WO2017164390A1 - Antibody capable of binding to undifferentiated germ cells of scombridae fish - Google Patents

Abstract

The present invention relates to a monoclonal antibody that specifically recognizes undifferentiated germ cells of scombridae fish, wherein the monoclonal antibody is an antibody against undifferentiated germ cells isolated from a testis or an ovary of scombridae fish. The present invention provides an antibody that can isolate undifferentiated germ cells of scombridae fish.

Description

Mackerel fish undifferentiated germ cell binding antibody Reference to related applications

This patent application is based on Japanese Patent Application No. 2016-60908 (filing date: March 24, 2016), which was previously filed in Japan, 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 an antibody that specifically recognizes an undifferentiated germ cell of a mackerel fish, a method for concentrating and inducing differentiation of an undifferentiated germ cell of a mackerel fish, and a mackerel using these methods. The present invention relates to a method for producing sperm or egg of a family fish and a method for producing a mackerel fish.

Background Art Bluefin tuna and other mackerel fish are important fisheries with high economic value. On the other hand, large-scale mackerel fish such as bluefin tuna and southern bluefin tuna, which are in high demand as high-grade fish, has a problem of a significant decrease in the amount of natural resources due to overfishing. Therefore, artificial seedling production and artificial seedling (fry) release for the purpose of increasing the amount of resources are being studied. In the production of artificial seeds and seeds, in order to avoid annihilation due to inbreeding and the onset of specific diseases, genetic diversity of individuals that become parent fish is required, and therefore it is necessary to grow a large number of parent fish. However, bluefin tuna, for example, weighs more than 100 kg, has a length of 1 m or more, matures only when it is 3-5 years of age, so it takes a long time to become a parent fish and requires a very large breeding space for swimming at high speed. And Furthermore, artificial ripening is technically difficult due to its weak handling stress, and the success rate of one-to-one mating is low. From such a background, it is extremely difficult in terms of cost and technology to raise a large number of parent fish under artificial management and collect sperm and eggs, including problems of feed and breeding space.

As an artificial seed production technology for fish, the present inventors have transplanted germ cells derived from different types of fish (donor fish) different from the host (recipient) fish into the abdominal cavity of the individual host fish before and after hatching. Have found that germ cells can be induced to differentiate into germ line (gametes), and have succeeded in inducing differentiation of gametes of target fish using heterologous host fish (for example, patents) Reference 1). This technique is also called surrogate parent fish technique or borrowed belly farming technique, and sperm and eggs of fish that are difficult to breed such as bluefin tuna are produced by heterogeneous host fish that are easy to breed and are mated at low cost and simple seedling production It is expected as a technology that enables

In this surrogate parent fish technique, it is necessary to isolate only undifferentiated germ cells from the gonad, testis or ovary of the donor fish and use it for transplantation in order to ensure sufficient transplantation efficiency for commercial mass production. is there. In Patent Document 1, in order to isolate only primordial germ cells, genetic recombination in which a GFP (Green Fluorescent Protein) gene is introduced into the transcriptional regulatory region of the vasa gene that is specifically expressed in primordial germ cells. Rainbow trout is used as a donor fish, and cells expressing the GFP gene are isolated as primordial germ cells. However, sperm and eggs obtained by inducing differentiation of primordial germ cells derived from such genetically modified fish are those into which the GFP gene has been introduced, and the same applies to fry produced using these sperm and eggs. Seedlings into which the GFP gene has been introduced. Therefore, it cannot be used as edible fish or released seedlings and is not suitable as a practical production technique.

As a method for separating undifferentiated germ cells such as primordial germ cells from donor fish without introducing a reporter gene such as GFP gene, a method for separating germ cells using an antibody that specifically binds to germ cells is examined. Has been. For example, Patent Document 2 discloses an anti-tuna vasa antibody that can specifically detect tuna-derived germ cells. However, because this antibody has an antigen recognition site in the cytoplasm, it can detect germ cells in fixed cells and tissues, but it cannot detect or isolate living cells. Impossible.

On the other hand, in salmonids such as rainbow trout, the present inventors selectively detect spermatogonia by antibodies obtained by immunizing mice with live spermatogonia isolated from GFP-transfected rainbow trout. It has been confirmed that germ cells can be separated even if they are not transgenic fish by using the antibody (see Non-Patent Document 1). However, in the method of Non-Patent Document 1, the spermatogonia that serve as the antigen are derived from transgenic fish, and it is difficult to establish transgenic fish in mackerel fish such as bluefin tuna. is there.

Japanese Patent No. 4300287 WO2010 / 035465

The present inventors used the rainbow trout antibody of Non-Patent Document 1 to selectively separate bluefin tuna spermatogonia. However, the rainbow trout antibody was able to detect a signal to the gonad cells of bluefin tuna, but could not specifically detect and isolate undifferentiated germ cells such as live spermatogonia of bluefin tuna.

As described above, as far as the inventors know, no antibody has been reported that is sufficiently satisfactory as an antibody used for the practical application of surrogate parent fish techniques for mackerel fish such as bluefin tuna.

The present inventors have so far succeeded in establishing a gate (fraction) containing many undifferentiated germ cells such as spermatogonia in bluefin tuna without using a gene transfer technique. Further, when a monoclonal antibody was prepared using the obtained fraction as an antigen, it was found that the monoclonal antibody specifically recognizes the surface of undifferentiated germ cells, particularly the undifferentiated germ cells. In addition, the inventors succeeded in specifically separating and concentrating undifferentiated germ cells of mackerel fish using the monoclonal antibody as a label. Furthermore, when the bluefin tuna undifferentiated germ cells concentrated with the monoclonal antibody and the testis dispersed cells (gonad cells) containing the undifferentiated bluefin tuna undifferentiated germ cells were transplanted into the peritoneal cavity of the nibe, respectively, Surprisingly, in the case of transplanting non-enriched bluefin tuna testis-dispersed cells, no engraftment of the transplanted cells in the host gonad was observed, whereas bluefin tuna undifferentiated reproduction enriched with the monoclonal antibody was not observed. In the case where the cells were transplanted, it was confirmed that the transplanted cells had engrafted in the nibe host gonad. From the above results, it has been found that the use of bluefin tuna undifferentiated germ cells concentrated with the monoclonal antibody as transplanted cells significantly increases the engraftment efficiency in the host gonad. The present invention is based on these findings.

That is, the present invention relates to a novel antibody specifically recognizing mackerel fish undifferentiated germ cells, a method of enriching mackerel fish undifferentiated germ cells, a method of inducing differentiation, and a method of producing mackerel fish using these methods. The purpose is to provide.

According to the present invention, the following inventions are provided.
(1) A monoclonal antibody that specifically recognizes undifferentiated germ cells of mackerel fish,
A monoclonal antibody, wherein the monoclonal antibody is an antibody against undifferentiated germ cells isolated from the testis or ovary of a mackerel fish.
(2) a monoclonal antibody that specifically recognizes undifferentiated germ cells of mackerel fish,
The monoclonal antibody is a hybridoma TA-No. 6-28 (NITE ABP-02222) or TA-No. The monoclonal antibody according to (1), which is produced by 15-1 (NITE ABP-02223).
(3) The monoclonal antibody according to (1) or (2), wherein the undifferentiated germ cells are primordial germ cells, A-type spermatogonia or oocytes.
(4) The monoclonal antibody according to any one of (1) to (3), wherein the mackerel fish is one selected from the group of Tuna fish, Suma fish, Bonito fish, and Mackerel fish.
(5) The monoclonal antibody according to any one of (1) to (4), which specifically recognizes the surface of undifferentiated germ cells of mackerel fish.
(6) Using the antibody according to any one of (1) to (5), from the testis or ovary of a mackerel fish comprising undifferentiated germ cells derived from a mackerel fish, undifferentiated from a mackerel fish A method for enriching undifferentiated germ cells of mackerel fish, comprising isolating germ cells.
(7) A method for inducing differentiation of undifferentiated germ cells into gametes, comprising transplanting undifferentiated germ cells isolated from the testis or ovary of a mackerel fish into the abdominal cavity of a host fish individual before and after hatching In
Before transplantation, using the antibody according to any one of (1) to (5), the undifferentiated reproduction of the mackerel fish from the testis or ovary of the mackerel fish comprising undifferentiated germ cells derived from the mackerel fish Isolate and concentrate cells,
A method for inducing differentiation of undifferentiated germ cells into gametes, comprising transplanting the separated and concentrated undifferentiated germ cells into the abdominal cavity of a host fish individual before and after hatching.
(8) The method for inducing differentiation of undifferentiated germ cells into gametes according to (7), wherein the mackerel fish that provides the undifferentiated germ cells to be transplanted is different from the host fish.
(9) The method for inducing differentiation of undifferentiated germ cells into gametes according to (7) or (8), wherein the mackerel fish that provides the undifferentiated germ cells to be transplanted is larger than the host individual.
(10) The host fish is one kind selected from mackerel fish, and the mackerel fish that provides undifferentiated germ cells to be transplanted is one kind selected from tuna, (7) to (9) The method for inducing differentiation of undifferentiated germ cells into gametes according to any one of the above.
(11) A method for producing sperm or egg of a mackerel fish comprising transplanting undifferentiated germ cells isolated from a testis or ovary of a mackerel fish into the abdominal cavity of a host fish individual before and after hatching. And
Before transplantation, using the antibody according to any one of (1) to (5), the undifferentiated reproduction of the mackerel fish from the testis or ovary of the mackerel fish comprising undifferentiated germ cells derived from the mackerel fish Isolate and concentrate cells,
Transplanting the separated and concentrated undifferentiated germ cells into the peritoneal cavity of a host fish individual before and after hatching,
A method for producing a sperm or egg of a mackerel fish, comprising inducing differentiation of the transplanted differentiated germ cell into a gamete to obtain a sperm or egg of a mackerel fish.
(12) A method for producing a mackerel fish comprising transplanting undifferentiated germ cells isolated from a testis or ovary of a mackerel fish into the abdominal cavity of a host fish individual before and after hatching,
Before transplantation, using the antibody according to any one of (1) to (5), the undifferentiated reproduction of the mackerel fish from the testis or ovary of the mackerel fish comprising undifferentiated germ cells derived from the mackerel fish Isolate and concentrate cells,
Transplanting the separated and concentrated undifferentiated germ cells into the peritoneal cavity of a host fish individual before and after hatching,
Inducing differentiation of transplanted undifferentiated germ cells into gametes,
A method for producing a mackerel fish comprising crossing the obtained sperm and egg.

By using the antibody of the present invention, it is possible to specifically recognize undifferentiated germ cells of mackerel fish. In addition, by using the antibody of the present invention, it is possible to specifically separate and concentrate mackerel fish undifferentiated germ cells.

FIG. 1a shows the results of HE staining and in situ hybridization on a bluefin tuna paraffin-embedded tissue section (testis tissue). FIG. 1b is a scatter plot of cells contained in a bluefin tuna cell suspension obtained by flow cytometry (FCM) analysis. FS represents the size of the cell, and SS represents the complexity of the internal structure of the cell. FIG. 1c shows photographs (biological microscope, Olympus) of the morphology of the cells contained in the AG gates (fractions) of the scatter plot, respectively. FIG. 2a shows a scatter diagram of cells contained in a bluefin tuna cell suspension obtained by FCM analysis in an immature individual (age 1), and in situ cells contained in the A to G gates of the scatter diagram, respectively. The result of hybridization is shown. FIG. 2b shows the percentage of vasa positive cells (vasa ⁺ ratio) for each gate relative to the total cells. FIG. 3a shows the results of testicular HE staining and in situ hybridization (vasa) in mature individuals (age 3). FIG. 3b shows the results of a scatter plot of cells contained in the bluefin tuna crude cell suspension obtained by FCM analysis. FIG. 3c shows the morphology of the isolated cells contained in the A gate and the results of in situ hybridization. FIG. 3d shows the percentage of vasa positive cells (vasa ⁺ ratio) for each gate relative to the total cells. FIG. 4 shows the results of immune cell staining in the secondary screening. a is a cell in which green fluorescence is clearly observed on the cell membrane (positive signal), b is a cell in which non-specific signals such as blood cells and debris are observed, and c is no signal. An example of a cultured cell is shown. FIG. 5a shows a scatter plot obtained by FCM analysis (FS-SS development) of all cells used in the tertiary screening. FIG. 5b shows a histogram of antibody positive (right arrow) and antibody negative (left arrow). The horizontal axis indicates the signal intensity of the antibody, and the vertical axis indicates the number of cells. FIG. 5c shows a scatter plot obtained by FCM analysis (FS-SS development) of an antibody positive cell population. 6 shows TA-No. The results of immune cell staining and in situ hybridization using a 6-28 hybridoma-producing antibody are shown. 7 shows TA-No. 6-28 hybridoma producing antibody (a) and TA-No. The results of in situ hybridization using the 15-1 hybridoma-producing antibody (b) are shown. Concentration rates were 85.2% and 78.9%, respectively. FIG. 8 shows HE staining in testicular tissue sections of bluefin tuna and TA-No. The result of immunohistochemical staining using 6-28 hybridoma-producing antibody is shown. FIG. 9 shows HE staining (left) in testicular tissue sections of bluefin tuna and TA-No. 6-28 hybridoma producing antibody (top) and TA-No. The results of immunohistochemical staining (right) using a 15-1 hybridoma-producing antibody (bottom) are shown. FIG. 10 shows TA-No. 6-28 hybridoma producing antibody (left) and TA-No. (A) Photograph of cell morphology (biological microscope, Olympus) (flow fraction (Flow) and elution fraction) of flow cytometric analysis of bluefin tuna MACS-enriched undifferentiated germ cells using 15-1 hybridoma-producing antibody (right) Minute (Elute)) and (b) photo of vasa in situｓ hybridization (biological microscope, Olympus) (flow fraction (Flow) and elution fraction (Elute)). FIG. 11 shows a laparotomy photograph of the transplanted nibe. Fluorescence field (fluorescence biological microscope, Olympus) and bright field photograph. FIG. 12 is a graph showing the engraftment rate of the transplanted cells to the Nibe host germ line. A vertical axis | shaft shows the ratio (%) of the individual in which the engraftment of the transplanted bluefin tuna undifferentiated germ cell was observed among all the observed panicle hosts.

Detailed description of the invention

According to one aspect of the present invention, there is provided a monoclonal antibody that specifically recognizes the surface of a mackerel fish undifferentiated germ cell, preferably a mackerel fish undifferentiated germ cell.

The term “mackerid fish” means a fish included in the periaceae mackerel family, for example, tuna fish, suma fish, bonito fish, mackerel fish, sawara fish, soda bonito, bonito genus, isola genus And preferred are tuna fish, suma fish, bonito fish and mackerel fish. As typical fish species of mackerelid fishes, for example, bluefin tuna (for example, bluefin bluefin tuna, bluefin tuna), southern bluefin tuna, bigeye tuna, yellowfin tuna, albacore tuna, Atlantic bluefin tuna, cochinaga. Examples of Suma fishes include Suma and Atlantic Yait. Examples of bonito fish include bonito. Mackerel and sesame mackerel are examples of mackerel fish. The fish species of the mackerel family of the present invention are preferably bluefin tuna, southern bluefin tuna, bigeye tuna, yellowfin tuna, albacore tuna, Atlantic bluefin tuna or cochinaga, which are sometimes collectively referred to as tuna. More preferably, the fish species of the mackerel family fish of the present invention is bluefin tuna, southern bluefin tuna, Atlantic bluefin tuna or suma in that resource protection by artificial seedling production is expected.

“Undifferentiated germ cells” are primordial germ cells, oocytes, spermatogonia, oocytes, spermatocytes, sperm cells, eggs and sperm, which are classified as germ cells, are hardly differentiated. It means a germ cell, and examples thereof include primordial germ cells, spermatogonia, and oocytes. Whether a germ cell is an undifferentiated germ cell can be determined by its morphological characteristics. For example, histological observation of type A spermatogonia is a germ cell that is surrounded by somatic cells in testis cells and is present alone, and should be identified by histological observation. Is possible. In addition, the oocyte is a cell population in which the first meiosis has not started among the small germ cells before the yolk formation phase, and therefore expresses meiosis marker genes such as Sycp3. Absence can also be an indicator for identifying oocyte cells. The undifferentiated germ cell of the present invention is preferably an undifferentiated germ cell having the engraftment ability to the host gonad, which has the engraftment ability when transplanted to the host fish gonad in the surrogate parent fish technique. Examples of the undifferentiated germ cells having engraftment ability to the host gonad include primordial germ cells, type A spermatogonia, or oocytes. The type A spermatogonia are undifferentiated germ cells that exist together with somatic cells in the undifferentiated testis. The type A spermatogonia are B-type spermatogonia, spermatocytes in the testis that has started to mature or mature testis. Cells that differentiate into cells, sperm cells, and sperm. An oocyte is an undifferentiated germ cell that exists together with a somatic cell in an undifferentiated ovary, and is a cell that differentiates into an oocyte or an egg as it matures. Whether or not a germ cell is an undifferentiated germ cell capable of engraftment in the host gonad can be determined by the presence or absence of engraftment when transplanted into the host fish gonad in the surrogate parent fish technique. Specifically, undifferentiated reproductive cells that have a high expression of the vasa gene and that have been engrafted in the gonad of the host fish in the surrogate parent fish technique have the ability to engraft in the gonad of the host. For example, Non-Patent Document 1 can be referred to.

“Specific recognition of maize fish undifferentiated germ cells” means that it specifically binds to undifferentiated germ cells of mackerel fish and is specific to differentiated germ cells and somatic cells of mackerel fish. Is preferably bound specifically to cell surface antigens of undifferentiated germ cells of mackerel fish. “Specifically recognizing mackerel fish undifferentiated germ cells” preferably refers to specifically recognizing the surface of maize fish undifferentiated germ cells, more preferably the germ cell membrane surface. In the present invention, “specific binding” includes binding preferentially.

“Monoclonal antibody” means an antibody (immunoglobulin molecule) obtained from a clone derived from a single antibody-producing cell, and is not particularly limited as an immunoglobulin class. For example, IgG, IgM, IgA, IgD and IgE can be mentioned, and IgG is preferable.

The monoclonal antibody of the present invention produces an antibody-producing hybridoma using an undifferentiated germ cell of a mackerel fish as an antigen, and detects that the antibody produced by the hybridoma is an antibody that recognizes an undifferentiated germ cell. You can get it.

The antigen for producing the monoclonal antibody of the present invention is preferably undifferentiated isolated from the testis or ovary of a mackerel fish in that it is easy to secure the number of undifferentiated germ cells used for transplantation of surrogate parent fish technique. It is a germ cell. In a preferred embodiment of the present invention, an undifferentiated germ cell separated from the testis or ovary is used as a cell population in which only undifferentiated germ cells are separated and concentrated from a cell suspension containing somatic cells derived from the testis or ovary. Specifically, when FS and SS are displayed as linear values for a fraction of a cell population containing many undifferentiated germ cells obtained by flow cytometry (FCM) analysis, A fraction having a large FS value and a small SS value can be used. More specifically, as shown in Example 1 of the Example described later, when an FCM analysis is performed with an Epics Altra (Beckman Coulter) equipped with a 488 nm argon laser, testis cells in a single state can be obtained by cell dispersion. When the FS-SS plot is developed under all detectable conditions, the fraction with a relatively large FS and a small SS, preferably about 1/3 the higher the FS value, the lower the SS value. A cell population that appears in about a third of the fraction can be used. Such a cell population containing a large number of undifferentiated germ cells can be prepared as an A gate in the method of Example 1 of the Examples described later. Thus, according to one aspect of the present invention, there is provided an antigen for preparing an antibody that specifically recognizes the undifferentiated germ cell of the present invention, and the antigen is preferably flow cytometry ( FCM) Fraction of cell population containing many undifferentiated germ cells obtained by analysis, when FS and SS are displayed as linear values, the fraction with relatively large FS value and small SS value It is.

According to a preferred embodiment of the present invention, the monoclonal antibody of the present invention is prepared using an undifferentiated germ cell isolated from a testicular or ovary of a mackerel fish as an antigen to produce an antibody-producing hybridoma, and for the antibody produced by the hybridoma, It can be obtained by detecting that the antibody recognizes undifferentiated germ cells. An antibody-producing hybridoma can be prepared according to a conventional method. For example, an antigen is administered to an animal (eg, mouse, rat, rabbit, etc.), immunized, and lymph node-derived cells obtained from the animal are used. It can be prepared by fusing with myeloma cells. Detection of an antibody recognizing an undifferentiated germ cell is performed by, for example, Cell ELISA method, immune cell staining, in situ hybridization described in Example 2 of Example described later, RT-PCR method for vasa gene, and the like. It can be carried out. Detection of an antibody recognizing an undifferentiated germ cell having an engraftment ability to the host gonad confirms the engraftment ability to the host gonad when transplanted to the host gonad by the surrogate parent fish technique (Non-patent Document 1). Can be performed.

According to a preferred embodiment of the present invention, the monoclonal antibody specifically recognizing the undifferentiated germ cell of the mackerel fish of the present invention is an antibody-producing hybridoma TA deposited at the Patent Evaluation Center of the National Institute of Technology and Evaluation. -No. 6-28 (reception number: NITE ABP-02222) or TA-No. 15-1 (reception number: NITE ABP-02223).

TA-No. 6-28 is a hybridoma that produces an antibody that specifically recognizes spermatogonial spermatogonia, dated March 22, 2016 (original deposit date), and deposited with a patent microorganism of the National Institute of Technology and Evaluation Technology The reception number is NITE ABP-02222 (transferred from domestic deposit NITE P-02222) at the center (Kazusa Kamashitsu 2-chome, 5-8-8, Kisarazu, Chiba, Japan) (Indication of identification: TA-No. 6-28) Has been deposited. TA-No. 6-28 is a hybridoma prepared by cell fusion of myeloma cells and antibody-producing B lymphocytes. The hybridoma is circular and weakly adherent, with a final concentration of 1% Penicillin-Streptomycin, Liquid ( Gibco) and growth in Hybridoma-SFM medium (Gibco, 12300-067) containing 10% fetal bovine serum (FBS) by incubation at 37 ° C., 5% CO2. It has mouse antibody production ability (IgG antibody), and its production amount is about 1 to 10 μg / ml.

TA-No. 15-1 is a hybridoma that produces an antibody that specifically recognizes spermatogonial spermatogonial cells, and was deposited on March 22, 2016 (original deposit date) by the National Institute of Technology and Evaluation Patent Microorganisms. The reception number is NITE ABP-02223 (transferred from domestic deposit NITE P-02223) at the center (Kazusa Kamashitsu 2-chome No. 5-8, Kisarazu City, Chiba, Japan) (Indication of identification: TA-No. 15-1) Has been deposited. TA-No. 15-1 is a hybridoma prepared by fusing myeloma cells and antibody-producing B lymphocytes into cells, the form of which is circular and weakly adhesive, with a final concentration of 1% Penicillin-Streptomycin, Liquid ( Gibco) and growth in Hybridoma-SFM medium (Gibco, 12300-067) containing 10% fetal bovine serum (FBS) by incubation at 37 ° C., 5% CO2. It has mouse antibody production ability (IgG antibody), and its production amount is about 1 to 10 μg / ml.

The monoclonal antibody of the present invention can separate undifferentiated germ cells of mackerel fish from the testes or ovaries of mackerel fish. The method for separating undifferentiated germ cells of mackerel fish using the monoclonal antibody of the present invention is not particularly limited as long as undifferentiated germ cells can be isolated while alive, and a known method can be used, for example, Cell separation (isolation) by flow cytometry analysis using a fluorescently labeled antibody such as FITC or magnetic cell separation using an antibody with magnetic beads can be used. The magnetic cell separation (MACS) method is preferable because it does not require sophisticated equipment or technology. The magnetic cell separation method is advantageous in that it can be easily processed even with a large amount of cells because it is separated by magnetic beads, compared to cell separation in flow cytometry analysis. In addition, the MACS method using the monoclonal antibody of the present invention is advantageous in that it does not need to use a cytotoxic solution unlike the density gradient centrifugation method using Percoll, so that it does not adversely affect the cell properties. In addition, it is advantageous in that a part of the cell population can be concentrated even in a cell population having a specific gravity close.

The undifferentiated germ cells can be concentrated by collecting the undifferentiated germ cells separated (isolated) by the monoclonal antibody of the present invention. Therefore, according to one embodiment of the present invention, an undifferentiated reproduction derived from a mackerel fish is obtained from a testis or ovary of a mackerel fish comprising undifferentiated germ cells derived from a mackerel fish using the antibody of the present invention. A method is provided for enriching maize undifferentiated germ cells comprising separating the cells. Since the concentrated undifferentiated germ cells obtained by the concentration method of the present invention are living cells, the use of the undifferentiated germ cells used for transplantation of the surrogate parent fish technique can be expected to improve transplantation efficiency. That is, according to one aspect of the present invention, an undifferentiated reproduction derived from a mackerel fish is obtained from a testis or ovary of a mackerel fish comprising undifferentiated germ cells derived from a mackerel fish using the antibody of the present invention. A method is provided for producing undifferentiated germ cells of mackerel fish comprising separating the cells.

By using undifferentiated germ cells separated (isolated) by the monoclonal antibody of the present invention, the transplanted undifferentiated germ cells can be induced to differentiate into gametes efficiently in the surrogate parent fish technique. Here, inducing differentiation of transplanted undifferentiated germ cells into gametes means that undifferentiated germ cells transplanted into the abdominal cavity move into the gonad of the transplanted host and engraft, and in the gonad of the host It means that it matures and is induced to differentiate into gametes. Therefore, the success or failure of induction of differentiation of transplanted undifferentiated germ cells into gametes can be determined by the presence or absence of transplanted undifferentiated germ cells in the gonad of the host (Okutsu, T., Suzuki, K. , Takeuchi, Y., Takeuchi, T & Yoshizaki, G. (2006) Testicular germ cells can colonize sexually undifferentiated embryonic gonad and produce functional eggs in fish. Proceedings of the National Academy of S -2729). If engraftment of transplanted undifferentiated germ cells can be confirmed in the gonad of the host, it can be said that the transplanted undifferentiated germ cells can be induced to differentiate into gametes with high probability. Thus, according to one aspect of the present invention, a surrogate surrogate fish technique, i.e., transplanting undifferentiated germ cells of a mackerel fish into the peritoneal cavity of a host fish individual before and after hatching, In the method for inducing differentiation of undifferentiated germ cells into gametes, using the antibody of the present invention before transplantation, the testis or ovary of a mackerel fish comprising undifferentiated germ cells derived from a mackerel fish Inducing differentiation of undifferentiated germ cells into gametes, comprising isolating and concentrating undifferentiated germ cells and transplanting the separated and enriched undifferentiated germ cells into the abdominal cavity of a host fish individual before and after hatching A method is provided.

According to one embodiment of the present invention, in the surrogate progeny technique of mackerel fish, i.e., undifferentiated germ cells isolated from the testes or ovaries of mackerel fish are transplanted into the peritoneal cavity of the individual host fish before and after hatching. In the method for inducing differentiation of undifferentiated germ cells into gametes, using the antibody of the present invention before transplantation, the testis of a mackerel fish comprising undifferentiated germ cells derived from a mackerel fish Alternatively, undifferentiated germ cells comprising separating and concentrating undifferentiated germ cells of mackerel fish from the ovary, and transplanting the separated and concentrated undifferentiated germ cells into the abdominal cavity of a host fish individual before and after hatching A method for inducing differentiation into a gamete is provided.

According to one aspect of the present invention, the testis or ovary of a mackerel fish comprising undifferentiated germ cells derived from a mackerel fish using the antibody of the present invention before transplantation in the differentiation inducing method of the present invention. And then isolating and concentrating undifferentiated germ cells of mackerel fish that have the ability to engraft the host gonads, and transplanting the separated and enriched undifferentiated germ cells into the peritoneal cavity of the individual host fish before and after hatching. A method for inducing differentiation of undifferentiated germ cells into gametes can be provided.

In the differentiation-inducing method of the present invention, the mackerel fish that provides undifferentiated germ cells to be transplanted as donor fish may be different from or same as the host fish, and preferably different. That the donor fish and the host fish are the same species means that the species of the mackerel fish that provides undifferentiated germ cells and the species of the host fish are the same. By making good varieties into donor fish, good varieties can be produced in large quantities in a short time without selective breeding. The fact that the donor fish and the host fish are heterogeneous means that the species of mackerel fish that provides undifferentiated germ cells differs from the species of the host fish, for example, the mackerel family that provides undifferentiated germ cells. The case where the fish species is a fish species different from the genus of the host fish and the case where the fish species are other fish species included in the same genus are included. When they are heterogeneous, the combination of donor fish and host fish can be determined in consideration of the similarity of their habitats, the size of the gonads, and the like.

The mackerel fish as donor fish is preferably larger fish than the host fish. Examples of large fishes of mackerel fish include bluefin tuna (for example, bluefin tuna, Atlantic bluefin tuna) and southern bluefin tuna. In addition, since fish grows larger with age, large fish includes parent fish that have grown enormously. By separating germ cells of large fish, transplanting them into the gonads of small host fish, and inducing differentiation into gametes, it is possible to reduce the feed and breeding space that are a problem for parent fish of large fish .

According to a more preferred embodiment of the present invention, in the differentiation induction method of the present invention, the host fish is one selected from mackerel fish, and the donor fish provides the undifferentiated germ cells to be transplanted as a mackerel fish. Is one selected from tuna, ie, bluefin tuna, southern bluefin tuna, bigeye tuna, yellowfin tuna, albacore tuna, Atlantic bluefin tuna or cocinaga. According to a further preferred embodiment of the present invention, the host fish is a mackerel or a mare, and the mackerel fish used as a donor for the transplanted undifferentiated germ cells is a bluefin tuna (eg, Atlantic bluefin tuna and Atlantic bluefin tuna) or southern bluefin tuna. More preferably, the combination of the host fish and the mackerel fish that provides the undifferentiated germ cells to be transplanted is a species that is difficult to breed, and can produce seeds in a small aquarium that is closely related to the donor. In terms of the combination of species, mackerel and Atlantic bluefin tuna, mackerel and southern bluefin tuna, mackerel and Atlantic bluefin tuna, suma and Thai bluefin tuna, suma and southern bluefin tuna, suma and southern bluefin tuna, suma and Atlantic bluefin tuna.

According to one aspect of the present invention, there is provided a method for producing sperm or eggs of a mackerel fish comprising the differentiation induction method of the present invention. That is, according to one aspect of the present invention, a mackerel fish comprising transplanting undifferentiated germ cells isolated from the testis or ovary of a mackerel fish into the peritoneal cavity of a host fish individual before and after hatching. A method for producing a sperm or egg of an animal from a testis or ovary of a mackerel fish comprising undifferentiated germ cells derived from a mackerel fish prior to transplantation using the antibody of the present invention. Isolating and concentrating undifferentiated germ cells of sebaceous fish having an ability to adhere, transplanting the separated and concentrated undifferentiated germ cells into the peritoneal cavity of the host fish individual before and after hatching, and transplanting the terminally differentiated germ cells into gametes A method for producing a sperm or egg of a mackerel fish is provided that comprises inducing differentiation into a sperm or egg of a mackerel fish.

According to another aspect of the present invention, the production of a mackerel fish comprising transplanting undifferentiated germ cells isolated from a testis or ovary of a mackerel fish into the peritoneal cavity of a host fish individual before and after hatching. The method is a mackerel family that has the ability to engraft the host gonad from the testis or ovary of a mackerel fish comprising undifferentiated germ cells derived from a mackerel fish using the antibody of the present invention prior to transplantation. Obtained by isolating and concentrating undifferentiated fish cells, transplanting the separated and enriched undifferentiated germ cells into the peritoneal cavity of the host fish individual before and after hatching, and inducing differentiation of the transplanted undifferentiated germ cells into gametes A method of producing mackerel fish is provided that comprises mating sperm and eggs.

The method for producing sperm or egg of mackerel fish and the method for producing mackerel fish according to the present invention can be carried out according to the contents described in the present specification for the concentration method and differentiation induction method of the present invention.

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

Example 1: Acquisition of undifferentiated germ cell fraction of mackerel fish (1) Extraction of the testis The testis is extracted from a single bluefin tuna (2 to 3 years old body weight 30 to 40 kg, gonad weight of about 100 to 200 g) and chopped into small pieces It is. Collagenase H (Roche) at a final concentration of 2 mg / ml, Dispase II (joint spirit) / L-15 medium (Invitrogen) (5% fetal bovine serum (FBS)) at a final concentration of 1.65 mg / ml 10 ml was added and incubated for 2 hours at 20 ° C. with physical dispersion by pipetting. The enzyme reaction was stopped by adding 1 ml of L-15 medium (Invitrogen). The obtained cell suspension (about 10 million cells / ml) was roughly purified by GE Healthcare density gradient centrifugation and used for flow cytometry (FCM) analysis and cell separation.

The testicular maturity used was confirmed by HE staining and in situ hybridization analysis of paraffin-embedded tissue sections (4 μm) of bluefin tuna testis tissue fixed with 4% paraformaldehyde (PFA) / PBS fixative. .

In Situ Hybridization Analysis In Situ Hybridization Analysis (Nagasawa, K., Takeuchi, Y., Miwa, M., Higuchi, K., Morita, T., Mitsuboshi, T., & Yoshizaki, G. (2009) cDNA cloning and expression analysis of avasa-like gene in Pacific bluefin tuna Thunnus orientalis. Fisheries Science, 75 (1), 71-79.). Specifically, the tissue sections were deparaffinized by xylene / ethanol series and hydration. The obtained sections were post-fixed with 4% paraformaldehyde and subjected to inactivation of endogenous alkaline phosphatase (AP) with 1% hydrogen peroxide, proteolysis with protease K (Wako), and acetylation. After standing for 1 hour at 65 ° C. in hybridization buffer (50% formamide, 2 × SSC, 50 ng / ml yeast tRNA, 50 ng / ml heparin, 0.02% SDS, 10% dextran sulfate), the bluefin tuna vasa probe: Hybridization was performed overnight at 65 ° C. in a hybridization buffer to which digoxigenin (DIG) -labeled RNA probe was added to a concentration of 1 ng / μl. The bluefin tuna vasa probe amplifies a 1085 bp fragment by PCR using a primer specific for the bluefin tuna vasa gene (vasa F 5′-CAACCAGGGAGCTCATCAACCAGA-3 ′ / vasa R 5′-GCAACTCAGGTCAATGCTGTGTGG-3 ′) Using the DIG Labeling kit (Roche) as a template, the plasmid DNA contained in was synthesized according to the standard method of DIG-labeled probes.

After sufficient washing, the non-specifically bound probe was removed with RNase A (Invitrogen). Masking was performed with a blocking solution (2% Blockin reagent (Roche) / TBST), and an AP-labeled anti-DIG antibody solution (Roche) (diluted 1: 500 with a blocking solution) was added dropwise. It left still at room temperature for 1 hour, and visualized using NBT / BCIP chromogenic substrate. The slide glass was sealed with ethanol using ethanol series xylene. Microscopic observation was performed and images were digitally recorded.

The results of the tissue sections are shown in FIG. 1a (immature individuals) and FIG. 3a (mature individuals).

(2) Flow cytometry (FCM) analysis Flow cytometry analysis was performed using an Epis Altra (Beckman Coulter) equipped with a 488 nm argon laser. Previous studies have revealed that fish spermatogonia have larger and rounder morphology than other germ cells, and when flow cytometric analysis was performed using vasa-GFP transgenic fish, It has been revealed that the spermatogonia population is fractionated into a large population with a simple internal structure. For this reason, it is assumed that fractionation is also possible in bluefin tuna spermatogonia, and the crudely purified cell suspension obtained by the Percoll density mating method is irradiated with laser, and the obtained scattered light is analyzed. Signals of cell size (Forward light scatter: FS) and internal structure complexity (side light scatter: SS) were measured. Based on the obtained scatter diagram, the fraction in which each cell population is densely distributed was divided into A to G gates (fractions), and cell separation was performed on each gate. The cell population of each separated gate was analyzed by morphology and in situ hybridization.

In the analysis of morphology, each cell was irradiated with a laser, and the cells were classified into seven types of gates A to G based on the characteristics of each cell obtained by the obtained scattered light. In general, spermatogonia are larger and rounder than other testicular cells. Thus, a large fraction with a simple internal structure was estimated as a fraction with many spermatogonia (A gate in FIG. 1c).

In situ hybridization analysis was performed according to the in situ hybridization analysis described in (1) above, except that cells of each gate were smeared on a slide glass instead of paraffin-embedded tissue sections, and 4% paraformaldehyde (PFA) / PBS. Fixed cell smears were used.

Scatter plot results are shown in FIG. 1b (immature individuals) and FIG. 2a (mature individuals), and analysis results are shown in FIG. 1c, FIG. 2 (immature individuals) and FIG. 3 (mature individuals).

As shown in FIGS. 2 and 3, it was confirmed that the A gate contains many undifferentiated germ cells such as spermatogonia. The ratio of undifferentiated germ cells such as spermatogonia contained in A gate is 80.7 ± 1.5% (standard error) with respect to the total number of cells in the case of immature individuals, It was 77.2 ± 4.9% (standard error).

Example 2: Acquisition of undifferentiated germ cell identifying antibody (1) Preparation of monoclonal antibody-producing hybridoma A mouse monoclonal antibody was prepared using the cell population contained in the A-gate obtained in Example 1 as an antigen. Specifically, immunization was carried out by dividing the A-gate cell population (5 × 10 ⁶ cells / ml) into 4 mice (Balb / c strain) in 5 portions. For immunization, a cell population of A gate was diluted 2 times with Freund's complete adjuvant (Sigma) with PBS, and 100 μl of each mouse foot was immunized.

On the 8th day after the 5th immunization, the enlarged lymph nodes were aseptically removed from the above-mentioned mouse 5 feet, and the lymph node cells were collected. The recovered lymph node cells and myeloma cells (P3U1, Tokyo Marine University) were mixed in the presence of 50% polyethylene glycol (PEG) to perform cell fusion. The obtained mixture of fused cells (hybridoma) was diluted with a HAT medium containing 15% FBS and seeded on 12 96-well plates.

(2) Primary screening: Cell ELISA
When incubated for 14 days (37 ° C.), formation of 1152 colonies was observed. Therefore, in order to clarify whether or not these hybridomas produce antibodies that recognize undifferentiated germ cells, screening by Cell ELISA was performed. Specifically, crude purified spermatogonia obtained by Percoll density gradient centrifugation were seeded on a plate (10 ⁵ cells / well), and using this plate, the titer of the antibody produced by the hybridoma against the immunized antigen was used. Was measured. An HRP color development system was used for the titer measurement. A clone having a cell ELISA signal value of 0.100 or more was selected as a positive clone having a high titer (high affinity).

(3) Secondary screening: immune cell staining (fluorescence microscope observation)
In order to clarify whether or not the antibody produced from the 384 clone that gave a positive signal in the primary screening can recognize the cell surface antigen of undifferentiated germ cells, screening by immune cell staining was performed. Testis isolated from bluefin tuna (2-3 years old, 2 tails) was subjected to enzyme digestion (20 ° C., 2 hours) with collagenase IV (Sigma), and the resulting dispersed cells were left alive and derived from the 384 clone. Bluefin tuna testis cells were indirectly labeled using a secondary antibody alexa flour 488 anti-mouse igG (life technology) labeled with a green fluorescent substance. Thereafter, the clones, which were observed under a fluorescence microscope and the green fluorescence was clearly observed on the cell membrane, were selected as clones capable of recognizing cell surface antigens. The results of observation under a fluorescence microscope in the secondary screening are shown in FIG.

(4) Tertiary screening: FCM analysis It is clarified whether the antibody produced by the 50 loan obtained in the secondary screening can specifically recognize A-gate cells, that is, undifferentiated germ cell fractions. In order to achieve this, FCM analysis was performed on whole cells and antibody-positive cells. Specifically, a scatter diagram (FIG. 5a) was obtained by FCM analysis (FS-SS development) of all cells, and the A gate rate was calculated. The whole cells were reacted with a fluorescently labeled secondary antibody and subjected to FCM analysis to obtain a histogram of antibody positive cell population and antibody negative cell population (FIG. 5b). A scatter diagram (FIG. 5c) was obtained by FCM analysis (FS-SS development) of the obtained antibody-positive cell population, and the A gate rate was calculated. The ratio of A-gate in the antibody-positive cell population thus obtained (A-gate ratio in the antibody-positive fraction) and A-gate in all cells including antibody-positive and antibody-negative cells. From the ratio (A gate rate in all cells), the concentration rate of undifferentiated germ cells by the antibody was calculated using the following formula.

[Formula 1]
Germ cell concentration rate by antibody = A gate rate in antibody positive fraction / A gate rate in whole cells

An antibody with a concentration ratio of 1.0 or more was selected as a positive clone. The results are shown in Table 1.

(4) Quaternary screening: in situ hybridization In order to clarify whether the bluefin tuna spermatogonia can be separated by FCM analysis using 20 antibodies from the 20 clones obtained by the tertiary screening, in situ. Screening was performed by hybridization analysis. In accordance with the method of Example 1 (1), each antibody was bound as a primary antibody to a crude cell suspension prepared from bluefin tuna testis, and then an antibody labeled with a green fluorescent substance was bound as a secondary antibody. FCM analysis was used to isolate an antibody positive fraction using green fluorescence as an index. Next, the isolated cells were smeared on a slide glass to prepare a cell smear. These cell smears were subjected to in situ hybridization analysis using an RNA probe of vasa gene known as an undifferentiated germ cell marker according to the method of Example 2 (2), and the percentage of vasa positive cells was determined. Asked.

The results are shown in FIGS.

As shown in FIG. 7, the two obtained clones, TA-No. 6-28 hybridoma and TA-No. The cell populations separated by the antibody produced from the 15-1 hybridoma contain vasa positive cells at the rate of 85.2% and 78.9%, respectively, ie, by using these antibodies, the bluefin tuna undifferentiated It was confirmed that germ cells could be separated (isolated) and concentrated at a high concentration.

FIG. 8 and FIG. 9 show the results of immunohistochemical staining using these two types of clone-produced antibodies. Immunohistochemical staining was performed according to the method of Example 1 (1).

As shown in FIG. 8 and FIG. 9, it was confirmed that these two types of antibodies specifically recognized the spermatogonia population, that is, the undifferentiated germ cell population, in the tissue section. In addition, these two types of antibodies have been confirmed to bind to the antibody by reacting the antibody solution with living cells that have not been fixed and digested with cell membranes. Was confirmed to specifically recognize the cell membrane surface.

Example 3: Concentration of tuna undifferentiated germ cells by MACS (Magnetic Activated Cell Sorting) method Using MACS method, undifferentiated from tuna testis cells using the two antibodies obtained in Example 2 above Germ cell enrichment was performed. Specifically, it is as follows. First, tuna testis cells were dispersed using collagenase H (Roche) having a final concentration of 2 mg / ml and dispase II (joint spirit) / L-15 medium (Invitrogen) having a final concentration of 1.65 mg / ml. Adjusted to 10 ⁶ cells. Therein, as a primary antibody, it was suspended in one step antibody biotination kit (Miltenyi biotech) and biotinylated by reacting at room temperature for 24 hours. 6-28 hybridoma and TA-No. 100 μl of antibody produced from the 15-1 hybridoma was added and reacted at 4 ° C. for 30 minutes. After completion of the reaction, centrifugation (200 × g, 5 minutes) was performed, and the resulting cell precipitate was washed with L-15 medium. Further, the cell precipitate was resuspended in 240 μl of L-15 medium, 60 μl of anti-biotin microbeads (manufactured by Miltenyi biotech) was added as a secondary antibody, and the mixture was reacted at 4 ° C. for 15 minutes. After completion of the reaction, centrifugation (200 × g, 5 minutes) was performed, the resulting cell precipitate was washed with L-15 medium, and the cell precipitate was further added to 500 μl of auto-MACS buffer (Miltenyi biotech In the product). From the suspension, magnetic beads-bound cells were recovered using a magnetic separator: Mini MACS separator (Miltenyi biotech) and MS column (Miltenyi biotech) to obtain MACS-enriched undifferentiated germ cells.

The obtained MACS-enriched undifferentiated germ cells were subjected to flow cytometry analysis and vasa in situ hybridization analysis according to the method of Example 1. The results are shown in FIG.

As shown in FIG. 10, testis cells concentrated by the MACS method contained many vasa positive cells.

Example 4: Transplantation of bluefin tuna-enriched undifferentiated germ cells into nibs (host) The bluefin tuna MACS-enriched undifferentiated germ cells obtained in Example 3 were transplanted into the peritoneal cavity of nibes, and the engraftment ability of the transplanted cells to the nibe gonad It was confirmed. Specifically, it is as follows.

As a nibe host, 246 individuals of 2-week-old nibe (produced by Tokyo University of Marine Science and Tateyama Station) were used. The bluefin tuna MACS-enriched undifferentiated germ cells obtained in Example 3 were stained with PKH26 (manufactured by SIGMA). Stained bluefin tuna MACS-enriched undifferentiated germ cells are microinjected (Yazawa, R., Takeuchi, Y., Higuchi, K., Yatabe, T., Kabeya, N) so as to obtain about 7000-10000 cells / tail. & Yoshizaki, Goro (2010) Chub mackerel gonads support colonization, survival, and proliferation of intraperitoneally transplanted xenogenic germ cells. Biology of reproduction, 82, 896-904). After 20 days from transplantation, laparotomy was performed and fluorescence analysis was performed. As a control (unconcentrated), bluefin tuna testis cells that had not been concentrated were treated with collagenase H (Roche) at a final concentration of 2 mg / ml, dispase II (joint spirit) / L-15 medium (final concentration of 1.65 mg / ml). Testis-dispersed cells obtained by dispersion using Invitrogen) were used. The results are shown in FIG.

As shown in FIG. 11, the nibs transplanted with bluefin tuna MACS-enriched undifferentiated germ cells were confirmed to be positive in the gonad in the fluorescence field, that is, the transplanted bluefin tuna MACS-enriched undifferentiated germ cells were engrafted in the nibe gonads. It was confirmed that On the other hand, no positive reaction was confirmed in the control (unconcentrated). The ratio of each engrafted individual is shown in FIG. 12 as the engraftment rate. The survival rate was calculated based on the following formula.

[Formula 2]
Engraftment rate (%) = Number of individuals engrafted with transplanted cells / Number of individuals who have undergone transplantation and opened

As shown in FIG. 12, the engraftment rate of bluefin tuna MACS-enriched undifferentiated germ cells markedly increased compared to the engraftment rate of unenriched cells.

Claims (12)

1. A monoclonal antibody that specifically recognizes undifferentiated germ cells of mackerel fish,
   A monoclonal antibody, wherein the monoclonal antibody is an antibody against undifferentiated germ cells isolated from the testis or ovary of a mackerel fish.
2. A monoclonal antibody that specifically recognizes undifferentiated germ cells of mackerel fish,
   The monoclonal antibody is an antibody producing hybridoma TA-No. 6-28 (NITE ABP-02222) or TA-No. The monoclonal antibody according to claim 1, which is produced by 15-1 (NITE ABP-02223).
3. 3. The monoclonal antibody according to claim 1 or 2, wherein the undifferentiated germ cells are primordial germ cells, type A spermatogonia or oocyte cells.
4. The monoclonal antibody according to any one of claims 1 to 3, wherein the mackerel fish is one selected from a tuna fish, a genus fish, a bonito fish, and a mackerel fish.
5. The monoclonal antibody according to any one of claims 1 to 4, which specifically recognizes the surface of undifferentiated germ cells of mackerel fish.
6. An undifferentiated germ cell derived from a mackerel fish is obtained from the testis or ovary of a mackerel fish comprising the undifferentiated germ cell derived from a mackerel fish using the antibody according to any one of claims 1 to 5. A method for enriching undifferentiated germ cells of mackerel fish, comprising separating.
7. In a method for inducing differentiation of undifferentiated germ cells into gametes, comprising transplanting undifferentiated germ cells isolated from a testis or ovary of a mackerel fish into the peritoneal cavity of a host fish individual before and after hatching,
   Before transplantation, using the antibody according to any one of claims 1 to 5, from the testis or ovary of a mackerel fish comprising undifferentiated germ cells derived from a mackerel fish, the maize fish undifferentiated reproduction Isolate and concentrate cells,
   A method for inducing differentiation of undifferentiated germ cells into gametes, comprising transplanting the separated and concentrated undifferentiated germ cells into the abdominal cavity of a host fish individual before and after hatching.
8. The method for inducing differentiation of undifferentiated germ cells into gametes according to claim 7, wherein the mackerel fish that provides the undifferentiated germ cells to be transplanted is different from the host fish.
9. 9. The method for inducing differentiation of undifferentiated germ cells into gametes according to claim 7 or 8, wherein the mackerel fish that provides the undifferentiated germ cells to be transplanted is a larger fish than the host fish.
10. 10. The host fish is one species selected from mackerel fish, and the mackerel fish that provides undifferentiated germ cells to be transplanted is one species selected from tuna. The method for inducing differentiation of undifferentiated germ cells into gametes as described.
11. A method for producing a sperm or egg of a mackerel fish comprising transplanting undifferentiated germ cells isolated from a testis or ovary of a mackerel fish into the peritoneal cavity of a host fish individual before and after hatching,
    Before transplantation, using the antibody according to any one of claims 1 to 5, from the testis or ovary of a mackerel fish comprising undifferentiated germ cells derived from a mackerel fish, the maize fish undifferentiated reproduction Isolate and concentrate cells,
    Transplanting the separated and concentrated undifferentiated germ cells into the peritoneal cavity of a host fish individual before and after hatching,
    A method for producing a sperm or egg of a mackerel fish, comprising inducing differentiation of the transplanted differentiated germ cell into a gamete to obtain a sperm or egg of a mackerel fish.
12. A method for producing a mackerel fish comprising transplanting undifferentiated germ cells isolated from a testis or ovary of a mackerel fish into the abdominal cavity of a host fish individual before and after hatching,
    Before transplantation, using the antibody according to any one of claims 1 to 5, from the testis or ovary of a mackerel fish comprising undifferentiated germ cells derived from a mackerel fish, the maize fish undifferentiated reproduction Isolate and concentrate cells,
    Transplanting the separated and concentrated undifferentiated germ cells into the peritoneal cavity of a host fish individual before and after hatching,
    Inducing differentiation of transplanted undifferentiated germ cells into gametes,
    A method for producing a mackerel fish comprising crossing the obtained sperm and egg.

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