WO2017164391A1 - Marker for identifying germ cells having engraftability at host gonad - Google Patents

Abstract

The present invention relates to a marker for identifying germ cells, comprising (I) a marker for identifying germ cells having engraftability at a host gonad and/or (II) a marker for identifying germ cells not having engraftability at a host gonad. The present invention provides a germ cell marker that can identify the presence or absence of engraftability at a host gonad in fish.

Description

Germ cell marker with engraftment to the host gonad Reference to related applications

This patent application is based on Japanese Patent Application No. 2016-60914 (filing date: March 24, 2016), which is a previously filed Japanese patent application, and claims the benefit of its priority. , Incorporated herein by reference in its entirety.

Background of the Invention

TECHNICAL FIELD The present invention relates to a surrogate parent fish technique, and in particular, to a germ cell identification marker for identifying the presence or absence of engraftment of isolated germ cells to be transplanted into a host gonad in the surrogate parent fish technique. The present invention also relates to a method for producing a germ cell having the ability to engraft the host gonad using the germ cell identification marker of the present invention.

Background Art Due to growing health consciousness in Europe and the United States and economic development in China and other countries, the consumption of edible marine products in the world continues to increase year by year. In particular, eels and bluefin tuna, which are in high demand as high-grade fish, have reached a state where they are threatened with extinction due to a significant decrease in the amount of natural resources due to overfishing. Therefore, seedling production and artificial seedling (fry) release for the purpose of increasing the amount of marine resources are being studied. In general, in seedling production, genetic diversity of individuals that become parent fish is required to avoid annihilation due to inbreeding and the onset of a specific disease, and thus it is necessary to grow a large number of parent fish. However, depending on the fish species, seed production is difficult. For example, in bluefin tuna, the parent fish has a large body weight of 100 kg or more, and requires a wide breeding environment due to the swimming method. Also, sturgeon takes a long time to mature sperm and eggs. Because there are some fish species that are technically difficult to artificially ripen, and those that are difficult to mate one-on-one, it is costly and difficult to grow a large number of parent fish under artificial control and collect sperm and eggs. Technically extremely difficult.

As a seed production technology for fish, the present inventors transplanted germ cells derived from different species of fish (donor fish) different from the host (recipient) fish into the peritoneal cavity of the host fish individual before and after hatching, It has been found that germ cells can be induced to differentiate into germ cells, and has succeeded in inducing differentiation of isolated germ cells into germ cells in heterologous host fish (see, for example, Patent Document 1). This technique, also called surrogate parent fish technique or borrowed belly culture technique, is produced at low cost and easily by producing and mating sperm and eggs of fish, which are problematic for breeding of parent fish, by heterogeneous host fish that are easy to breed. It is expected to be a technology that enables seedling production.

In Patent Document 1, primordial germ cells are used as germ cells to be transplanted. Specifically, the ridges of reproductive tissues obtained from hatched embryos of transgenic rainbow trout with GFP (Green Fluorescent Protein) introduced into the regulatory region of the vasa gene that is specifically expressed in primordial germ cells Using a suspension as a raw material, cell populations with strong fluorescence intensity are separated as primordial germ cells by flow cytometry analysis using green fluorescence as an index. However, primordial germ cells are derived from very young embryos before and after hatching, and the number thereof is as small as about 60 per hatched fry, so it is not suitable for commercial mass production.

In order to secure a sufficient number of isolated germ cells to be transplanted for the purpose of commercial mass production, for example, Non-Patent Document 1 discloses that germ cells are separated from a cell suspension of testis which is a gonad of donor fish. However, a method of transplanting the isolated germ cells into the peritoneal cavity of a host fish individual using the surrogate parent fish technique is disclosed. However, the proportion of individuals engrafted with transplanted cells in the host gonad when using isolated testicular germ cells as transplanted germ cells was lower than when using primordial germ cells.

Non-patent document 2 describes that when rainbow trout spermatogonia are transplanted into the peritoneal cavity of hatched fry, only a part of type A spermatogonia (A-SG) engraft in the host gonad, and a functional gamete derived from a donor. Based on the idea that type A spermatogonia with high engraftability are spermatogonial stem cells (SSC), the host focused on SideＳｉPopulation (SP) of spermatogonial stem cells. A method for concentrating type A spermatogonia having high engraftment ability to the gonads is disclosed. However, in this method, the concentration rate of spermatogonial stem cells is insufficient, and a germ cell identification marker having engraftment ability to the host gonad has not been obtained. Therefore, in the surrogate parent fish technique, in order to determine the engraftment ability of the isolated germ cells to be transplanted into the host gonad, there is a method of identifying other than by transplanting to the host fish individual before and after actual hatching. do not do.

Japanese Patent No. 4300287

In this way, germ cell markers that can distinguish the presence or absence of fish engraftment into the host gonad, especially germ cells that have the engraftment ability into the host gonad, which are necessary for the practical application of surrogate parent fish techniques, are introduced before transplantation. As far as the inventors know, no germ cell markers that can be detected and separated have been reported.

The inventors of the present invention have recently confirmed that transplanted germ cells derived from the testes of donor fish were transplanted into the abdominal cavity of the individual host fish and engrafted in the host gonad. Germ cells that have the ability to survive (i.e., engraftment cells), and the gene expression in these cells was comprehensively analyzed. We have succeeded in developing germ cell identification markers that can detect germ cells that do not have engraftment ability. The present invention is based on these findings.

That is, an object of the present invention is to provide a novel germ cell identification marker, particularly a germ cell identification marker for identifying the presence or absence of engraftment of the isolated germ cell to be transplanted into the host gonad. Another object of the present invention is to provide a method for producing a germ cell having the ability to engraft the host gonad using the germ cell identification marker of the present invention.

According to the present invention, the following inventions are provided.
(1) A germ cell comprising the following (I) germ cell identification marker having engraftment ability to the host gonad and / or (II) a germ cell identification marker not having engraftment ability to the host gonad Identification marker:
(I) A germ cell identification marker having engraftment ability to the host gonad comprising one or more polynucleotides selected from the following (a) to (f):
(A) a polynucleotide comprising one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 1 to 74,
(B) a polynucleotide comprising one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 1 to 74, wherein the polynucleotide has a germ cell identification function capable of engraftment in the host gonad ,
(C) a nucleotide sequence in which one or several bases are deleted, added, inserted or substituted in one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 1 to 74, and / or one of them, or A polynucleotide represented by a nucleotide sequence to which bases are added at both ends, and having a germ cell identification function having engraftment ability to the host gonad,
(D) a polynucleotide represented by a nucleotide sequence having at least 70% identity with one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 1 to 74, and having the ability to engraft in the host gonad A polynucleotide having a germ cell identification function,
(E) a polynucleotide encoding an ortholog gene of a gene comprising one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 1 to 74, and (f) any one of (b) to (e) above A polynucleotide that hybridizes under high stringency conditions with a polynucleotide comprising a complementary sequence of a polynucleotide having the nucleotide sequence shown, and has a germ cell identification function capable of engraftment in the host gonad,
(II) Differentiated germ cell identification marker having no engraftment ability to the host gonad consisting of one or more polynucleotides selected from the following (g) to (l):
(G) a polynucleotide comprising one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 75 to 101,
(H) a polynucleotide comprising one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 75 to 101 and having a germ cell identification function having no engraftment ability in the host gonad Polynucleotides,
(I) a nucleotide sequence in which one or several bases are deleted, added, inserted or substituted in one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 75 to 101, and / or one of them, or A polynucleotide represented by a nucleotide sequence to which bases are added to both ends, and having a germ cell identification function having no engraftment ability to the host gonad,
(J) a polynucleotide represented by a base sequence having at least 70% identity with one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 75 to 101, and having the ability to engraft in the host gonad A polynucleotide having a germ cell identification function,
(K) a polynucleotide encoding an orthologous gene of a gene consisting of one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 75 to 101, and (l) any one of (g) to (k) above A polynucleotide that hybridizes under high stringency conditions with a polynucleotide comprising a complementary sequence of a polynucleotide having the nucleotide sequence shown, and has a germ cell identification function that does not have the ability to engraft the host gonad. nucleotide.
(2) The marker according to (1), wherein the germ cell identification marker is a germ cell identification marker for identifying the presence or absence of engraftment in the host gonad.
(3) The germ cell identification marker comprising one or more polynucleotides selected from (a) to (f) of (1) is a germ cell identification marker having engraftment ability to the host gonad ( The marker according to 1) or (2).
(4) The marker according to any one of (1) to (3), wherein the germ cell identification marker having engraftment ability to the host gonad is a spermatogonial stem cell (SSC) marker.
(5) A primer or probe for detecting an SSC gene, which can hybridize to the polynucleotide according to (4) or a polynucleotide comprising a complementary sequence of the polynucleotide.
(6) The germ cell identification marker comprising one or more polynucleotides selected from (g) to (l) of (1) is a germ cell identification marker having no engraftment ability in the host gonad. The marker according to (1) or (2).
(7) Using the germ cell identification marker having engraftment ability to the host gonad as described in any one of (1) to (4), the gene of the germ cell identification marker having engraftment ability to the host gonad Detect the presence and / or degree of expression,
A method for producing a germ cell capable of engraftment in a host gonad, comprising separating a cell having expression or high expression from a testis or ovary of a fish.
(8) A germ cell having no engraftment ability to the host gonad using the germ cell identification marker having no engraftment ability to the host gonad as described in (1), (2) or (6) Detect the presence and / or degree of expression of the identification marker gene,
The method for producing undifferentiated germ cells according to (7), further comprising isolating cells that are expressed or highly expressed from the testis or ovary of fish.
(9) The method for producing a germ cell having engraftment ability to the host gonad according to (7) or (8), wherein the fish is a salmonid fish.
(10) Reproduction having engraftment ability to the host gonad that binds to a part of the protein encoded by the germ cell identification marker having engraftment ability to the host gonad according to any one of (1) to (4) Cell identification antibody.

By using the germ cell identification marker of the present invention, it is possible to identify the status of germ cells, in particular, the presence or absence of engraftment of the isolated germ cells to be transplanted into the host gonad before transplantation in the surrogate parent fish technique. In addition, by using the germ cell identification marker of the present invention, germ cells capable of engraftment in the host gonad can be efficiently produced.

It is the scheme of the data processing of the sequence result obtained by Quartz-seq analysis. The number of total reads in all samples (94 transplanted cells and 14 engrafted cells) in data processing is shown. The GC content of all samples (94 transplanted cells and 14 engrafted cells) in data processing is shown. The mapping ratio of all samples (94 transplanted cells and 14 engrafted cells) in data processing is shown. The result of PCA-pair plot of PCA analysis is shown. The ranking of factor loading of PCA analysis is shown. A comparison of SP array and Quartz-seq analysis is shown. The result of Blast search is shown. a) Results of a Blast search on puffer fish with a sequence of 74 transcripts characterizing engrafted cells b) Blast search results for tilapia with a sequence of 74 transcripts characterizing engrafted cells. c) Blast search results for tuna with 74 sequences of transcripts characterizing engrafted cells. d) Results of a Blast search on puffers with 27 sequences of transcripts that characterize non-engrafting cells. e) Blast search results for tilapia with a sequence of 27 transcripts characterizing non-engrafting cells. f) Blast search results for tuna with 27 sequences of transcripts characterizing non-engrafting cells. The result of the expression level of each transcript in SP cells and non-SP cells of qRT-PCR is shown. * And # in the graph indicate P values calculated by Student's t-test, * indicates P value <0.05 (a significant difference is observed between SP cells and non-SP cells), and # indicates P values. Values> 0.1 (no significant difference is observed between SP cells and non-SP cells). The result of immunohistochemical staining is shown. Fluorescence micrograph (left), HE-stained photo (right).

Detailed description of the invention

According to the present invention, a germ cell identification marker comprising a germ cell identification marker capable of engrafting in the host gonad and / or a germ cell identification marker not capable of engrafting in the host gonad is provided. . According to a preferred embodiment of the present invention, the germ cell identification marker is a germ cell identification marker that identifies the presence or absence of engraftment in the host gonad.

The “surrogate parent fish technique” is a method in which isolated germ cells isolated from donor fish are transplanted into the abdominal cavity of the host (recipient) fish before and after hatching, and the transplanted germ cells are germ cells in the gonad of the host fish individual. This means induction of differentiation into a lineage (gamete) (see Patent Document 1).

“Reproductive cells” means gametes for sexual reproduction and cells that are the basis for them. Germ cells include in the case of fish, for example, primordial germ cells, oocytes, spermatogonia, oocytes, spermatocytes, sperm cells, eggs and sperm.

A “germ cell capable of engrafting the host gonad” is a separated germ cell derived from a donor fish that is transplanted into the abdominal cavity of the individual host fish in the surrogate parent fish technique, and can be engrafted in the host gonad after transplantation. It means isolated germ cells, also called engraftment germ cells. Such germ cells include primordial germ cells, some type A spermatogonia and some oocytes. Among type A spermatogonia, type A spermatogonia that have the ability to engraft the host gonads are said to be less than 1%. Whether or not a germ cell is a germ cell capable of engrafting into the host gonad is determined by the germ cell marker having the ability to reproduce into the host gonad of the present invention, or in the surrogate fish technique, It can be judged by the presence or absence of engraftment when transplanted to the gonad.

“Type A spermatogonia” are undifferentiated germ cells that exist with somatic cells in an undifferentiated testis (immature testis). Type spermatogonia, spermatocytes, sperm cells, cells that differentiate into sperm. Type A spermatogonia include spermatogonial stem cells (SSC) that continue to replicate spermatogonia. Further, SSC is concentrated to a fraction having a low staining property by staining with Hoechst 33342, which is a cell membrane-permeable nuclear staining reagent. In the present invention, the type A spermatogonia as germ cells capable of engrafting the host gonads are preferably spermatogonial stem cells.

An “egg cell” is an undifferentiated germ cell that is present together with somatic cells in an undifferentiated ovary (immature ovary), and is a cell that differentiates into an oocyte or egg as it matures. The oocyte includes an oocyte stem cell (OSC) that continues to replicate the oocyte. In the present invention, the oocyte as a germ cell capable of engrafting the host gonad is preferably an oocyte stem cell.

By using the germ cell identification marker of the present invention, it is possible to identify the presence or absence of the engraftment ability of the isolated germ cell to be transplanted into the host gonad in the surrogate parent fish technique. That is, according to one embodiment of the present invention, a germ cell identification marker having engraftment ability to the host gonad is provided. According to another embodiment, a germ cell identification marker that does not have the ability to engraft the host gonad is provided.

According to one embodiment of the present invention, the germ cell identification marker having the engraftment ability to the host gonad of the present invention comprises one or more polynucleotides selected from the following (a) to (f): Is:
(A) a polynucleotide comprising one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 1 to 74,
(B) a polynucleotide comprising one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 1 to 74, wherein the polynucleotide has a germ cell identification function capable of engraftment in the host gonad ,
(C) a nucleotide sequence in which one or several bases are deleted, added, inserted or substituted in one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 1 to 74, and / or one of them, or A polynucleotide represented by a nucleotide sequence to which bases are added at both ends, and having a germ cell identification function having engraftment ability to the host gonad,
(D) a polynucleotide represented by a nucleotide sequence having at least 70% identity with one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 1 to 74, and having the ability to engraft in the host gonad A polynucleotide having a germ cell identification function,
(E) a polynucleotide encoding an ortholog gene of a gene consisting of one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 1 to 74, and (f) any one of (a) to (e) above A polynucleotide having a germ cell identification function capable of engrafting in a host gonad, which hybridizes under a highly stringent condition with a polynucleotide comprising a complementary sequence of the polynucleotide having the nucleotide sequence shown.

According to a preferred embodiment of the present invention, the germ cell identification marker capable of engrafting the host gonad of the present invention is a polynucleotide comprising one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 1 to 74. It is. According to a preferred embodiment of the present invention, the germ cell identification marker having the engraftment ability to the host gonad of the present invention consists of one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 1 to 74. It is a polynucleotide having a germ cell identification function having an engraftment ability in the host gonad substantially equivalent to the polynucleotide.

Examples of the polynucleotide having a germ cell identification function having an engraftment ability to the host gonad substantially equivalent to a polynucleotide consisting of one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 1 to 74 are as described above. Examples include polynucleotides selected from (b) to (f).

In the polynucleotide (b), the polynucleotide comprising one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 1 to 74 is 1 selected from the nucleotide sequences of SEQ ID NOs: 1 to 74 Alternatively, other nucleotide sequences may be included as long as expression of two or more nucleotide sequences is not hindered, and other nucleotide sequences can be appropriately selected by those skilled in the art. The length of the polynucleotide sequence of (b) is preferably 500%, more preferably 400%, relative to the length of one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 1 to 74, More preferably 300%, more preferably 200%, more preferably 100%, more preferably 70%, more preferably 50%, more preferably 30%, still more preferably 10%, more preferably 5%, still more preferably May be 1% longer.

In the polynucleotide of (c), the “nucleotide sequence in which one or several bases have been deleted, added, inserted or substituted” refers to a well-known technical method such as site-directed mutagenesis or the like. This means that the modification has been made, for example, by substitution of one to a plurality of bases to the extent that can occur. The number of base modifications is, for example, 1 to 20, preferably 1 to 10, more preferably 1 to 5, more preferably 1 to 4, further preferably 1 to 3, particularly preferably 1 to 5. Two. In the polynucleotide (c), “the addition of a base to one or both ends thereof” means that the expression of one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID Nos. 1 to 74 is hindered. Unless otherwise specified, other nucleotide sequences may be added to one or both ends of the nucleotide sequence, and other nucleotide sequences can be appropriately selected by those skilled in the art. The other nucleotide sequence is preferably 500%, more preferably 400%, even more preferably 300%, relative to the length of one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 1 to 74 More preferably, the length is 200%, more preferably 100%, more preferably 70%, more preferably 50%, more preferably 30%, more preferably 10%, more preferably 5%, and even more preferably 1%. The arrangement of

The modification of the polynucleotide (c) is preferably performed at a site where the number of differences when comparing the cDNA sequences of puffer fish, tilapia and tuna in FIG.

The polynucleotide (d) is composed of a nucleotide sequence having at least 70% identity with one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 1 to 74. This sequence identity is preferably at least preferably 75% or higher, more preferably at least 85% or higher, more preferably 88% or higher, still more preferably 89% or higher, still more preferably 90% or higher, still more preferably 91% or higher. More preferably 92% or more, still more preferably 93% or more, further preferably 94% or more, more preferably 95% or more, still more preferably 96% or more, still more preferably 97% or more, particularly preferably 98% or more, Most preferably, it is 99% or more.

The sequence identity is determined by a known method. Examples of such a method include BLAST search (Altschul, SF, Gish, W., Miller, W., Myers) described in Example 2 of the Examples. , “EW” & “Lipman”, “DJ” (1990) “Basic” local ”alignment” search “tool.” “J. Mol. Biol. 215: 403-410.), Etc. Moreover, BLASTN and BLASTX based on this algorithm BLAST can also be used.

In the polynucleotide (e), the “orthologous gene” is also called an orthologous gene, and is a homologous gene that shares the same function among other species with common ancestor genes. A polynucleotide encoding an ortholog gene of a gene consisting of one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 1 to 74 is one or more selected from the nucleotide sequences of SEQ ID NOs: 1 to 74 As long as it has a function equivalent to that of the nucleotide sequence, it is not particularly limited, and those skilled in the art can easily identify it by a known method. For example, it can be identified by performing phylogenetic tree analysis on a homologous gene identified by BLASTP search using an amino acid sequence encoded by BLASTX or polynucleotide.

According to a preferred embodiment, in the polynucleotide of (e), the “orthologous gene” is preferably at least 70%, more preferably one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 1 to 74 Is at least 75%, more preferably at least 85%, more preferably at least 88%, more preferably at least 89%, even more preferably at least 90%, even more preferably at least 91%, even more preferably at least 92%, Preferably 93% or more, more preferably 94% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, particularly preferably 98% or more, most preferably 99% or more. It consists of a nucleotide sequence having

The polynucleotide of (f) can be selected from those that hybridize under high stringency conditions to a polynucleotide comprising a complementary sequence of the polynucleotide having the nucleotide sequence shown in any of (a) to (f). . Here, “hybridizes under highly stringent conditions” means that the polynucleotide hybridizes to the target polynucleotide under highly stringent conditions and does not hybridize to nucleotides other than the target nucleotide. “High stringency conditions” can be determined depending on the melting temperature (° C.) of the double strand of the polynucleotide and the target nucleotide, the salt concentration of the solution, and the like. It is a technique well known to those skilled in the art to select a target sequence and then set a stringent condition accordingly (for example, Molecular Cloning 2nd edition, Cold Spring Harbor Laboratory (1989)).

The hybridization method can be performed according to a known method. Moreover, when using a commercially available library, it can carry out according to the method as described in an attached instruction manual.

According to a preferred embodiment of the present invention, the germ cell identification marker having the engraftment ability to the host gonad of the present invention comprises one or more polynucleotides selected from the following (a) to (f): is there:
(A) 1 selected from the nucleotide sequences of SEQ ID NOs: 1-7, 9, 11-13, 18-28, 31-33, 35-39, 42-49, 51-60, 63-66, 70, 73 Or a polynucleotide comprising two or more nucleotide sequences,
(B) selected from the nucleotide sequences of SEQ ID NOS: 1-7, 9, 11-13, 18-28, 31-33, 35-39, 42-49, 51-60, 63-66, 70, 73 A polynucleotide comprising one or more nucleotide sequences, wherein the polynucleotide has a germ cell identification function capable of engraftment in the host gonad,
(C) 1 selected from the nucleotide sequences of SEQ ID NOs: 1-7, 9, 11-13, 18-28, 31-33, 35-39, 42-49, 51-60, 63-66, 70, 73 Alternatively, in two or more nucleotide sequences, one or several (for example, 1 to 20, preferably 1 to 10, more preferably 1 to 5, more preferably 1 to 4, further preferably 1 to 3) (Particularly preferably 1 to 2) of a polynucleotide represented by a nucleotide sequence in which bases have been deleted, added, inserted or substituted, and / or a nucleotide sequence in which bases are added to one or both ends thereof. A polynucleotide having a germ cell identification function capable of engraftment in the host gonad,
(D) 1 selected from the nucleotide sequences of SEQ ID NOs: 1-7, 9, 11-13, 18-28, 31-33, 35-39, 42-49, 51-60, 63-66, 70, 73 Or a polynucleotide represented by a nucleotide sequence having at least 70% identity (preferably at least 85%, more preferably at least 90%, even more preferably at least 95% identity) with two or more nucleotide sequences, A polynucleotide having a germ cell identification function capable of engraftment in the host gonad,
(E) 1 selected from the nucleotide sequences of SEQ ID NOs: 1-7, 9, 11-13, 18-28, 31-33, 35-39, 42-49, 51-60, 63-66, 70, 73 Or a polynucleotide encoding an ortholog gene of a gene consisting of two or more nucleotide sequences, and (f) a polynucleotide consisting of a complementary sequence of a polynucleotide having the nucleotide sequence shown in any of (a) to (e) above A polynucleotide having a function of recognizing a germ cell, which hybridizes under highly stringent conditions and has an ability to engraft a host gonad.

According to a further preferred aspect of the present invention, the germ cell identification marker having engraftment ability to the host gonad of the present invention comprises one or more polynucleotides selected from the following (a) to (f): Is:
(A) SEQ ID NOs: 1, 3, 4, 6, 9, 11, 12, 18, 21, 22, 24, 25, 26, 27, 28, 31, 32, 33, 35, 37, 38, 43, 44 , 45, 46, 47, 49, 51, 52, 53, 54, 56, 57, 58, 60, 63, 65, 70, 73. A polynucleotide comprising one or more nucleotide sequences selected from nucleotide sequences ,
(B) Sequence application No. 1, 3, 4, 6, 9, 11, 12, 18, 21, 22, 24, 25, 26, 27, 28, 31, 32, 33, 35, 37, 38, 43, Comprising one or more nucleotide sequences selected from nucleotide sequences 44, 45, 46, 47, 49, 51, 52, 53, 54, 56, 57, 58, 60, 63, 65, 70, 73 A polynucleotide having a germ cell identification function capable of engrafting in the host gonad,
(C) SEQ ID NOs: 1, 3, 4, 6, 9, 11, 12, 18, 21, 22, 24, 25, 26, 27, 28, 31, 32, 33, 35, 37, 38, 43, 44 , 45, 46, 47, 49, 51, 52, 53, 54, 56, 57, 58, 60, 63, 65, 70, 73 in one or more nucleotide sequences, 1 or Several (for example, 1 to 20, preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 4, further preferably 1 to 3, particularly preferably 1 to 2) A polynucleotide represented by a nucleotide sequence in which a base is deleted, added, inserted or substituted, and / or a nucleotide sequence in which a base is added to one or both ends thereof, and is engrafted in the host gonad Raw Polynucleotide having a cell identification function,
(D) SEQ ID NOs: 1, 3, 4, 6, 9, 11, 12, 18, 21, 22, 24, 25, 26, 27, 28, 31, 32, 33, 35, 37, 38, 43, 44 45, 46, 47, 49, 51, 52, 53, 54, 56, 57, 58, 60, 63, 65, 70, 73 and at least 70% of the nucleotide sequence. A polynucleotide represented by a nucleotide sequence having the identity of (preferably at least 85%, more preferably at least 90%, and even more preferably at least 95%), and has the ability to engraft the host gonad A polynucleotide having a germ cell identification function,
(E) SEQ ID NOs: 1, 3, 4, 6, 9, 11, 12, 18, 21, 22, 24, 25, 26, 27, 28, 31, 32, 33, 35, 37, 38, 43, 44 45, 46, 47, 49, 51, 52, 53, 54, 56, 57, 58, 60, 63, 65, 70, of a gene consisting of one or more nucleotide sequences selected from nucleotide sequences A polynucleotide encoding an ortholog gene, and (f) hybridizing under a highly stringent condition with a polynucleotide comprising a complementary sequence of the polynucleotide having the nucleotide sequence shown in any of (a) to (e) above A polynucleotide having a germ cell identification function, which is a polynucleotide and has engraftment ability to a host gonad.

According to a further preferred aspect of the present invention, the germ cell identification marker having engraftment ability to the host gonad of the present invention comprises one or more polynucleotides selected from the following (a) to (f): Is:
(A) consisting of one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 9, 11, 18, 25, 28, 32, 35, 37, 38, 44, 47, 49, 52, 65, 70 Polynucleotides,
(B) one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 9, 11, 18, 25, 28, 32, 35, 37, 38, 44, 47, 49, 52, 65, 70 A polynucleotide comprising a germ cell identification function having engraftment ability to the host gonad,
(C) in one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 9, 11, 18, 25, 28, 32, 35, 37, 38, 44, 47, 49, 52, 65, 70, 1 or several (for example, 1 to 20, preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 4, further preferably 1 to 3, particularly preferably 1 to 2) ) In which nucleotides have been deleted, added, inserted or substituted, and / or nucleotide sequences to which one or both of the ends have been added. A polynucleotide having a germ cell identification function,
(D) one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 9, 11, 18, 25, 28, 32, 35, 37, 38, 44, 47, 49, 52, 65, 70 and at least A polynucleotide represented by a nucleotide sequence having 70% identity (preferably at least 85%, more preferably at least 90%, and even more preferably at least 95% identity), the engraftment ability in the host gonad A polynucleotide having a germ cell identification function,
(E) consisting of one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 9, 11, 18, 25, 28, 32, 35, 37, 38, 44, 47, 49, 52, 65, 70 A polynucleotide encoding a gene orthologous gene, and (f) hybridizing under high stringency conditions with a polynucleotide comprising a complementary sequence of the polynucleotide having the nucleotide sequence shown in any of (a) to (e) above A polynucleotide that is a soybean polynucleotide and has a germ cell identification function capable of engraftment in the host gonad.

According to a further preferred aspect of the present invention, the germ cell identification marker having engraftment ability to the host gonad of the present invention comprises one or more polynucleotides selected from the following (a) to (f): Is:
(A) a polynucleotide comprising one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 9, 11, 25, 28, 35, 37, 44, 47, 49, 52, 65,
(B) a polynucleotide comprising one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 9, 11, 25, 28, 35, 37, 44, 47, 49, 52, 65, A polynucleotide having a germ cell identification function capable of engraftment in the host gonad,
(C) one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 9, 11, 25, 28, 35, 37, 44, 47, 49, 52, 65 (for example, 1 -20 bases, preferably 1-10 bases, more preferably 1-5 bases, further preferably 1-4 bases, more preferably 1-3 bases, and most preferably 1-2 bases). A germ cell discriminating function having the ability to engraft the host gonad, comprising a nucleotide sequence inserted or substituted, and / or a nucleotide sequence to which a base is added to one or both ends thereof A polynucleotide having
(D) at least 70% identity with one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 9, 11, 25, 28, 35, 37, 44, 47, 49, 52, 65 (preferably A polynucleotide represented by a nucleotide sequence having an identity of at least 85%, more preferably at least 90%, and even more preferably at least 95%, and having a germ cell identification function capable of engraftment in the host gonad Polynucleotides,
(E) a poly encoding an orthologous gene of a gene comprising one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 9, 11, 25, 28, 35, 37, 44, 47, 49, 52, 65 A polynucleotide that hybridizes under high stringency conditions with a nucleotide, and (f) a polynucleotide consisting of a complementary sequence of the polynucleotide having the nucleotide sequence shown in any of (a) to (e) above, A polynucleotide having a germ cell identification function capable of engrafting a host gonad.

According to a further preferred aspect of the present invention, the germ cell identification marker having engraftment ability to the host gonad of the present invention comprises one or more polynucleotides selected from the following (a) to (f): Is:
(A) a polynucleotide comprising one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 25, 28 and 47;
(B) a polynucleotide comprising one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 25, 28 and 47, and having a germ cell identification function capable of engraftment in the host gonad Polynucleotides,
(C) 1 or several (for example, 1 to 20, preferably 1 to 10, more preferably 1 to 1) nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 25, 28 and 47 (5, more preferably 1 to 4, more preferably 1 to 3, particularly preferably 1 to 2) nucleotide sequences deleted, added, inserted or substituted, and / or one or both of them. A polynucleotide represented by a nucleotide sequence to which a base is added at the end, which has a germ cell identification function capable of engraftment in the host gonad,
(D) at least 70% identity (preferably at least 85%, more preferably at least 90%, even more preferably at least 95) with one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 25, 28, 47 A polynucleotide having a germ cell identification function having engraftment ability to the host gonad,
(E) a polynucleotide encoding an ortholog gene of a gene consisting of one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 25, 28, and 47, and (f) any one of (a) to (e) above A polynucleotide that hybridizes under high stringency conditions with a polynucleotide comprising a complementary sequence of the polynucleotide having the nucleotide sequence shown above, and has a germ cell identification function capable of engrafting in the host gonad. nucleotide.

According to a more preferred embodiment of the present invention, the germ cell identification marker having the engraftment ability to the host gonad of the present invention is SEQ ID NO: 1-7, 9, 11-13, 18-28, 31-33, 35- A polynucleotide comprising one or more nucleotide sequences selected from nucleotide sequences of 39, 42 to 49, 51 to 60, 63 to 66, 70, 73, more preferably SEQ ID NOs: 1, 3, 4, 6, 9, 11, 12, 18, 21, 22, 24, 25, 26, 27, 28, 31, 32, 33, 35, 37, 38, 43, 44, 45, 46, 47, 49, 51, 52, 53, 54, 56, 57, 58, 60, 63, 65, 70, 73, more preferably SEQ ID NOs: 9, 11, 18, 25, 28, 32, 35, 37, 38, 44, 47, 49, 52, 6 A 70, more preferably is SEQ ID NO: 9,11,25,28,35,37,44,47,49,52,65, particularly preferably SEQ ID NO: 25,28,47. According to a preferred embodiment of the present invention, the germ cell identification marker having the engraftment ability to the host gonad of the present invention is a polynucleotide comprising one or more nucleotide sequences selected from the nucleotide sequences of the above SEQ ID NOs. It is a polynucleotide having a germ cell discrimination function having an engraftment ability in the host gonad that is substantially equivalent to the above. The term of a polynucleotide having a germ cell discriminating function having an engraftment ability to the host gonad substantially equivalent to a polynucleotide comprising one or more nucleotide sequences selected from the nucleotide sequence of SEQ ID NO: In the description of the present specification in which the terminology of the polynucleotides b) to (f) is explained, it can be understood by substituting the respective SEQ ID NOs for the SEQ ID NOs that are the subject of the polynucleotide.

The germ cell identification marker having the engraftment ability to the host gonad of the present invention is also called an engraftment germ cell identification marker. By using the germ cell identification marker having the engraftment ability to the host gonad of the present invention, in the surrogate parent fish technique, the germ cell having the engraftment ability to the host gonad can be detected and separated as a transplanted cell. In addition, by using the germ cell identification marker having the engraftment ability to the host gonad of the present invention, a type A spermatogonia having the engraftment ability to the host gonad as a germ cell having the engraftment ability to the host gonad. It is possible to detect and isolate (including spermatogonial stem cells) and oocyte cells (including oocyte stem cells) having the ability to engraft the host gonads. That is, according to one embodiment of the present invention, a type A spermatogonia identification marker having engraftment ability to the host gonad (ie, type A having engraftment ability to the host gonad in type A spermatogonia). A marker for identifying spermatogonia), a spermatogonial stem cell identification marker, an oocyte identification marker capable of engraftment in the host gonad, and an oocyte stem cell identification marker. According to a preferred embodiment of the present invention, the germ cell identification marker having engraftment ability to the host gonad is a type A spermatogonia discrimination marker having engraftment ability to the host gonad.

According to another embodiment of the present invention, the germ cell identification marker having no engraftment ability in the host gonad of the present invention is one or more polynucleotides selected from the following (g) to (l): It consists of:
(G) a polynucleotide comprising one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 75 to 101,
(H) a polynucleotide comprising one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 75 to 101 and having a germ cell identification function having no engraftment ability in the host gonad Polynucleotides,
(I) a nucleotide sequence in which one or several bases are deleted, added, inserted or substituted in one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 75 to 101, and / or one of them, or A polynucleotide represented by a nucleotide sequence to which bases are added to both ends, and having a germ cell identification function having no engraftment ability to the host gonad,
(J) a polynucleotide represented by a base sequence having at least 70% identity with one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 75 to 101, and having the ability to engraft in the host gonad A polynucleotide having a germ cell identification function,
(K) a polynucleotide encoding an orthologous gene of a gene consisting of one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 75 to 101, and (l) any one of (g) to (k) above A polynucleotide that hybridizes under high stringency conditions with a polynucleotide comprising a complementary sequence of a polynucleotide having the nucleotide sequence shown, and has a germ cell identification function that does not have the ability to engraft the host gonad. nucleotide.

According to a preferred embodiment of the present invention, the germ cell identification marker not capable of engrafting the host gonad of the present invention consists of one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 75 to 101. It is a polynucleotide. According to a preferred embodiment of the present invention, the germ cell identification marker having no engraftment ability in the host gonad of the present invention is one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 75 to 101. A polynucleotide having a germ cell identification function that does not have the same ability to engraft the host gonad as a polynucleotide comprising

As a polynucleotide having a germ cell discriminating function having no engraftment ability to the host gonad substantially equivalent to a polynucleotide comprising one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 75 to 101, And polynucleotides selected from the above (g) to (l).

The terms (h) to (i) refer to the sequence numbers and functions that are the subject of the polynucleotide in the contents of the present specification explained for the terms (b) to (f) above. It can be understood by substituting SEQ ID NOs: 75 to 101 and a germ cell discriminating function that does not have the ability to engraft the host gonads.

According to a preferred embodiment of the present invention, the germ cell identification marker having no engraftment ability in the host gonad of the present invention comprises one or more polynucleotides selected from the following (g) to (l): Is:
(G) a polynucleotide comprising one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 75 to 84, 86, 88, 91 to 99,
(H) a polynucleotide comprising one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 75-84, 86, 88, 91-99, and having engraftment ability in the host gonad A polynucleotide having a germ cell identification function,
(I) 1 or several (for example, 1 to 20, preferably 1 to 10) of one or more nucleotide sequences selected from nucleotide sequences of SEQ ID NOs: 75 to 84, 86, 88, 91 to 99 More preferably 1 to 5, more preferably 1 to 4, further preferably 1 to 3, particularly preferably 1 to 2) nucleotide sequences deleted, added, inserted or substituted, and A polynucleotide represented by a nucleotide sequence to which a base is added to one or both ends thereof, and having a germ cell identification function having no engraftment ability to the host gonad,
(J) at least 70% identity (preferably at least 85%, more preferably at least 90%) with one or more nucleotide sequences selected from nucleotide sequences of SEQ ID NOs: 75-84, 86, 88, 91-99 A polynucleotide represented by a base sequence having at least 95% identity), and having a germ cell identification function that does not have the ability to engraft the host gonad,
(K) a polynucleotide encoding an ortholog gene of a gene consisting of one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 75 to 84, 86, 88, 91 to 99, and (l) said (g) A polynucleotide that hybridizes under high stringency conditions with a polynucleotide comprising a complementary sequence of the polynucleotide having the nucleotide sequence shown in any one of (k) and has the ability to engraft the host gonad. A polynucleotide having no germ cell discrimination function.

According to a further preferred aspect of the present invention, the germ cell identification marker having no engraftment ability in the host gonad of the present invention is one or more polynucleotides selected from the following (g) to (l): Is:
(G) 1 selected from the nucleotide sequences of SEQ ID NOs: 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 86, 88, 91, 92, 93, 95, 96, 97, 98 Or a polynucleotide comprising two or more nucleotide sequences,
(H) selected from the nucleotide sequences of SEQ ID NOs: 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 86, 88, 91, 92, 93, 95, 96, 97, 98 A polynucleotide comprising one or more nucleotide sequences, wherein the polynucleotide has a germ cell identification function that does not have the ability to engraft the host gonad,
(I) 1 selected from the nucleotide sequences of SEQ ID NOs: 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 86, 88, 91, 92, 93, 95, 96, 97, 98 Alternatively, in two or more nucleotide sequences, one or several (for example, 1 to 20, preferably 1 to 10, more preferably 1 to 5, more preferably 1 to 4, further preferably 1 to 3) (Particularly preferably 1 to 2) of a polynucleotide represented by a nucleotide sequence in which bases have been deleted, added, inserted or substituted, and / or a nucleotide sequence in which bases are added to one or both ends thereof. A polynucleotide having a germ cell identification function having no engraftment ability in the host gonad,
(J) 1 selected from the nucleotide sequences of SEQ ID NOs: 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 86, 88, 91, 92, 93, 95, 96, 97, 98 Or a polynucleotide represented by a base sequence having at least 70% identity (preferably at least 85%, more preferably at least 90%, even more preferably at least 95% identity) with two or more nucleotide sequences, , A polynucleotide having a germ cell identification function having no engraftment ability to the host gonad,
(K) 1 selected from the nucleotide sequences of SEQ ID NOs: 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 86, 88, 91, 92, 93, 95, 96, 97, 98 Or a polynucleotide encoding an ortholog gene of a gene consisting of two or more nucleotide sequences, and (1) a polynucleotide consisting of a complementary sequence of a polynucleotide having the nucleotide sequence shown in any of (g) to (k) above A polynucleotide having a function of recognizing a germ cell, which hybridizes under highly stringent conditions and has no ability to engraft the host gonad.

According to a further preferred aspect of the present invention, the germ cell identification marker having no engraftment ability in the host gonad of the present invention is one or more polynucleotides selected from the following (g) to (l): Is:
(G) a polynucleotide comprising one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 75, 78, 81, 83, 86, 88, 98;
(H) a polynucleotide comprising one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 75, 78, 81, 83, 86, 88, 98, and engraftment in the host gonad A polynucleotide having a germ cell identification function,
(I) one or several (for example, 1 to 20, preferably 1 to 2) nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 75, 78, 81, 83, 86, 88, 98 A nucleotide sequence in which 10 bases, more preferably 1 to 5, further preferably 1 to 4, further preferably 1 to 3, particularly preferably 1 to 2 bases are deleted, added, inserted or substituted. And / or a polynucleotide represented by a nucleotide sequence to which a base is added to one or both ends thereof, and a polynucleotide having a germ cell identification function having no engraftment ability to the host gonad,
(J) at least 70% identity (preferably at least 85%, more preferably at least 85%) with one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 75, 78, 81, 83, 86, 88, 98 A polynucleotide represented by a nucleotide sequence having 90%, more preferably at least 95% identity), and having a germ cell identification function not capable of engraftment in the host gonad,
(K) a polynucleotide encoding an orthologous gene of a gene consisting of one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 75, 78, 81, 83, 86, 88, 98; g) a polynucleotide that hybridizes under high stringency conditions with a polynucleotide comprising a complementary sequence of the polynucleotide having the nucleotide sequence shown in any of (k) to (k), and has the ability to engraft the host gonad. A polynucleotide having a germ cell identification function which does not have.

According to a further preferred aspect of the present invention, the germ cell identification marker having no engraftment ability in the host gonad of the present invention is one or more polynucleotides selected from the following (g) to (l): Is:
(G) a polynucleotide comprising one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 78, 81, 83, 86, 88;
(H) a polynucleotide comprising one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 78, 81, 83, 86, 88, and having no engraftment ability in the host gonad A polynucleotide having a germ cell identification function,
(I) In one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 78, 81, 83, 86, 88, one or several (for example, 1 to 20, preferably 1 to 10, more Preferably 1 to 5, more preferably 1 to 4, more preferably 1 to 3, particularly preferably 1 to 2 nucleotide sequences deleted and added, inserted or substituted, and / or A polynucleotide represented by a nucleotide sequence to which a base is added to one or both ends thereof, and having a germ cell identification function that does not have the ability to engraft the host gonad,
(J) at least 70% identity (preferably at least 85%, more preferably at least 90%, further with one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 78, 81, 83, 86, 88; A polynucleotide represented by a base sequence having preferably an identity of at least 95%) and having a germ cell identification function having no engraftment ability in the host gonad,
(K) a polynucleotide encoding an ortholog gene of a gene consisting of one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 78, 81, 83, 86, 88, and (l) the above (g) to ( k) a polynucleotide that hybridizes under high stringency conditions with a polynucleotide comprising a complementary sequence of the polynucleotide having the nucleotide sequence shown in any one of the above, and has no ability to engraft the host gonad A polynucleotide having a cell identification function.

According to a further preferred aspect of the present invention, the germ cell identification marker having no engraftment ability in the host gonad of the present invention is one or more polynucleotides selected from the following (g) to (l): Is:
(G) a polynucleotide comprising one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 78 and 86;
(H) a polynucleotide comprising one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 78 and 86, and having a germ cell identification function having no engraftment ability in the host gonad Polynucleotides,
(I) 1 or 2 or more (for example, 1 to 20, preferably 1 to 10, more preferably 1 to 5) of one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 78 and 86 More preferably 1 to 4, more preferably 1 to 3, particularly preferably 1 to 2) nucleotide sequence deleted, added, inserted or substituted, and / or one or both ends thereof. A polynucleotide represented by a nucleotide sequence to which a base is added and having a germ cell identification function having no engraftment ability in the host gonad,
(J) at least 70% identity (preferably at least 85%, more preferably at least 90%, even more preferably at least 95%) with one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 78, 86 A polynucleotide represented by a nucleotide sequence having identity) and having a germ cell identification function having no engraftment ability in the host gonad,
(K) a polynucleotide encoding an ortholog gene of a gene consisting of one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 78 and 86, and (1) any one of (g) to (k) above A polynucleotide that hybridizes under high stringency conditions with a polynucleotide comprising a complementary sequence of a polynucleotide having the nucleotide sequence shown, and has a germ cell identification function that does not have the ability to engraft the host gonad. nucleotide.

According to a more preferred embodiment of the present invention, the germ cell identification marker capable of engraftment in the host gonad of the present invention is selected from the nucleotide sequences of SEQ ID NOs: 75 to 84, 86, 88, 91 to 99 or A polynucleotide comprising two or more nucleotide sequences, more preferably SEQ ID NOs: 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 86, 88, 91, 92, 93, 95, 96, 97, 98, more preferably SEQ ID NO: 75, 78, 81, 83, 86, 88, 98, still more preferably SEQ ID NO: 78, 81, 83, 86, 88, particularly preferable. Are SEQ ID NOs: 78, 86. According to a preferred embodiment of the present invention, the germ cell identification marker having the engraftment ability to the host gonad of the present invention is a polynucleotide comprising one or more nucleotide sequences selected from the nucleotide sequences of the above SEQ ID NOs. It is a polynucleotide having a germ cell discrimination function having an engraftment ability in the host gonad that is substantially equivalent to the above. The term of a polynucleotide having a germ cell discriminating function having an engraftment ability to the host gonad substantially equivalent to a polynucleotide comprising one or more nucleotide sequences selected from the nucleotide sequence of SEQ ID NO: In the contents of the present specification in which the terms of the polynucleotides h) to (i) are explained, it can be understood by substituting the respective SEQ ID NOs for the SEQ ID NOs that are the subject of the polynucleotides.

The germ cell identification marker having no engraftment ability to the host gonad of the present invention is also called a non-engraftment germ cell identification marker. By using the germ cell identification marker that does not have the ability to engraft the host gonad of the present invention, in the surrogate parent fish technique, a germ cell that does not have the ability to engraft the host gonad is detected and separated as a transplanted cell. Can do. By using the germ cell identification marker having no engraftment ability to the host gonad of the present invention as a negative marker, germ cells having engraftment ability to the host gonad can be isolated as transplanted cells.

According to another aspect of the present invention, primers and probes as germ cell identification markers are provided.

The primer and probe according to the present invention specifically hybridize with a polynucleotide according to the present invention or a polynucleotide comprising a complementary sequence of the polynucleotide, and all or part of the germ cell identification gene of the present invention is obtained by a nucleic acid amplification method. Can be amplified. Accordingly, the primers and probes according to the present invention are primers and probes as germ cell gene detection markers that have engraftment ability to the host gonad or germ cell gene detection markers (negative marker) that do not have engraftment ability to the host gonad. Can be used as In addition, the primer and probe according to the present invention include a type A spermatogonia gene detection marker that has engraftment ability to the host gonad, a spermatogonial stem cell gene detection marker, and an oocyte gene detection marker that has engraftment ability to the host gonad. Alternatively, it can be used as a marker for detecting oocyte stem cell genes.

The primers and probes according to the present invention mean those composed of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and the like.

The primer according to the present invention only needs to be able to amplify all or part of the germ cell identification gene of the present invention by a nucleic acid amplification method. For example, a polynucleotide comprising at least 10, preferably at least 15, more preferably at least 20, even more preferably at least 21 base sequences of the nucleotide sequence of the polynucleotide according to the present invention or a complementary sequence thereof. The thing which consists of is mentioned. The primer according to the present invention comprises a nucleotide sequence of 10-30, 12-30, 15-30, 20-30, or 21-30 consecutive nucleotide sequences of the polynucleotide according to the present invention or its complementary sequence. It may consist of a polynucleotide comprising.

The length of the primer according to the present invention can be at least 10 bases, preferably at least 15 bases, more preferably at least 20 bases, and even more preferably at least 21 bases. The length of the primer according to the present invention may be 12-30 bases, 20-30 bases, or 21-30 bases.

The primer according to the present invention may be used as a primer set formed by combining two or more kinds of primers according to the present invention.

Primers and primer sets according to the present invention are obtained by PCR method (Saiki, RK, Bugawan, TL, et al. (1986) Nature, 324, 163-166), NASBA method (Comptom, J. (1991) Nature, 650, 91-92). ), TMA method (Kacian, DL, and Fultz, TJ (1995) US. Patent 5,399, 491), SDA method (Walker, GT, Little, MC, et al (1992) Proc. Natl. Acad. Sci. USA, 89, 392- 396), PCR-SSCP method (Orita, M., Iwahara, H., et al. (1989) Proc. Natl. Acad. Sci. USA, 86, 2776-2770), etc. It can be used as a primer and primer set according to the method.

Examples of the probe according to the present invention include those capable of hybridizing to the base sequence of the polynucleotide according to the present invention.

The length of the probe according to the present invention is at least 10 bases. The length of the probe according to the present invention can be appropriately adjusted depending on the use of the probe, and is preferably 100 to 500 bases when used in an in situ hybridization method.

The probe according to the present invention can be used as a probe according to a conventional method in known methods such as Northern blotting, Southern blotting, in situ hybridization and the like.

Primers and probes according to the present invention can be chemically synthesized based on the base sequences disclosed herein. Primer preparation methods are well known and can be performed according to conventional methods.

According to another aspect of the present invention, there is provided an antibody that specifically binds to germ cells, particularly germ cells having the ability to engraft the host gonads, using the germ cell identification marker of the present invention. Methods for preparing antibodies are well known and can be performed according to conventional methods. For example, such an antibody includes a germ cell identification antibody having the ability to engraft the host gonad, which binds to a part of the protein encoded by the germ cell identification marker of the present invention. A peptide corresponding to a part of the protein encoded by the cell identification marker can be synthesized, and the peptide can be immunized to obtain an antiserum. The peptide is preferably PKWETDSKISECEQT (SEQ ID NO: 308).

According to another aspect of the present invention, there is provided a germ cell identification kit comprising the germ cell identification marker of the present invention, particularly a germ cell identification kit having engraftment ability to the host gonad. The method for preparing the kit is well known and can be carried out according to a conventional method.

According to one aspect of the present invention, the presence or absence of expression of a gene of a germ cell identification marker capable of engrafting in the host gonad using the germ cell identification marker capable of engrafting in the host gonad of the present invention. A method of producing a germ cell capable of engraftment in the host gonad is provided, comprising detecting and / or extent to isolate cells that are expressed or highly expressed from the testis or ovary of a fish. . That is, by using the germ cell identification marker having the engraftment ability to the host gonad of the present invention, the presence or absence and / or the degree of expression of the gene of the germ cell identification marker having the engraftment ability to the host gonad is detected. There is provided a method of enriching germ cells having the ability to engraft into the host gonad, comprising isolating cells that are expressed or highly expressed from the testis or ovary of a fish.

According to a preferred embodiment of the present invention, the method for producing a germ cell capable of engrafting the host gonad of the present invention uses the germ cell identification marker not capable of engrafting the host gonad of the present invention. To detect the presence or absence and / or degree of expression of a germ cell identification marker gene not capable of engraftment in the host gonad, and to isolate cells that are expressed or highly expressed from the testis or ovary of fish Further comprising. As such a method, for example, using a germ cell identification marker having no engraftment ability to the host gonad of the present invention as a negative marker, a germ cell having engraftment ability to the host gonad is converted into a host gonad. For example, separation and removal of cells having no engraftment ability can be mentioned.

In the method for producing a germ cell having the engraftment ability to the host gonad of the present invention, separation using the germ cell identification marker having the engraftment ability to the host gonad of the present invention as a positive marker, and to the host gonad of the present invention Separation using a germ cell identification marker having no engraftment ability as a negative marker may be performed simultaneously, separately or sequentially according to the separation method. May be either. According to a preferred embodiment of the present invention, there is a method of concentrating with a positive marker after removing germ cells that do not have the ability to engraft the host gonads with a negative marker. *

In the present invention, germ cell separation methods using the germ cell marker of the present invention include flow cytometry analysis, density gradient centrifugation, analysis using cell properties such as motility and adhesion ability, and cell surface antigens. Examples include cell separation by analysis using antibodies.

According to one embodiment of the present invention, the germ cells obtained by the method for producing germ cells having the engraftment ability to the host gonad of the present invention are used as isolated germ cells derived from donor fish, and the individual host fish before and after hatching. There is provided a method for inducing differentiation of isolated germ cells into germ line, comprising transplanting into the peritoneal cavity.

The types of host fish and donor fish in the differentiation induction method of the present invention may be the same or different. Examples of different types of fish include the same species of different species and different species of fish. For example, when the donor fish is a rainbow trout, the same species of different species of recipient fish are yamame trout, and the different species of recipient fish are brown trout and char. When the donor fish is bluefin tuna, the recipient fishes of the same genus and different species are, for example, bigeye tuna, albacore tuna, yellowfin tuna, bigeye tuna, etc. In the case where the donor fish is yellowtail or amberjack, examples of the recipient fish of different genera include horse mackerel and nibs. In addition, when the donor fish are groupers or eels, it is possible to use dissimilar fish that are easily reared. When heterogeneous fish are used, for example, immature germ cells derived from donor fish use immature germ cells of fish that are relatively expensive and labor-intensive to grow up to adult fish (for example, larger fish than recipient fish). When using fish that are relatively low cost and low labor to grow up to adult fish (for example, smaller fish than donor fish), the recipient fish eggs are grown by growing the recipient fish after transplantation. It is possible to induce differentiation of sperm and sperm at a relatively low cost and with low labor, for example, using a relatively small water tank. As a preferable combination of donor fish and recipient fish to enjoy this merit, a combination of bluefin tuna (donor) and chub mackerel (recipient) can be exemplified.

In the present invention, the fish is not particularly limited as long as it is a fish that specifically expresses the polynucleotide of the present invention. For example, salmonid fish (for example, rainbow trout, salmon, scallop, chinook salmon), azirid fish (for example, yellowtail) ), Puffer fishes (eg, tiger puffer fish), mackerel fishes (eg, bluefin tuna, southern bluefin tuna), Thai fishes (eg red sea bream), cichlid fishes (eg tilapia), eels, coelacanthes, etc. It is done. In the present invention, the fish may be a saltwater fish or a freshwater fish.

The method of inducing differentiation of the present invention comprises forming sperm and / or eggs derived from donor fish. That is, according to one aspect of the present invention, the germ cells obtained by the method for producing germ cells having engraftment ability in the host gonads of the present invention are used as isolated germ cells derived from donor fish, and the host fish before and after hatching. Provided is a method for producing sperm or egg of a donor fish comprising transplanting into the abdominal cavity of an individual to obtain donor-derived sperm or egg. Further, according to one aspect of the present invention, the germ cells obtained by the method for producing germ cells having the engraftment ability to the host gonads of the present invention are used as isolated germ cells derived from donor fish, and the host fish before and after hatching. Transplanting into the abdominal cavity of an individual, obtaining a sperm or egg from the donor, producing a fertilized egg using the egg or sperm, and hatching it to create a new individual of the donor fish, A method for producing donor fish is provided.

As an example of the use of the differentiation induction method of the present invention, it may be used for the preservation of fish genetic resources. Specifically, if immature germ cells of a rare or endangered species are cryopreserved and transplanted to a host of a related species that is easy to breed when needed, these hosts are rare or endangered species ( In some cases, it becomes possible to produce eggs and sperm derived from already extinct species). In addition, when various useful strains and varieties are produced by techniques such as gene transfer in the future, it is possible to maintain them without subcultivating individuals, and when necessary, individuals can be maintained by transplanting to host embryos. Can be modified.

The present invention will be described in detail by the following examples, but the present invention is not limited thereto.

Example 1: Production of germ cell identification marker In order to create a germ cell identification marker that can identify the presence or absence of engraftment in the host gonad, a germ cell capable of engraftment in the host gonad is isolated and specific to this cell. The gene that was expressed in a comprehensive manner was comprehensively analyzed to obtain a germ cell identification marker. Specifically, it is as follows.

(1) Isolation of germ cells (engraftment cells) capable of engraftment in the host gonads According to the method of Non-patent document 1, the testis was extracted from 15 rainbow trout (male) introduced with vasa-GFP gene as donor fish After dispersion by treatment with trypsin enzyme (manufactured by Worthington Biochemical Corpotaion), a cell population containing type A spermatogonia was obtained. The obtained cell population was used as isolated germ cells (transplanted cells) to be transplanted into individual host fish in the surrogate parent fish technique. As individual host fish, 230 rainbow trout larvae (29 days after fertilization) were used.

The obtained isolated germ cells were transplanted by a fixed number of cells into the peritoneal cavity of the host fish individual according to the method of Citation 1. On the 14th day after transplantation, the host gonads in which engraftment of the transplanted GFP-positive germ cells was confirmed using a fluorescence microscope was isolated using a tweezers under a stereomicroscope. The isolated gonads were dispersed by treatment with trypsin enzyme (manufactured by Worthington Biochemical Corp.), and then engrafted transplanted germ cells were isolated and collected using GFP fluorescence as an index.

(2) Analysis by Quartz-seq method In order to perform comprehensive gene expression analysis from isolated engrafted cells, the Quartz-seq method (Sasagawa et. Al., Quartz-Seq: a highly The analysis was performed according to the reproducible and sensitive single-cell RNA sequencing method, reveals non-genetic gene-expression heterogeneity. Genome Biol. 2013 Apr 17; 14 (4)). Specifically, 14 engrafted cells obtained by (1) above and 94 cells (transplanted cells) contained in the cell population obtained from the testis used for transplantation were subjected to the Quartz-seq method. Comparison of gene expression was performed.

The Quartz-seq method is one of 1-cell RNA sequencing methods. Specifically, the obtained cells were used as a cell lysate, and 10% NP-40 (manufactured by ThermocientScientific) diluted to a final concentration of 0.5% and 0.4 unit (1 unit / μl) of RNasin Plus RNase Inhibitor. (Promega Co., Ltd.) is used to melt, and cDNA complementary to the mRNA is obtained by reverse transcription reaction. Poly-A is added to the 3 ′ end of the reverse transcribed cDNA using terminal transferase. The complementary strand (second strand) is synthesized by an extension reaction from the tagging primer bound to the poly-A strand, and the cDNA derived from the target RNA is amplified by suppression PCR. Purified.

The obtained purified cDNA was sequenced and data processing was performed according to the scheme of FIG. Specifically, data processing was performed in the order of removal of the sequence adapter sequence and quantification of the gene expression level. The results are shown in FIGS.

As shown in FIG. 2, some of the number of reads was observed in the engrafted cells. As shown in FIG. 3, the GC content of the sequence was uniform. As shown in FIG. 4, the mapping ratio was uniform.

Based on these results, WTA (whole transcription amplification) and / or 1 engrafted cell that is considered to affect the survival of the somatic cell was removed from the analysis target, and the gene expression difference was analyzed. As a result, the total number of transcripts of rainbow trout used in the analysis was 175,285, and it was found that there were 2,904 transcripts that had a significant difference in expression between transplanted cells and engrafted cells. It was.

Thereafter, PCA (principal component analysis) was performed. PCA is an analysis in which multivariate is subjected to dimensional compression in a low dimension, and it is understood how cells are classified by performing this analysis. In addition, genes that have a large functional contribution to the classified cells can be obtained from the magnitude of factor loading. Specifically, 94 cells of transplanted cells and 13 cells of engrafted cells, a total of 107 cells, based on the expression information of 2904 gene that had a significant difference in expression between the transplanted cells and the engrafted cells. A pair plot was drawn. The result of the plot is shown in FIG. Also, factor loading ranking in PCA. As shown in FIG. As shown in FIG. 6, genes having a large contribution to the first main component can be seen.

As a result of PCA analysis, 74 transcripts characterizing engrafted cells and 27 transcripts characterizing non-engrafted cells were obtained.

(3) Comparison with SP microarray As disclosed in Non-Patent Document 1, type A spermatogonia having high engraftment ability are considered to be spermatogonial stem cells. Since the engrafted cells obtained in (1) above are cells that have just engrafted in the host gonad, it is considered that the gene as a spermatogonial stem cell (SSC) itself is expressed. Therefore, the results of the microarray analysis that is significantly expressed in the SP cells of interest in spermatogonial stem cells and the microarray analysis that is significantly expressed in non-SP cells were compared with the results of the Quartz-seq analysis obtained in (2) above. .

The results of microarray analysis significantly expressed in SP cells and microarray analysis significantly expressed in non-SP cells were those disclosed in Non-Patent Document 2. Here, SP (Side® Population) cells are a cell population with low staining ability when stained with Hoechst 33342, which is a cell membrane-permeable nuclear staining reagent, and cells with high engraftment ability to the host gonad are concentrated. It is known that Specifically, according to the enrichment method of Non-Patent Document 2, these microarrays are based on the development of Hoechst blue and Hoechred by flow cytometry analysis, and the SP cell population with high engraftment ability and low engraftment ability. It was obtained by separating a non-SP cell population and analyzing gene expression by microarray between SP cells and non-SP cells. There are 8350 microarray transcripts that are significantly strongly expressed in SP cells and 7798 microarray transcripts that are significantly strongly expressed in non-SP cells.

FIG. 7 shows the result of comparison between microarray analysis using SP cells and Quartz-seq analysis. As a result, 14 of the transcripts that characterize the engrafted cells obtained by the Quartz-seq analysis overlap with genes that are significantly expressed in the SP cells in the microarray analysis, and the translip that characterizes the non-engrafted cells. 8 of them overlapped with non-SP significantly expressed genes in microarray analysis.

Example 2: Analysis of sequence similarity with other fish species 74 transcripts characterizing engraft cells obtained as a result of the Quartz-seq analysis of Example 1 (2) above, transcript 27 characterizing non-engraft cells Blast search was performed on cDNA sequences of tilapia, puffer fish and tuna as fish species other than rainbow trout. The tilapia cDNA sequence is from the Ensemble database (ftp://ftp.ensembl.org/pub/release-84/fasta/oreochromis_niloticus/), while the puffer's cDNA sequence is from the Ensemble database (ftp://ftp.ensembl.org) / pub / release-84 / fasta / tetraodon_nigroviridis /) The tuna cDNA sequence is available from the Central Fisheries Research Institute database (http://nrifs.fra.affrc.go.jp/ResearchCenter/5_AG/genomes/Tuna_DNAmicroarray/index_j. html). Blast search used blastn 2.2.31.

The results are shown in FIGS. 8a to f, and the summary is shown in Tables 1 and 2 below.

As shown in Tables 1 and 2, about 30% of the Blast searches out of 74 transcripts that characterize engrafted cells resulted in about 30% of the Blast searches out of 27 transcripts that characterized non-engrafted cells. The result was a little less than 20%. The identity of the hit sequence was 70% or more.

The sequence numbers 1 to 74 and 75 to 101 and the corresponding transcript sequence names are shown in Tables 3 and 4 below, respectively.

Example 3: Analysis by qRT-PCR Concentration of non-engrafted cells on the sequence of 74 transcripts (SEQ ID NOs: 1 to 74) characterizing the engrafted cells obtained as a result of the Quartz-seq analysis in Example 1 (2) above In order to ascertain whether or not it is highly expressed in type A spermatogonia (SP cells) in the engrafted cell-enriched fraction compared to fractional type A spermatogonia (nonSP cells (non-SP cells)), and The non-engrafting cell enriched fraction of the 27 transcript sequences (SEQ ID NOs: 75 to 101) that characterize non-engrafting cells is compared to the engrafted cell-enriched fraction type A spermatogonia (SP cells). Real-time quantitative PCR (qRT-PCR, qPCR (quantitative real-time PCR)) was performed in order to confirm whether it was highly expressed in type A spermatogonia (non-SP cells).

QRT-PCR was performed according to the following method. First, immature rainbow trout at 10 months, 11 months, and 15 months, respectively, using a flow-sert meter, Hayashi, Makoto, et al. "Enrichment of spermatogonial stem cells using side population in teleost." Biology of reproduction 91.1 (2014): SP cells and non-SP cells were sorted according to the method of 23. RNA was purified from the sorted cells using RNeasy mini Kit (Qiagen), and then subjected to reverse transcription to synthesize cDNA. Oligo- (dT) primer (5'-d (T) 25VN-3 ') and SuperScript III First-Strand Synthesis System (Thermo Fisher Fisher Scientific) were used for cDNA synthesis.

Using the obtained cDNA, the gene expression level was analyzed by qRT-PCR. qRT-PCR was performed using LightCycler 480 System II (Nippon Genetics Co., Ltd.) and QuantTect SYBR Green PCR Kit (Qiagen). Corresponds to the sequence of 74 transcripts that characterize engraft cells (SEQ ID NOs 1 to 74) and 27 sequences that characterize non-engraft cells (SEQ ID NOs 75 to 101) used in qRT-PCR. The primers are shown in Tables 5 and 6 below. Vasa was used as a representative gene that is known to be uniformly expressed in all type A spermatogonia and whose expression level does not correlate with the engraftment ability of type A spermatogonia. The b-actin gene was used to correct the RNA amount between samples. The primers for vasa and b-actin are shown in Table 7.

PCR was performed at 95 ° C. for 15 minutes, and then the reaction of “95 ° C. for 15 seconds, 60 ° C. for 1 minute” was performed 45 times. Data analysis is performed using Microsoft Excel (Microsoft) and the ΔΔCt method (Livak, Kenneth J., and Thomas D. Schmittgen. "Analysis of relative gene expression data using real-time quantitative PCR and the 2- ΔΔCTmethod. methods 25.4 (2001): 402-408.)

Based on the results of qRT-PCR, the expression levels of each transcript in SP cells and non-SP cells were compared. A significant difference test was also performed by Student's t-test. The comparison by qRT-PCR can be analyzed with higher accuracy than the comparison with Example 1 (3) SP microarray in which association by blast is performed. In addition, in Example 1 (3) SP microarray, only a limited number of genes plotted on the array could be analyzed, whereas qTR-PCR can analyze all candidate genes. . As a result, the expression levels of SP cells and non-SP cells differed with respect to the 74 transcript sequences (SEQ ID NOs: 1 to 74) that characterize engrafted cells, and SP cells were compared to non-SP cells. 39 transcripts (SEQ ID NOs: 1, 3, 4, 6, 9, 11, 12, 18, 21, 22, 24, 25, 26, 27, 28, 31, 32, 33, 35, 37, 38, 43, 44, 45, 46, 47, 49, 51, 52, 53, 54, 56, 57, 58, 60, 63, 65, 70, 73). Among these, 15 transcripts (corresponding to SEQ ID NOs: 9, 11, 18, 25, 28, 32, 35, 37, 38, 44, 47, 49, 52, 65, 70) differ in expression level. Was confirmed to be 2 times or more, or the expression level was significantly high in SP cells although the difference in expression level was 2 times or less. Eleven transcripts (corresponding to SEQ ID NOs: 9, 11, 25, 28, 35, 37, 44, 47, 49, 52, 65) were confirmed to have a difference in expression level of 2 times or more. It is. Among these, in particular, three transcripts (corresponding to SEQ ID NOs: 25, 28, and 47) are confirmed to have a difference in expression level of 2 times or more, and may be significantly highly expressed in SP cells. Admitted.

In addition, the expression levels of SP cells and non-SP cells differed from the 27 transcript sequences (SEQ ID NOs: 75 to 101) that characterize non-engrafting cells, and non-SP cells compared to SP cells. 19 transcripts (SEQ ID NOs: 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 86, 88, 91, 92, 93, 95, 96, 97, 98) Corresponding) was obtained. Among these, 7 transcripts (corresponding to SEQ ID NOs: 75, 78, 81, 83, 86, 88, 98) were confirmed to have a difference in expression level of 2 times or more, or Although the difference was 2 times or less, it was found that the expression was significantly high in non-SP cells. Five transcripts (corresponding to SEQ ID NOs: 78, 81, 83, 86, 88) were confirmed to have a difference in expression level of 2 times or more. Among these, in particular, two transcripts (corresponding to SEQ ID NOs: 78 and 86) were confirmed to have a difference in expression level of 2 times or more, and were also found to be significantly highly expressed in non-SP cells. It was.

Three transcripts (SEQ ID NO: 25 (TCONS_00162389), SEQ ID NO: 28 (TCONS_00095523), which characterize the engrafted cells, in which the difference in expression level was confirmed to be twice or more and a significant difference was also observed. The results for SEQ ID NO: 47 (TCONS — 00101612)) and two transcripts (TCONS — 00173676, TCONS — 00002882) and vasa characterizing non-engrafting cells are shown in FIG.

Example 4: Immunohistochemical qRT-PCR method of dyeing Example 3, a significant difference was observed in the expression between SP cells and non-SP cells, and was most highly expressed in SP cells compared to non-SP cells sequence In order to ascertain whether or not the transcript corresponding to number 28 is highly expressed in some immature testis cells in immature testis, immunohistochemical staining was performed. First, in order to produce an antibody against a part of the protein encoded by SEQ ID NO: 28, a peptide (PKWETDSKISISCEQT (SEQ ID NO: 308)) was synthesized, and an antiserum (GP8023) was obtained by immunizing a guinea pig.

In order to perform immunohistochemical staining, testis of 12.5 months old immature rainbow trout was fixed with 4% paraformaldehyde phosphate buffer (PFA), and paraffin sections were prepared according to a conventional method. The prepared paraffin section was subjected to deparaffinization, hydrophilic treatment, antigen activation with HistoVT ONE (manufactured by Nacalai Tesque), and blocking with Block Ace (manufactured by Snow Brand Megmilk), followed by primary antibody reaction. . The primary antibody reaction was performed at 4 ° C. for 16 hours using the above antibody (GP8023) diluted 1000 times using Can Get Signal Solution B (TOYOBO). The secondary antibody reaction was performed at room temperature for 1 hour using Alexa Fluor 488 conjugated anti-guinea pig IgG diluted 200-fold with Can Get Signal Solution A (TOYOBO).

After observing the expression of the protein encoded by the transcript corresponding to SEQ ID NO: 28 using a fluorescent microscope (Olympus), it was subjected to hematoxylin eosin (HE) staining, and the cell morphology was observed by bright field observation. The results are shown in FIG.

As shown in FIG. 10, the antibody binding to a part of the protein encoded by SEQ ID NO: 28 showed a strong signal in some A-type restricted cells in the immature testis. That is, it was confirmed that the protein encoded by the transcript corresponding to SEQ ID NO: 28 was strongly expressed in some A-type spermatogonia in immature testis. The type A spermatogonia in which this strong expression was observed are considered to be germ cells having engraftment ability to the host gonad.

Claims (10)

1. A germ cell identification marker comprising the following (I) germ cell identification marker having engraftment ability to the host gonad and / or (II) germ cell identification marker having no engraftment ability to the host gonad:
   (I) Germ cell identification marker comprising one or more polynucleotides selected from the following (a) to (f):
   (A) a polynucleotide comprising one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 1 to 74,
   (B) a polynucleotide comprising one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 1 to 74, wherein the polynucleotide has a germ cell identification function capable of engraftment in the host gonad ,
   (C) a nucleotide sequence in which one or several bases are deleted, added, inserted or substituted in one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 1 to 74, and / or one of them, or A polynucleotide represented by a nucleotide sequence to which bases are added at both ends, and having a germ cell identification function having engraftment ability to the host gonad,
   (D) a polynucleotide represented by a nucleotide sequence having at least 70% identity with one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 1 to 74, and having the ability to engraft in the host gonad A polynucleotide having a germ cell identification function,
   (E) a polynucleotide encoding an ortholog gene of a gene consisting of one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 1 to 74, and (f) any one of (a) to (e) above A polynucleotide that hybridizes under high stringency conditions with a polynucleotide comprising a complementary sequence of a polynucleotide having the nucleotide sequence shown, and has a germ cell identification function capable of engraftment in the host gonad,
   (II) A germ cell identification marker having no engraftment ability to the host gonad consisting of one or more polynucleotides selected from the following (g) to (l):
   (G) a polynucleotide comprising one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 75 to 101,
   (H) a polynucleotide comprising one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 75 to 101 and having a germ cell identification function having no engraftment ability in the host gonad Polynucleotides,
   (I) a nucleotide sequence in which one or several bases are deleted, added, inserted or substituted in one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 75 to 101, and / or one of them, or A polynucleotide represented by a nucleotide sequence to which bases are added to both ends, and having a germ cell identification function having no engraftment ability to the host gonad,
   (J) a polynucleotide represented by a base sequence having at least 70% identity with one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 75 to 101, and having the ability to engraft in the host gonad A polynucleotide having a germ cell identification function,
   (K) a polynucleotide encoding an orthologous gene of a gene consisting of one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 75 to 101, and (l) any one of (g) to (k) above A polynucleotide that hybridizes under high stringency conditions with a polynucleotide comprising a complementary sequence of a polynucleotide having the nucleotide sequence shown, and has a germ cell identification function that does not have the ability to engraft the host gonad. nucleotide.
2. The marker according to claim 1, wherein the germ cell identification marker is a germ cell identification marker for identifying the presence or absence of engraftment in the host gonad.
3. The germ cell identification marker comprising one or more polynucleotides selected from the following (a) to (f) is a germ cell identification marker having engraftment ability to the host gonads: Markers listed:
   (A) a polynucleotide comprising one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 1 to 74,
   (B) a polynucleotide comprising one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 1 to 74, wherein the polynucleotide has a germ cell identification function capable of engraftment in the host gonad ,
   (C) a nucleotide sequence in which one or several bases are deleted, added, inserted or substituted in one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 1 to 74, and / or one of them, or A polynucleotide represented by a nucleotide sequence to which bases are added at both ends, and having a germ cell identification function having engraftment ability to the host gonad,
   (D) a polynucleotide represented by a nucleotide sequence having at least 70% identity with one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 1 to 74, and having the ability to engraft in the host gonad A polynucleotide having a germ cell identification function,
   (E) a polynucleotide encoding an ortholog gene of a gene consisting of one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 1 to 74, and (f) any one of (a) to (e) above A polynucleotide having the function of recognizing a germ cell, which hybridizes with a polynucleotide having the nucleotide sequence shown under highly stringent conditions and has the ability to engraft the host gonad.
4. The marker according to any one of claims 1 to 3, wherein the germ cell identification marker having engraftment ability to the host gonad is a spermatogonial stem cell (SSC) marker.
5. A primer or probe for detecting an SSC gene, which can hybridize to the polynucleotide according to claim 4 or a polynucleotide comprising a complementary sequence of the polynucleotide.
6. The germ cell identification marker comprising one or more polynucleotides selected from the following (g) to (l) is a germ cell identification marker having no engraftment ability in the host gonad: Marker according to 2:
   (G) a polynucleotide comprising one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 75 to 101,
   (H) a polynucleotide comprising one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 75 to 101 and having a germ cell identification function having no engraftment ability in the host gonad Polynucleotides,
   (I) a nucleotide sequence in which one or several bases are deleted, added, inserted or substituted in one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 75 to 101, and / or one of them, or A polynucleotide represented by a nucleotide sequence to which bases are added to both ends, and having a germ cell identification function having no engraftment ability to the host gonad,
   (J) a polynucleotide represented by a base sequence having at least 70% identity with one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 75 to 101, and having the ability to engraft in the host gonad A polynucleotide having a germ cell identification function,
   (K) a polynucleotide encoding an orthologous gene of a gene consisting of one or more nucleotide sequences selected from the nucleotide sequences of SEQ ID NOs: 75 to 101, and (l) any one of (g) to (k) above A polynucleotide that hybridizes under high stringency conditions with a polynucleotide comprising a complementary sequence of a polynucleotide having the nucleotide sequence shown, and has a germ cell identification function that does not have the ability to engraft the host gonad. nucleotide.
7. Use of the germ cell identification marker having engraftment ability to the host gonad according to any one of claims 1 to 4, and presence or absence of expression of a gene of the germ cell identification marker having engraftment ability to the host gonad And / or detect the degree
   A method for producing a germ cell capable of engraftment in a host gonad, comprising separating a cell having expression or high expression from a testis or ovary of a fish.
8. The expression of a gene of a germ cell identification marker that does not have the ability to engraft the host gonad using the germ cell identification marker that does not have the ability to engraft the host gonad according to claim 1, 2, or 6. Detect presence and / or degree,
   The method for producing a germ cell having an engraftment ability in a host gonad according to claim 7, further comprising separating a cell having a high expression or a high expression from a testis or ovary of a fish.
9. The method for producing a germ cell having an engraftment ability in the host gonad according to claim 7 or 8, wherein the fish is a salmonid fish or a mackerel fish.
10. 5. A germ cell identification antibody having engraftment ability to the host gonad that binds to a part of a protein encoded by the germ cell identification marker having engraftment ability to the host gonad according to any one of claims 1 to 4. .

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