as other genus fish species, and allows easy use of fish species such as other genus breeding as recipients in surrogate parent fish technology. It is possible to select a fish species that is suitable as an ent, and it is possible to provide technology for practical use as a production method of fish gametes (sperm and eggs) for the production of cultured fish using surrogate parent fish did.

Claims

Gametes of fish using surrogate parent fish, including transplanting germ cells derived from recipient fish and transplanting germ cells of a different or different lineage from recipient fish into recipient fish In the production method of
(A) selecting a donor fish species that supplies isolated germ cells;
(B) selecting a fish of a first species that can be a surrogate parent for the donor species;
(C) selecting a fish of a second species that cannot be a surrogate parent for the donor species;
(D) The fish of the first species selected in (B) and the fish of the second species selected in (C) are hybridized to become a surrogate parent for the donor species. Creating hybrid fish species with the ability to gain,
(E) Using the hybrid fish species produced in (D) as a recipient, transplanting the isolated germ cells of the donor fish species to induce differentiation into the germ line,
Method for producing fish gametes using surrogate parent fish including
The fish gamete production method using the surrogate parent fish according to claim 1, wherein the fish gamete is a sperm or egg of a donor fish species.
The fish of the first species that can be a surrogate parent for the donor species is a fish that has the ability to engraft the isolated germ cells of the transplanted donor species, and The fish of the second fish species that cannot be a surrogate parent fish is a fish of a fish species that does not have the ability to engraft the isolated germ cells of the transplanted donor fish species. Or the production method of the fish gametes using the surrogate parent fish of 2 description.
The fish using the surrogate parent fish according to any one of claims 1 to 3, wherein the fish of the first fish species that can be a surrogate parent fish for the donor fish species belongs to the same genus as the donor fish species How to produce gametes.
The surrogate parent fish according to any one of claims 1 to 4, wherein the fish of the second fish species that cannot be a surrogate parent fish for the donor fish species is different from the donor fish species. Production method of fish gametes.
The method for producing fish gametes using the surrogate parent fish according to any one of claims 1 to 5, wherein the donor fish species is genus Ivana, Yellowtail or Tuna.
A hybrid fish species capable of being a surrogate parent for a donor fish species has less than half the genome per cell of a first fish species that can be a surrogate parent for a donor fish species Item 7. A fish gamete production method using the surrogate parent fish according to any one of Items 1 to 6.
A hybrid fish species capable of becoming a surrogate parent fish for a donor fish species has a genome of a first fish species that can be a surrogate parent fish for a donor fish species at 1/3 or less per cell. A method for producing fish gametes using the surrogate parent fish according to claim 7.
The hybrid fish species capable of becoming a surrogate parent fish for the donor fish species are heteroploid, and are infertile hybrid fish species that do not produce gametes derived from the hybrid fish species. A method for producing fish gametes using the surrogate parent fish according to any one of 1 to 8.
A hybrid fish species that has the ability to be a surrogate parent to a donor species is an allopolyploid, and an infertile hybrid species that does not produce sperm and eggs from the hybrid fish species is triploid. A method for producing fish gametes using the surrogate parent fish according to claim 9.
In the method of producing hybrid fish as a recipient,
(A) selecting a donor fish species that supplies isolated germ cells;
(B) selecting a fish of a first species that can be a surrogate parent for the donor species;
(C) selecting a fish of a second species that cannot be a surrogate parent for the donor species;
(D) The fish of the first species selected in (B) and the fish of the second species selected in (C) are hybridized to become a surrogate parent for the donor species. Creating hybrid fish species with the ability to gain,
Of hybrid fish including
Using recipient fish, transplanting the isolated germ cells of a donor fish species of a different or different species from the recipient fish to the recipient fish to induce differentiation into germ line In the method for inducing differentiation of a separated germ cell into a germ line, the hybrid fish species produced in claim 11 is used as a recipient fish to transplant and induce differentiation of a separated germ cell of a donor fish species. A method for inducing differentiation of a separated germ cell into a germ line using a surrogate parent fish.
Hybrid fish produced by the method according to claim 11.
An egg or sperm of a donor fish species produced by the method for inducing differentiation of a separated germ cell into a germ line using the surrogate parent fish according to any one of claims 1 to 10.
A seedling for aquaculture of a donor fish species produced from an egg or sperm of the donor fish species according to claim 14.
An adult donor fish species obtained by cultivating the seedling for breeding a donor fish species according to claim 15.
Recipient fish, and transplantation of fish using surrogate parent fish including transplanting germ cells of a different or different lineage from recipient fish into recipient fish and inducing differentiation into germline. In the production method,
(A) selecting a donor fish species that supplies isolated germ cells;
(B) selecting a fish of a first species that can be a surrogate parent for the donor species;
(C) selecting a fish of a second species that cannot be a surrogate parent for the donor species;
(D) The fish of the first species selected in (B) and the fish of the second species selected in (C) are hybridized to become a surrogate parent for the donor species. Creating hybrid fish species with the ability to gain,
(E) A method for producing transplanted fish using a surrogate parent fish, comprising transplanting the isolated germ cells of a donor fish species using the hybrid fish species produced in (D) as a recipient.
The fish of the first species that can be a surrogate parent for the donor species is a fish that has the ability to engraft the isolated germ cells of the transplanted donor species, and The second fish species that cannot be a surrogate parent fish is a fish of a fish species that does not have the ability to engraft the isolated germ cells of the transplanted donor fish species into the gonads. A method for producing transplanted fish using the surrogate parent fish described in 1.
The method for producing a transplanted fish using a surrogate parent fish according to claim 17 or 18, wherein the fish of the first fish species that can be a surrogate parent fish for the donor fish species belongs to the same genus as the donor fish species. .
The surrogate parent fish according to any one of claims 17 to 19, wherein the fish of the second fish species that cannot be a surrogate parent fish for the donor fish species is different from the donor fish species. There was a production method of transplanted fish.
The method for producing transplanted fish using the surrogate parent fish according to any one of claims 17 to 20, wherein the donor fish species is genus Ivana, Yellowtail or Tuna.
A hybrid fish species capable of being a surrogate parent for a donor fish species has less than half the genome per cell of a first fish species that can be a surrogate parent for a donor fish species Item 22. A method for producing transplanted fish using the surrogate parent fish according to any one of Items 17 to 21.
A hybrid fish species capable of becoming a surrogate parent fish for a donor fish species has a genome of a first fish species that can be a surrogate parent fish for a donor fish species at 1/3 or less per cell. A method for producing fish gametes using the surrogate parent fish according to claim 22.
The hybrid fish species capable of becoming a surrogate parent fish for the donor fish species are heteroploid, and are infertile hybrid fish species that do not produce gametes derived from the hybrid fish species. 24. A method for producing transplanted fish using the surrogate parent fish according to any one of items 1 to 23.
A hybrid fish species that has the ability to be a surrogate parent to a donor species is an allopolyploid, and an infertile hybrid species that does not produce sperm and eggs from the hybrid fish species is triploid. 25. A method for producing fish gametes using the surrogate parent fish according to claim 24.