JP2003235558A - Method for differentiation and induction to germ line by transplantation of separated primordial germ cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for transplanting a genetically modified or a separated cell to a host individual and differentiating and inducing the cell into a germ line in a vertebrate such as fishes and a method for propagating or breeding the vertebrate such as the fishes by using the method for differentiation and induction. <P>SOLUTION: The differentiation and induction of a primordial germ cell derived from the vertebrate such as the fishes into the germ line can be carried out by transplanting the separated primordial germ cell into the early germ of a host vertebrate, especially transplanting thereof to the back side of an intraperitoneal intestinal tract membrane of the host vertebrate in the early development stage. The primordial germ cell is differentiated and induced to oocytes or spermatogonia in the vertebrate individual and further differentiated and induced to ova or sperms by using the method for differentiation and induction of the separated primordial germ cell to the germ line. Thereby, the differentiation and induction of the primordial germ cell to the germ line are carried out by the method and the vertebrate can be propagated and bred by using the method. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel breeding and breeding method in vertebrates, and a method for inducing germ line differentiation using isolated primordial germ cells, particularly in a thermophilic vertebrate such as fish, and The present invention relates to a method for breeding or breeding vertebrates using a differentiation induction method.

[0002]

2. Description of the Related Art In recent years, for example, in mammals,
For the production of transgenic animals and cloned animals, various gene introduction techniques and propagation techniques have been developed as methods for modifying and cloning animal individuals. The most widely used method for introducing a foreign gene into a mammalian individual is the injection of DNA into the pronucleus of the fertilized egg, which is used in many animal species including livestock. . Although this method is a relatively reliable method, it has a low gene transfer rate and requires a large cost particularly when applied to large animals. In addition, a method of introducing a gene by treating sperm with DNA for fertilization, for example, by mixing mouse frozen sperm and DNA and then performing fertilization by an intraovular injection method, the gene is introduced at a high rate. It has been reported that mice can be obtained. This method is not yet applicable to a wide range of strains of mice and other animal species.

As a method for genetically modifying mammals and the like, a vector having a homologous portion with an endogenous gene is introduced,
There is a method of performing genetic modification by replacing the target gene on the chromosome with a foreign vector. For example, it is a method of carrying out targeted recombination (gene targeting) for the purpose of genetic modification or gene disruption, and selecting those that have undergone homologous gene recombination. Furthermore, at present, after carrying out gene transfer and selection using an embryonic stem cell (ES cell) line, a chimeric mouse in which a germline gene has been introduced using the embryonic stem cell is prepared, and a target gene-modified mouse is obtained by mating. A method of making an individual has been reported. However, at present, ES cell lines are present in only a few mouse strains, making it difficult to apply the method to a wide range of animals. Therefore, it has been attempted at present in domestic animals and the like that ES cell lines do not exist. Target gene recombination is carried out by inducing homologous gene recombination in somatic cell lines and then performing animal cloning by nuclear transfer to enucleated ova. It is a method of making animals. In addition, in recent years, a gene transfer method to spermatogenic cells present in the testis has also been proposed for the purpose of producing a highly transformed animal that can be applied to large animals such as livestock (Japanese Patent Laid-Open No. 2001-2001). No. 309736). As mentioned above, in an individual such as a mammal,
For the production of transgenic animals and cloned animals, various gene introduction techniques and propagation techniques have been developed as methods for modifying and cloning animal individuals.

Various methods for modifying and cloning individual animals for producing transgenic animals and cloned animals have been tried in vertebrates such as birds and fish. In recent years, a technique for producing germline chimeric individuals using primordial germ cells in birds such as chickens has been reported (J. Reprod. Fert., 96 (1992) 521-528). In this method, in birds, primordial germ cells are present in the blood of the embryo and circulate, and since they are huge cells, the germ cells are separated from the blood and the separated cells are transplanted. The simple method of putting it back into the blood is adopted. However, this method is applicable only to birds in which primordial germ cells are present in the blood of the embryo and is a huge cell, and is not applicable to other vertebrates. is there. Various methods for gene modification and cloning of individual animals have been attempted even in vertebrate animals such as fish. For vertebrates like fish,
Usually, a large amount of formed eggs and sperms are fertilized outside the individual, and proliferation is performed in a system that gives rise to a new individual, and ES cells such as in mammals have not been obtained. Therefore, in order to develop a method for modifying or cloning an individual animal by applying it as a practical method in a vertebrate such as a fish, there is a technical problem different from that in the mammal as described above. Exists.

Since the first successful case of introducing a foreign gene into a fish was reported in the mid-1980s, gene transfer research in fish has recently been carried out in model experimental fish such as zebrafish and medaka fish. Gene function analysis using ATP has been actively attempted (Transgenic A
nimals, Generation and Use. Harwood Academic Publ
ishers, Amsterdam. 1997; 387-395). Medaka and zebrafish are prolific and have a short maturation period. Furthermore, since the early embryo is transparent, it has the advantage that it is suitable for observation of early development, and in recent years, it has rapidly begun to be used as a model animal for basic biology.
In addition, a large-scale screening system for mutants has been established in these fish species, and we are vigorously proceeding with the genome project, and it is expected that the frequency of use as a model animal will increase in the future. (Protein nucleic acid enzyme, 45-17 (2000), 2667-2982).

The following are currently proposed as techniques for gene transfer into fish. First, the most widely used method for gene transfer into fish is the microinjection method into fertilized eggs. After egg fertilization (after contact with environmental water depending on the species), the surface protoplasts of fish eggs accumulate on the polar side of the animal and form the scutellum. This method is a method in which a foreign gene is injected into the cytoplasm of the scutellum using a glass fine pipette having an outer diameter of 5 to 10 μm at the tip.
Fertilized eggs of fish have large diameters of 0.7 mm (zebrafish and many marine fish) to 6 mm (salmonidae), and thus gene injection work is usually performed under a stereomicroscope. However, in the presence of a huge yolk, visualization of the nucleus existing in the scutellum is difficult, so that the foreign gene is injected into the cytoplasm. Approximately 50 to 20 depending on fish species
By injecting a very large amount of foreign gene of 0 pg, it is possible to produce transgenic fish without injection into the nucleus.

[0007] In some cases, the roe that develops outside the mother has a very strong egg membrane or a thick and low-transparent egg membrane to protect the embryo. Such an egg membrane becomes a major obstacle in microinjection. In the case of Cyprinidae, it is possible to produce transgenic fish by physically removing the egg membrane using proteolytic enzymes or tweezers, injecting the gene into naked eggs, and then culturing in salt solution. On the other hand, the egg membrane of salmonids is extremely strong and cannot be removed by commercially available proteolytic enzymes. Therefore, methods such as gene injection into an egg immediately after fertilization and before hardening of the egg membrane, and insertion of a fine pipette from the ovale have been developed (“Fish DNA” (Koseisha Koseikaku), 1997, 80-98).

[0008] Yoshizaki et al. Succeeded in suppressing the hardening of the egg membrane by fertilizing rainbow trout eggs in a reduced glutathione solution. When microinjecting a foreign gene into these fertilized eggs, about half of the treated eggs were treated. We have found that we will become transgenic individuals (Nippon Suisan Gakkaishi,
57 (1991), 819-824). In addition, Ozato et al. Took out oocytes before maturation in vitro and produced a gene-transfer medaka by a method of directly microinjecting a foreign gene into the nucleus (Cell Differ, 19 (1986), 237-4).
Four). Although this method is complicated to operate, the oocyte has a large egg membrane and a huge nucleus called a follicle vesicle, so it is an extremely rational method that solves the above two problems all at once. is there. In addition, in a fish species having a thin egg membrane such as zebrafish, it is easy to perform microinjection on a normal fertilized egg. In this way, the microinjection method can be applied to many fish species by devising it according to the target fish species.

On the other hand, the electroporation method is also considered useful as a method for producing transgenic fish. This is a method in which an electric pulse is applied to sperm before fertilization or a fertilized egg to form a transient pore in the cell membrane, and a foreign gene is allowed to flow into the cell from there. This method has the advantage that a large amount of individuals can be treated at one time and the operation method is easy, but since the amount of foreign gene that can be introduced per cell is smaller than that of the microinjection method, It is customary that the efficiency and production efficiency of transgenic fish lines are not very high. Recently, a new method utilizing the difference in osmotic pressure has been developed in order to improve the efficiency of introducing a foreign gene into sperm by electroporation. Spermatozoa of the carp family begin to move when the osmotic pressure of the external environment decreases, and then lose fertility in tens of seconds to minutes. Therefore, first, the spoiled carp sperm is immersed in a higher harmonic solution to dehydrate the sperm. After that, by returning the dehydrated sperm to the isotonic liquid, the sperm suddenly start absorbing water. By carrying out this water absorption in a foreign gene solution and applying an electric pulse at the same time, it became possible to efficiently flow the foreign gene into the sperm. In fact, it was reported that the efficiency of transgenic fish production was increased to more than 3 times that of the conventional method by using this method (Aquaculture, 173 (1
999), 297-307).

Recently, Lin et al. Have shown that retroviruses modified to carry the G-protein of vesicular stomatitis virus capable of infecting many animal species infect zebrafish. We have succeeded in producing transgenic zebrafish using this viral vector (Science. 265 (1994), 666-669). The system using these viral vectors has the advantage that the foreign gene does not form a concatemer and the foreign gene is not easily modified in the host cell. In addition, a method using gene cancer and a method of introducing a gene by directly injecting a foreign gene into a living tissue have been developed.

The behavior of the foreign gene in the fish body when the gene is introduced by the microinjection method into the fertilized egg or oocyte or the electroporation method into the sperm or fertilized egg shows that the foreign gene is the host individual. It is not immediately integrated into the chromosome, and may exist in an extrachromosomal free state during the early stages of development. When a foreign gene is introduced into a rainbow trout fertilized egg by the microinjection method, the amount of the foreign gene per cell sharply decreases by the blastula stage, and then slowly decreases until the hatching period. It is considered that the foreign gene is integrated into the chromosome of the host cell after the cleavage progresses to some extent, and that this integration phenomenon occurs independently in each blastomere (“DNA of fish” (Stellars). Koseikaku) 1997, 80-98). In many cases, the foreign gene forms a concatemer in which several to several hundred copies are linked,
It is integrated at one site to several sites on the host chromosome.

As described above, since the integration of the foreign gene into the host chromosome occurs after the initiation of development, cells having the foreign gene and cells not having the foreign gene are mixed in a mosaic pattern in the host individual. . Furthermore, since integration of a foreign gene occurs independently in each blastomere, it is often observed that cells that carry the foreign gene at different positions on different chromosomes are mixed in a mosaic pattern. This mosaic integration of a foreign gene is a phenomenon that has always been observed using any of the methods tried to date (Transgenic Res. 5 (1996), 147-16.
6). Normally, a foreign gene integrated into the host chromosome is inherited to the next generation through the germline, but in the first generation (founder generation) of transgenic fish, the foreign gene is also mosaic in this germline. Exists. In some cases, a foreign gene is often integrated into the somatic cell line, but is not integrated into the germ line, and the foreign gene is not transmitted to the F1 generation at all. Many reports show that transgenic parental fish are F1
When a generation is created, the proportion of transgenic F1 individuals in it is 10% or less. Thus, a problem with the current technique for producing transgenic fish is low efficiency in systemizing transgenic fish.

On the other hand, analysis of promoters derived from fish has progressed remarkably in the last 10 years, and techniques for accurately controlling the expression of foreign genes in transgenic fish have been steadily making progress. Expression of endogenous genes in fish is rapidly activated from the middle stage of blastula. When a ubiquitously expressed type of promoter is used in the production of transgenic fish, the foreign gene also shows transient and extremely high level expression immediately after the middle stage of the blastula. Although the biological significance of this transient expression is not clear, a large amount of foreign genes are usually introduced into fish eggs, and these foreign genes are not methylated. It does not work, so to speak, it is thought that gene expression is in a runaway state (Nucleic
Acids Res, 26 (1998), 4454-4461).

In recent years, promoter analysis of various genes has been particularly advanced using zebrafish, and promoters that are specifically expressed in erythroblast cells or brain neurons, or promoters whose expression is induced by various stresses, etc. Isolated (protein nucleic acid enzyme, 45 (200
0), 2954-2961). In rainbow trout, it has been reported that the vasa gene promoter is specifically activated in the germ line (J. Dev. Biol. 44 (2000), 323-.
326). A similar vasa promoter was subsequently isolated from medaka, and a similar activity was reported (protein nucleic acid enzyme, 45 (2000), 2954-2961). In the future, it is expected that many time-specific and tissue-specific promoters will be isolated centering on zebrafish and medaka fish. It becomes an important issue for controlling the expression of genes.

A gene targeting technique has been proposed as a technique for a genetic modification method in fish. This technique is a technique for destroying or modifying only the gene of interest, but it is already established in mammalian mice. The production of gene-targeted animals requires cultured cells that can be modified into individuals. In mice, as cells that meet these conditions,
Embryonic stem cell ES cells are used. Mouse E has been used for more than 10 years in fish.
Technical development has been carried out with the aim of establishing a cell line having characteristics similar to S cells. An excellent review of fish ES cell studies by Hong (Fish Physiol Biochem, 22
(2000), 165-170).

[0016] Many researchers have attempted to establish an ES cell line of fish, and a cell line similar in morphological and biochemical characteristics to mouse-derived ES cells was medaka (protein nucleic acid enzyme, 40, 2249-2256, 1995; Fish Phys. Biochem.
22, 165-170, 2000), Zebrafish (Methods Cel
Biol. 59: 29-37, 1999), and European sea bream (Bi
o Molecular Engineering, 15, 125-129, 1999). It has already been confirmed that when these cells are transplanted into a host embryo around the blastula stage, the transplanted cells are differentiated into various somatic cells. As mentioned above, E of fish
Attempts to establish an S cell line have been made by many researchers, but no paper has been published that fish ES-like cells differentiated into germ cell lines and contributed to the generation of the next generation. In fact, cells that have been cultivated in vitro for only a few days also differentiate into the germ line (Proc. Natl. Ac
ad. Sci. USA, 98, 2261-2266, 2001), when the culture period is extended, the ability to differentiate into germ cells suddenly disappears.

The mechanism by which fish germ cells differentiate from other somatic cells is not yet clear, but in recent years, R accumulated in the maturing egg in the parent ovary has been investigated.
Data suggesting that maternal factors such as NA and proteins may play important roles in germline determination have been published (Monthly Ocean, 31-5, 266-271, 1999). Since these maternal factors are heterogeneously present in the fertilized egg, only some blastomeres will receive this factor due to cell division. As a result, it is believed that only some of the cells that receive the maternal factor will differentiate into germline in the future. On the other hand, in mice where ES cells are known to differentiate into germ cells, it is considered that a cell population that maintains an undifferentiated state is differentiated into germ cells by stimulation from peripheral cells. (Protein nucleic acid enzyme, 43, 405-411, 1998). Considering the germline determination mechanism in this way, the condition required for a germline-differentiating cell line in mice is “undifferentiated”, but in the case of fish, it is “germline-differentiating”. Including a maternal factor that determines differentiation ”. This is consistent with the above-mentioned phenomenon that ES cells of undifferentiated mice differentiate into germ cells, but ES-like cells of fish differentiate only into somatic cells.

As described above, in vertebrates such as fish, attempts have been made so far to modify or clone individual animals to produce transgenic animals or cloned animals. However, in the case of vertebrates such as fish, such genetically modified or cloned cells are transplanted into a host individual, which is induced to differentiate and converted into a new individual. The technology is not established. Therefore, in vertebrates such as fish, in order to genetically modify or clone the individual, to breed or to produce a cloned animal,
The establishment of a technique for transplanting a modified or separated cell into a host individual, inducing differentiation of the host individual, and converting it as a new individual is an important issue.

[0019]

The object of the present invention is to provide a novel method for propagation or breeding in vertebrates, in particular, in genetically modified or isolated cells in anthermic vertebrates such as fish. It is intended to provide a method of transplanting to a host individual and inducing differentiation into a germ cell line, and a method of growing or breeding vertebrates such as fish using the differentiation induction method.

[0020]

Means for Solving the Problems The present inventor has proposed a method for transplanting a genetically modified or isolated cell in a vertebrate such as a fish to a host individual and inducing differentiation into a germ cell line. As a result of intensive research, (1) in mice in which ES cells are known to differentiate into germ cells, the cell population that maintains an undifferentiated state is transformed into germ cells by stimulation from peripheral cells. While it is considered to differentiate, in vertebrates such as fish, as described above, maternal factors such as RNA and protein accumulated in the maturing egg in the parent ovary are , Play an important role in germ line determination, and because of the heterogeneity of these maternal factors in fertilized eggs, only some blastomeres receive this factor due to cell division. Result, motherhood It is thought that only a part of the cells that receive the offspring will be differentiated into germ line in the future. (2) Considering such a germ line determination mechanism, in mouse, it is differentiated into germ line. Although the condition required for a possible cell line is "to be undifferentiated",
In the case of vertebrates such as fish, "containing a maternal factor that determines the differentiation into germ cells" (3) Considering the above facts, vertebrates such as fish are Cells that have been modified or separated into a host individual and are to be used when inducing differentiation into a germline (that is, after transplantation into a host individual, differentiate into an egg or sperm, and transform into a next-generation individual) Potential cells) are not undifferentiated embryonic cells, but stem cells of germ cells that are determined to differentiate into germ cells in the future, that is, primordial germ cells, and the primordial germ cells are identified. , It has been found that primordial germ cells can be induced to differentiate into germ cells by transplantation into early vertebrate embryos such as fish. That is, in the present invention, isolated primordial germ cells derived from vertebrates such as fish are transplanted into the early embryo of the host vertebrate, particularly, by transplanting into the abdominal lining of the abdominal cavity of the host vertebrate in the early developmental stage. Have found that it is possible to induce differentiation of the primordial germ cells into germ line.

In inducing the differentiation of the isolated primordial germ cells into the germ cell line in fish, the primordial germ cells isolated from the embryos before and after hatching are used as the isolated primordial germ cells, and transplanted to the host early embryo before and after hatching. It can be suitably carried out by transplanting the host into the abdominal lining of the abdominal cavity of the host in the developmental stage. Using the method for inducing germ cell differentiation of isolated primordial germ cells of the present invention, in a vertebrate individual, primordial germ cells are induced to differentiate into oocytes or spermatogonia, and then to ova or sperm. Thereby inducing differentiation of primordial germ cells into germ line, and using the method,
Propagation and breeding of vertebrates can be performed.

That is, the present invention is a method for inducing differentiation of a separated primordial germ cell into a germ cell line, which comprises transplanting a separated primordial germ cell derived from a vertebrate into an early embryo of a host vertebrate (claim 1). Or to the germ line of the isolated primordial germ cell according to claim 1, wherein the transplantation into the early embryo of the host vertebrate is transplantation into the abdominal lining of the abdominal cavity of the host vertebrate in the early developmental stage. Differentiation induction method (Claim 2)
Or a primordial germ cell derived from a vertebrate, which is a primordial germ cell obtained by visualizing a primordial germ cell of a vertebrate, to the germ line of the isolated primordial germ cell according to claim 1 or 2. The method of inducing differentiation of the bacterium (Claim 3) or the visualization of primordial germ cells encodes a protein capable of visualizing primordial germ cells in a living body, in the regulatory region of a gene specifically expressed in vertebrate germ cells. The method for inducing differentiation of an isolated primordial germ cell into a germ cell line according to claim 3, which is carried out by incorporating a plasmid into which a marker gene is incorporated into the cytoplasm of a vertebrate fertilized egg. 4) and the gene specifically expressed in vertebrate germ cells is the vasa gene, and the marker gene encoding a protein capable of visualizing primordial germ cells in the living body is Method for inducing differentiation into germ line (Claim 5) and the separation primordial germ cells of claim 4, wherein the a gene encoding a photoprotein FP,
Induction of differentiation of isolated primordial germ cells into germline cells, induction of differentiation of primordial germ cells into oocytes or spermatogonia,
Alternatively, the method for inducing differentiation into germ cells of isolated primordial germ cells according to any one of claims 1 to 5 (claim 6), or vertebrate origin, which is characterized by further inducing differentiation into eggs or sperms. 7. The isolated primordial germ cell of claim 1 is a primordial germ cell derived from a vertebrate different from or different from the host vertebrate. Differentiation induction method (Claim 7)
Alternatively, the isolated primordial germ cell derived from vertebrate is a genetically modified primordial germ cell isolated from a vertebrate, and the germ cell of the isolated primordial germ cell according to any one of claims 1 to 7. The method for inducing differentiation into a lineage (claim 8) or the isolated primordial germ cells derived from vertebrates are isolated primordial germ cells derived from fish, and the transplantation of the isolated primordial germ cells into the early embryos of the host vertebrate is performed in fish. 9. A method for inducing differentiation of isolated primordial germ cells into a germ cell line according to any one of claims 1 to 8, which is transplantation into a host early embryo.
Alternatively, the isolated primordial germ cells are primordial germ cells separated from the embryos before and after hatching, and / or the transplantation to the early embryos of the host can be performed to the back of the peritoneal intestinal membrane of the host in the developmental stage before and after hatching. The method for inducing differentiation of an isolated primordial germ cell into a germ cell line according to claim 9 (claim 10)
Consists of.

Further, the present invention uses the method for inducing differentiation according to claim 9 or 10 for forming eggs and / or sperm of a heterogeneous individual of fish to prepare germ cells of fish, thereby proliferating fish. The method (Claim 11) or the method of inducing differentiation according to Claim 9 or 10 is applied to a primordial germ cell of a genetically modified fish to form a genetically modified egg and / or sperm of the fish. And a method of breeding a fish (claim 12), characterized in that a genetically modified fish is prepared.

[0024]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention uses isolated primordial germ cells derived from vertebrates, transplanting the cells into an early embryo of a host vertebrate, and transplanting the isolated primordial germ cells in a host vertebrate individual. It consists of inducing differentiation into the germ line. The transfer of the isolated primordial germ cells to the early embryo of the host vertebrate can be carried out by transferring the host vertebrate in the early development stage to the inside of the abdominal cavity of the intestinal tract. According to the method of the present invention, when isolated primordial germ cells are transplanted into the abdominal cavity of a hatched embryo of a host vertebrate by a method such as a microinjection method, the transplanted primordial germ cells spontaneously enter the host gonad. It can move and induce proliferation and differentiation there. In vertebrates such as fish, primordial germ cells used for transplantation can be primordial germ cells isolated from embryos before and after hatching, and transplantation into early embryos of the host can be performed at the developmental stage before and after hatching. Any host can be used.

In the present invention, when primordial germ cells are used for transplantation, it is necessary to separate and purify the cells and obtain them. Primordial germ cells are a cell population that appears only at a limited stage of early development, for example, in fish, at a limited stage of early development before and after hatching. It is necessary to obtain it so that it can be separated and purified. For visualization of primordial germ cells, the method reported by the present inventors (Int. J. Dev. Biol. 4
4: 323-326, 2000) can be used. That is, for example, in order to visualize primordial germ cells of vertebrates such as fish, first, obtain a gene that is specifically expressed in germ cells of vertebrates (Mol. Reprod. Develop. 55: 364-371. ,
2000), using that regulatory region. In fish,
The vasa gene can be used. vasa was isolated as a causative gene of a mutation that makes the F1 generation infertile (F2 generation cannot be taken) in Drosophila,
A gene that is said to have RNA helicase activity involved in translation of mRNA in germ cells (protein nucleic acid enzyme, 43, 356-363, 1998), and is specifically expressed in primordial germ cells in fish such as rainbow trout. Therefore, the expression of this gene can be used for visualization of primordial germ cells.

In visualizing the primordial germ cells, in order to label and isolate the primordial germ cells in the living body by using the expression of the gene specifically expressed in the primordial germ cells, spinal cords like the vasa gene are used. For example, a plasmid in which a marker gene encoding a protein capable of visualizing primordial germ cells in the living body is incorporated into the regulatory region of a gene specifically expressed in animal germ cells is used for the fertilized egg of a vertebrate. It can be carried out by incorporating it into the cytoplasm. Examples of the marker gene include a gene encoding a fluorescent protein FP such as green fluorescent protein (GFP) derived from Onwan jellyfish and E
A marker gene such as GFP (Enhanced Green Fluorecent Protein) can be used.

In order to separate the visualized primordial germ cells from germ cell tissues, a known method can be appropriately used. For example, reproductive ridges containing primordial germ cells can be collected from hatched embryos, dissociated with proteolytic enzymes until they are dissociated, and further subjected to cell sorter for fractionation. Since the primordial germ cells are fluorescently labeled by the expression of the marker gene, the germ cells that fluoresce and other somatic cells that do not fluoresce can be easily separated by the cell sorter. .

The obtained primordial germ cells are transplanted to the early embryos of the host vertebrate such as the abdominal lining of the abdominal cavity of the host vertebrate in the early developmental stage by an appropriate method such as microinjection. , Primordial germ cells can spontaneously move into the host gonad to induce differentiation into oocytes or spermatogonia, and further into eggs or sperms. The method of inducing differentiation of the isolated primordial germ cells into the germ line of the present invention can be used for the production of new individuals, and can be used for the method of breeding and breeding vertebrates.

The use of the method of inducing differentiation of the isolated primordial germ cells into the germ cell line of the present invention to the method of growing vertebrates includes the method of inducing the differentiation of eggs and / or sperm of heterogeneous individuals such as fish. It can be used for large-scale breeding of fish and the like. The following can be said as characteristics of the proliferation method of the present invention. That is, as described above,
In mammalian mice and the like, a method for transplanting spermatogonia into a host individual to differentiate into sperm has been developed. Testis with spermatogonia retains these cells in astronomical quantities. Therefore, for example, after transplanting dozens or hundreds of foreign cells, the ratio of sperm formed in the host gonad is extremely low, and the technique cannot be put to practical use.
However, since the method of the present invention uses very young embryos,
The host holds only about 50 primordial germ cells.
By transplanting 10 or more cells (in principle, about 100 cells can be transplanted) into the host gonad, the proportion of eggs and sperm derived from the primordial germ cells transferred becomes extremely high.

In addition, since primordial germ cells are derived from extremely young embryos before and after hatching, their proliferative ability is very high originally,
It has been confirmed that the growth in the host individual after transplantation is also extremely fast. Furthermore, although spermatogonial cells can be produced only by transplantation of spermatogonial cells, primordial germ cells are cells before sex differentiation, and therefore both eggs and sperm can be formed. By the way, because the cryopreservation technology for eggs has not been completed in fish, it can be said that this technology is epoch-making from the viewpoint of preserving the gene progenitor if cryopreservation of primary primordial germ cells is followed by transplantation to produce eggs and sperm.

The following are specific examples of the use of the method of inducing differentiation of the present invention for the method of growing vertebrates. (1) Utilization as a litter of vertebrates such as fish: By transferring the isolated primordial germ cells to a host animal of a different strain or different species, eggs and sperm derived from different strains or different species within the host individual can be obtained. Made For example, primordial germ cells of rainbow trout can be transplanted into yamame trout, and cell proliferation and differentiation can be performed. In particular, when the parent fish transplants primordial germ cells of a huge species such as tuna to a small closely related species, it is possible to produce tuna seedlings in a small aquarium.

(2) Utilization for preservation of genetic resources of vertebrates such as fish: If primordial germ cells of rare species and endangered species are cryopreserved and transplanted to related species which are easy to raise when necessary,
These hosts can produce eggs and sperm derived from rare species or endangered species (in some cases already extinct species). In addition, when various useful strains and varieties are created by techniques such as gene transfer in the future, it is possible to maintain these without subculture of the individual, and when necessary, by transplanting to the host embryo, the individual can be maintained. Can be modified to (3) Utilization of isolated primordial germ cells for molecular biological / biochemical analysis: proteins from isolated primordial germ cells,
By extracting and analyzing the nucleic acid, the gene / protein expressed in this cell can be identified. It is particularly useful as an analysis system for stem cells, which is drawing attention in regenerative medicine.

The following are specific examples of the use of the method of inducing differentiation of the present invention for the method of growing vertebrates. (1) Utilization for breeding (breeding) of vertebrates such as fish:
After the isolated primordial germ cells are genetically modified in vitro, they are transplanted into a host individual, differentiated into eggs and sperms, and fertilized to efficiently produce the target genetically modified individual. can do. In order to genetically modify the primordial germ cells, as described above, the known gene introduction methods and gene modification methods that have already been used in vertebrates such as fish can be used. Examples of the method include a microinjection method and an electroporation method, which are widely used as a method for introducing a gene into fish or the like.

(2) Utilization for gene function analysis of vertebrates such as fish: When primordial germ cells can be cultured in vitro, gene targeting is performed on these cells, and then the above method is performed. By converting primordial germ cells into individuals, it is possible to produce, for example, a knockout fish in which the function of the gene is disrupted or a knockin fish in which the sequence of the gene is modified. That is, it can be used as a substitute for embryonic stem cells (ES cells) used in mice, which makes it possible to clarify the role of a novel gene whose function is unknown.

[0035]

The present invention will be described in more detail with reference to the following examples, but the technical scope of the present invention is not limited to these examples. As an example of the present invention, an example using rainbow trout will be shown to explain the present invention. [Separation of Primordial Germ Cells by Visualization] (Expression of vasa gene in rainbow trout primordial germ cells) In order to visualize primordial germ cells of rainbow trout, some kind of molecular marker for labeling the cells is necessary. Since specific antibodies against various cell surface antigens have already been produced in higher vertebrates, it has become possible to identify live primordial germ cells. However, such a simple molecular marker has not been known in fish. Therefore, we have focused on the vasa gene, which is known to be specifically expressed in the germ line over a wide range of animal species from Drosophila to human. vasa was isolated as a causative gene of a mutation that causes infertility in F1 generation (F2 generation cannot be taken) in Drosophila, and is said to have RNA helicase activity involved in translation of mRNA in germ cells (protein nucleic acid enzyme). ,Four
3, 356-363, 1998).

In order to clone the vasa cDNA of rainbow trout, using known sequence information, PCR was carried out using the cDNA method.
When A amplification was performed, v
A homologue of the asa cDNA could be isolated.
Next, the expression status of this gene was examined by Northern blot analysis using each tissue of adult fish, and strong expression was observed specifically in ovary and testis. Furthermore, the expression of the vasa gene in the ocular embryo was whole-mounted in situ.
When investigated by u hybridization method, it was found that
It was revealed that the a gene is expressed in about 60 cells arranged in two rows on the dorsal side of the abdominal cavity (Fig. 1).
(See Reference Photo 1): vasa gene-expressing cells in rainbow trout eyed embryos. As a result of whole-mount in situ hybridization performed using vasa antisense RNA as a probe, 2
Only cells lined up in a row stain specifically blue-black).

The distribution of these cells was determined by the location of primordial germ cells (Bull Japan Soc Sci F
ish. 46, 1317-1322, 1980). Therefore, when tissue sections were prepared from hatched embryos and attempts were made to identify cells expressing these vasa genes, the vasa genes were incorporated into a pair of reproductive ridges (undifferentiated gonads) suspended from the dorsal side of the abdominal cavity. It was revealed that it was specifically expressed in large round cells (Fig. 2 (see reference photo 2)): vasa mRNA was expressed only in primordial germ cells (arrows) in the genital ridge in rainbow trout hatching embryos. In situ hybridization performed on a tissue section using vasa antisense RNA as a probe, MD: mesonephric canal, G: intestinal canal. The location of these cells,
And morphology are identical to the characteristics of known primordial germ cells,
It was revealed that the vasa gene was specifically expressed in primordial germ cells in the rainbow trout embryo, and that the expression of this gene was useful as a marker for primordial germ cells.

(Visualization of rainbow trout primordial germ cells) All previously reported methods for identifying rainbow trout primordial germ cells using the expression of the vasa gene were carried out using fixed dead embryos. Therefore, the next step was to visualize the primordial germ cells of living rainbow trout embryos. va
The fact that the sa gene is expressed only in primordial germ cells means that the transcriptional regulatory region (promoter, enhancer) of vasa gene is activated only in primordial germ cells and promotes transcription of vasa mRNA.
Therefore, we cloned the transcriptional regulatory region of this vasa gene and connected it to the gene of green fluorescent protein (GFP, a protein that emits green fluorescence when blue light was applied) derived from Aequorea (Fig. 3: Structure of recombinant gene used for visualization of primordial germ cells, expression of GFP gene is regulated by transcriptional regulatory region of vasa gene (UTR: untranslated region),
This recombinant gene was introduced into rainbow trout. That is, in the primordial germ cells, the transcriptional regulatory region derived from the vasa gene was specifically activated to produce GFP, and it was expected that the primordial germ cells specifically emit green fluorescence.

When a foreign gene is introduced into fish, the gene is usually injected into a fertilized egg with a thin glass pipette (microinjection method). When cleavage progresses thereafter, the injected foreign gene is integrated into the host chromosome in some blastomers, but is eliminated as a foreign substance in many blastomeres.
As a result, in the transgenic fish produced by the microinjection method, cells having foreign genes and cells not having foreign genes coexist in a mosaic pattern. As expected, the transgenic rainbow trout produced by this method yielded mosaic individuals in which primordial germ cells that fluoresce green and those that did not coexist were mixed. However, because our ultimate goal is to purify large numbers of primordial germ cells and use them in culture,
It is extremely inefficient to perform microinjection on each fertilized egg and to visualize only some primordial germ cells. Fortunately, in transgenic fish, the foreign gene integrated into the host chromosome is inherited to the next generation together with the host chromosome.

Then, by using these transgenic rainbow trouts as parents, F1 and F2 generations were produced to mass-produce transgenic rainbow trouts not having a mosaic in which all cells carry the foreign gene. Fig. 4 (Fig. 4 (Reference photograph 3
): GF regulated by the transcriptional regulatory region of the vasa gene
Fluorescent photographs of F1 individuals were shown in the hatched juveniles of transgenic rainbow trouts in which the primordial germ cells specifically emit green fluorescence due to the introduction of the P gene. In these individuals, all of the primordial germ cells are strong as expected. It emits green fluorescence and was successful in visualizing live primordial germ cells in live rainbow trout embryos. This green fluorescence is not limited to primordial germ cells,
Similar to the expression pattern of the vasa gene, its expression was observed in oocytes / spermatogonia, and also in oocytes (Fig. 5 (see reference photo 4): regulated by the transcriptional regulatory region of the vasa gene). The gonads of rainbow trout 1-year-old fish into which the GFP gene has been introduced (left: ovary, right: testis). Germ cells emit green fluorescence. *, Unmarked gonads from transgenic and normal individuals, respectively. As described above, by using the vasa gene as a molecular marker, it became possible to visualize and identify not only fixed embryos but also primordial germ cells in living embryos. Therefore, in the next section, we outline the isolation and purification of these green fluorescent primordial germ cells.

(Separation of visualized rainbow trout primordial germ cells) As described above, in order to culture the primordial germ cells like ES cells and utilize them in the "stem cell-mediated gene transfer technique", a large amount of Cells need to be isolated and purified in a living state. Therefore, using a device called a cell sorter that can sort cells using the fluorescence intensity emitted by the cells as an index, it is possible to sort germ cells that are positive for green fluorescence and other somatic cells that do not fluoresce. I tried. Since there are only about 60 rainbow trout primordial germ cells per hatched juvenile, as a preliminary experiment, the fluorescence intensity analysis by a cell sorter was performed using the testis of a 1-year-old fish containing a large amount of fluorescence-labeled germ cells. First, the testis was dissociated and then tested on a flow cytometer to analyze its fluorescence intensity distribution.
As a result, as shown in FIG. 6 (FIG. 6: Fluorescence intensity distribution of dissociated testis cells. Only germ cells express the GFP gene to specifically fluoresce to form a fluorescence-positive cell population). A clear graph was obtained, and a cell population with a clearly high fluorescence intensity was recognized.

Then, when only those cells that emit strong fluorescence were collected, green fluorescence was observed in all cells, suggesting that these cells were germ cells (Fig. 7 (reference photograph). 5)): Testicular-derived germ cells isolated by a cell sorter, all cells fluoresce; left: bright field image, right: fluorescence image). Next, we attempted to purify the primordial germ cells using the conditions set using testis. In cell sorting using a cell sorter, the higher the concentration of the target cells (primer germ cells in this case) in the cell suspension to be tested, the higher the recovery efficiency can be expected. So instead of using the whole rainbow trout embryo,
Only large numbers of genital ridges containing primordial germ cells were recovered from hatched embryos. This reproductive ridge is an extremely fine tissue, but as described above, the rainbow trout hatching embryo is extremely large compared to other fish species (rainbow trout eggs are the same size as salmon eggs, salmon roe, Fry is about 1.5 cm in total length) and can be dissected under a stereoscopic microscope. Furthermore, since the primordial germ cells contained in the genital ridge emit green fluorescence, it was possible to easily isolate the genital ridge using the green fluorescence as a marker using a stereoscopic fluorescence microscope (Fig. 8; reference photograph). 6).

The resulting genital ridges were dissociated with a protease to separate them, and then subjected to a cell sorter.
When the cells were sorted by the same method as the experiment performed on the testis cells, a cell population with a high fluorescence intensity was recognized like the testis cells, and thus this population was sorted. The obtained cells were large round cells containing a large amount of granules, and showed well the morphological characteristics of primordial germ cells having large nuclei. Furthermore, as a result of extracting mRNA from these fluorescence positive cells and negative cells and examining the expression of vasa gene, the expression of vasa gene was observed only in the fluorescence positive cells. As described above, in view of its morphology and gene expression pattern, the cells obtained in this experiment were definitely considered to be primordial germ cells. By the method of this example, about 500 primordial germ cells can be purified from about 20 rainbow trout hatching juveniles.

[Induction of Differentiation of Isolated Primordial Germ Cells in Early Rainbow Trout Embryos] (Introduction of Isolated Primordial Germ Cells into Early Rainbow Trout Embryos) The GFP derived from Owan jellyfish is introduced into the expression control region of the vasa gene of rainbow trout isolated by the above method. The primordial germ cells of the rainbow trout, which were connected with genes and visualized, were introduced into the early embryo of the host rainbow trout. The rainbow trout introduced with this GFP gene can be visualized in a living individual because the primordial germ cells specifically emit green fluorescence. The introduction of primordial germ cells into the early embryos of rainbow trout is carried out from the embryos of the rainbow trout before and after hatching (in case of breeding at a water temperature of 10 ° C., embryos 30-40 days after fertilization, hatching 32 days after fertilization) by the above-mentioned method. About 10 isolated primordial germ cells were aspirated with a glass micropipette attached to a microinjector and transplanted to the inside of the peritoneal intestinal membrane of a rainbow trout individual at the same developmental stage (see FIG. 9; reference photograph 7). The embryo at this stage retains about 50 primordial germ cells. At this time, if transplanted on the front side of the intestinal membrane, the transplanted cells leak out of the embryo due to internal pressure in the abdominal cavity, resulting in poor transplant efficiency.

(Induction of Differentiation of Transplanted Primordial Germ Cells into Germ Lines) The primordial germ cells transplanted to the lining of the peritoneal intestinal membrane of rainbow trout individuals at an early stage of development spontaneously start migration toward the immature gonad. , And then taken into the gonad (see FIG. 10; reference photograph 8). At 30 days after transplantation, it was observed that the transplanted primordial germ cells migrated into the gonad (FIG. 11; see reference photograph 9). After that, it was observed that the transplanted primordial germ cells were present in one place in the gonad, being solidified and undergoing cell division (see FIG. 12; reference photograph 10). Furthermore, the transplanted primordial germ cells proliferate efficiently in the host gonads, and after 6 months, become spermatogonia in the testis (see FIG. 13; reference photograph 11) and oocytes / oocytes in the ovary ( (See Figure 14; Reference Photo 12)
It was observed that differentiation was induced. Since these oocytes and spermatogonia differentiate into eggs or sperms, new rainbow trout individuals can be prepared by using these germ cells.

[Induction of Differentiation of Separated Primordial Germ Cells in Early Chickpea Embryos] (Introduction of Separated Primordial Germ Cells into Early Chickpea Embryos) In the same manner as the above method, the expression control region of the rainbow trout vasa gene was derived from Owan jellyfish. The primordial germ cells of the rainbow trout, which were ligated with the GFP gene and visualized, were introduced into the early embryo of the host yamame trout, which is a heterogeneous individual. Since the primordial germ cells into which the GFP gene is introduced specifically emits green fluorescence, it can be visualized in a living yamame trout. The introduction of primordial germ cells into the early embryos of yamame trout is carried out from the embryos of the rainbow trout before and after hatching (in case of breeding at a water temperature of 10 ° C, embryos 30-40 days after fertilization, hatching 32 days after fertilization) by the above-mentioned method About 10 isolated primordial germ cells were aspirated with a glass micropipette attached to a microinjector, and transplanted to the inside of the peritoneal intestinal membrane of a yamame individual at the same early stage of development.

(Induction of Differentiation of Transplanted Primordial Germ Cells into Germ Lines) The primordial germ cells transplanted to the lining of the intestinal membrane of the abdominal trout at the early stage of development spontaneously started to migrate toward the immature gonad. , Where it is taken up in the gonad. At 60 days after transplantation, it was observed that the transplanted primordial germ cells migrated to the genital ridge in the gonad, were taken into the genital ridge, and were further proliferating (see FIG. 15 (reference photograph 13)). The arrow in the figure indicates a rainbow trout-derived germ cell observed in the gonad of the trout host.). Then, it was observed that the transplanted primordial germ cells proliferated efficiently in the host gonads and were induced to differentiate into spermatogonia in the testis and oocyte / oocyte in the ovary. Since these oocytes and spermatogonia differentiate into eggs or sperms within the yamame trout individuals, new rainbow trout individuals can be created by using these germ cells.

[0048]

INDUSTRIAL APPLICABILITY The method of the present invention uses a primordial germ cell in a vertebrate such as a fish, which is difficult to differentiate from an embryonic stem cell (ES cell) into a germline, and uses this as an early vertebrate animal. By transplanting into an embryo, even in a vertebrate such as a fish, it is possible to induce differentiation into a germ cell line and further form an individual. By using the method of the present invention, vertebrates such as fish can be propagated and bred, and can be used for various techniques. For example, (1) the isolated primordial germ cells can be transplanted to a heterologous strain or a heterogeneous host individual to be used as a litter of fish or the like. By this method, it is possible to produce tuna seedlings in a small aquarium by transplanting primordial germ cells of a huge species such as tuna into a small related species.

(2) To preserve the genetic resources of vertebrates such as fish by cryopreserving primordial germ cells of rare species and endangered species and transplanting them to related species that can be easily reared when necessary. Can be available. Also, (3)
By extracting and analyzing proteins and nucleic acids from the isolated primordial germ cells, it becomes possible to identify the genes and proteins expressed in these cells, and the molecular biology and biochemistry of the isolated primordial germ cells can be identified. It can be used for analysis. In addition, (4) the isolated primordial germ cells in vivo
After genetic modification in tro, transplanted into the host individual,
By differentiating into eggs / sperms and fertilization, it can be used for breeding (breeding improvement) of vertebrates such as fish. Furthermore, (5) by performing gene targeting on the primordial germ cells and then converting the primordial germ cells to an individual by the above-mentioned method, for example, knockout fish in which the function of the gene is disrupted, and further, the gene It becomes possible to produce a knock-in fish in which the sequence of is modified, and it can be used for gene function analysis of vertebrates such as fish.

[Brief description of drawings]

FIG. 1 shows a whole mount in which vasa antisense RNA was used as a probe in a vasa gene-expressing cell in a rainbow trout eyed embryo in an example of the present invention.
It is a figure which shows the result of in situ hybridization.

[Fig. 2] In the example of the present invention, in order to confirm the expression of vasa mRNA in rainbow trout hatching embryos, va
It is a figure which shows the result of having performed in situ hybridization with respect to a tissue section using sa antisense RNA as a probe.

FIG. 3 is a diagram showing the structure of a recombinant gene used for visualization of primordial germ cells in the example of the present invention.

FIG. 4 is a diagram showing a photograph of hatched fry of transgenic rainbow trout in which primordial germ cells specifically emit green fluorescence due to the introduction of a GFP gene controlled by the transcriptional regulatory region of the vasa gene in the example of the present invention. Is.

FIG. 5 shows that in the example of the present invention, germ cells emit green fluorescence in the gonads (left: ovary, right: testis) of a rainbow trout first year fish introduced with a GFP gene controlled by the transcriptional regulatory region of the vasa gene. It is a figure showing the state where it is.

FIG. 6 is a diagram showing a situation of fluorescence intensity distribution of dissociated testis cells in the example of the present invention.

FIG. 7 is a diagram showing a situation in which testicular-derived germ cells isolated by a cell sorter emit whole-cell fluorescence (left: bright-field image, right: fluorescence image) in the example of the present invention.

FIG. 8 is a stereoscopic fluorescence microscope image showing that the primordial germ cells contained in the genital ridge emit green fluorescence in the example of the present invention.

FIG. 9 is a diagram showing a situation in which primordial germ cells are aspirated by a glass micropipette attached to a microinjector and transplanted to the inside of the peritoneal intestinal membrane of a developing rainbow trout individual in an example of the present invention. Is.

FIG. 10 shows that in the example of the present invention, primordial germ cells transplanted to the lining of the abdominal cavity of the rainbow trout individual,
It is a figure explaining the situation which spontaneously starts migration toward the immature gonad and is taken into the gonad there.

FIG. 11 shows that in the example of the present invention, the primordial germ cells transplanted to the inner side of the abdominal cavity of the abdominal cavity started to migrate toward the immature gonad, and 30 days after the transplantation, the primordial germ cells transplanted into the gonad. It is a figure which shows the photograph of the situation which is moving.

FIG. 12 is a diagram showing a photograph of a situation in which transplanted primordial germ cells are present in one group in the gonad and are undergoing cell division in the example of the present invention.

FIG. 13 is a diagram showing a photograph of a situation in which transplanted primordial germ cells efficiently proliferate in the host gonad and induce differentiation into spermatogonia in testis after 6 months in the example of the present invention. is there.

FIG. 14 is a photograph showing a situation in which transplanted primordial germ cells efficiently proliferate in the host gonad and induce differentiation into oocytes / oocytes in the ovaries after 6 months in the example of the present invention. FIG.

FIG. 15 shows that, in the example of the present invention, rainbow trout primordial germ cells transplanted to the inside of the abdominal cavity of the yamame trout migrate to the genital ridge in the yamame embryo and are taken into the genital ridge and further proliferated. It is a photograph showing the situation.

Claims (12)

[Claims] 1. A method for inducing differentiation of a separated primordial germ cell into a germ cell line, which comprises transplanting a separated primordial germ cell derived from a vertebrate into an early embryo of a host vertebrate. 2. Reproduction of isolated primordial germ cells according to claim 1, wherein the transplantation into the early embryo of the host vertebrate is transplantation into the abdominal lining of the abdominal cavity of the host vertebrate in the early developmental stage. A method for inducing differentiation into a cell lineage. 3. Reproduction of isolated primordial germ cells according to claim 1 or 2, wherein the isolated primordial germ cells derived from vertebrates are primordial germ cells obtained by visualizing vertebrate primordial germ cells. A method for inducing differentiation into a cell lineage. 4. A marker gene encoding a protein capable of visualizing a primordial germ cell in a living body is incorporated into a regulatory region of a gene specifically expressed in a vertebrate germ cell for visualization of a primordial germ cell. 4. The method for inducing differentiation of isolated primordial germ cells into germ line according to claim 3, wherein said method is carried out by incorporating the said plasmid into the cytoplasm of fertilized vertebrate eggs. 5. The gene specifically expressed in vertebrate germ cells is vasa gene, and the marker gene encoding a protein capable of visualizing primordial germ cells in the living body encodes fluorescent protein FP. The method for inducing differentiation of isolated primordial germ cells into germ cells according to claim 4, which is a gene. 6. The method for inducing differentiation of a separated primordial germ cell into a germ cell line is to induce differentiation of a primordial germ cell into an oocyte or spermatogonia, or further into an egg or sperm. The method for inducing differentiation of isolated primordial germ cells into a germ cell line according to any one of claims 1 to 5. 7. The isolated primordial germ cell derived from a vertebrate is a primordial germ cell derived from a vertebrate of a different strain or a different species from the host vertebrate. A method for inducing differentiation of primordial germ cells into germ cells. 8. The isolated primordial germ cell according to claim 1, wherein the isolated primordial germ cell derived from a vertebrate is a genetically modified primordial germ cell isolated from a vertebrate. Method for inducing germ line differentiation into the germ line. 9. The isolated primordial germ cell derived from a vertebrate is an isolated primordial germ cell derived from a fish, and the transplantation of the isolated primordial germ cell into the early embryo of the host vertebrate is performed in the early embryo of a fish host. The method for inducing differentiation of the isolated primordial germ cells into a germ cell line according to any one of claims 1 to 8. 10. The isolated primordial germ cells are primordial germ cells separated from embryos before and after hatching, and / or transplantation to the early embryos of the host is to the inside of the peritoneal intestinal membrane of the host in the developing stage before and after hatching. 10. The method for inducing differentiation of isolated primordial germ cells into germ line according to claim 9, wherein 11. A method for proliferating fish, which comprises using the method for inducing differentiation according to claim 9 or 10 for forming eggs and / or sperm of a heterogeneous individual of fish to prepare germ cells of fish. 12. The method for inducing differentiation according to claim 9 or 10 is applied to primordial germ cells of genetically modified fish to form genetically modified ova and / or sperm of fish,
A method for breeding a fish, which comprises preparing a genetically modified fish.