JP6083760B2 - Improving the survival rate of isolated germ cells to the gonads of host fish - Google Patents

Description

  The present invention uses a host fish, transplants a separated germ cell of a fish different from the host fish into the host fish, and induces differentiation into a germ cell line. The present invention relates to a method for improving engraftment efficiency (engraftment rate) and increasing transplantation efficiency in inducing differentiation into germline by transplanting isolated germ cells.

  First, the present inventors transplanted isolated cells or genetically modified isolated cells into a host individual in fish, and induced differentiation of these into germline, and the differentiation induction method. In the course of studying methods for breeding or breeding fish, it is possible to induce the differentiation of the isolated germ cells into germ cell lines by transplanting the isolated germ cells into individual fish before and after hatching the fish. That is, by transferring the isolated germ cells derived from fish to the fish individual before and after hatching of the host vertebrate, in particular, by transplanting to the back side of the peritoneal mesentery of the fish individual in the developmental stage before and after hatching, The inventors found that it is possible to induce differentiation of germ cells into germ line, and succeeded in establishing a method for inducing differentiation of isolated germ cells of fish into germ lines (Japanese Patent No. 4300287) .

  Therefore, by using the above-described method for inducing differentiation of isolated fish germ cells into germ line, the host fish is used, and the separated germ cells of the fish different from the host fish are transplanted into the host fish to differentiate into the germ cell line. When using the method of inducing and proliferating fish for surrogate parent fish farming or the like, it is important to improve the survival rate of transplanted germ cells to the host gonad.

  In vertebrates other than fish, a method of transplanting isolated cells into a host individual and inducing differentiation into a germ cell line is known, and a method of promoting the engraftment of isolated cells transplanted into the host individual Several methods are also disclosed. For example, Japanese Patent Publication No. 2000-500327 discloses that a sample containing sperm is brought into contact with a solution containing a polysaccharide containing arabinose, galactose, and / or hexuronic acid, and the possibility of fertilization of sperm during sperm recovery is described. A method for enhancing is disclosed. JP-A-8-27011 discloses a fetal growth abnormality and prevention of dropping by using a fetal colonization enhancing agent for pregnant animals, which contains Bifidobacterium having an IgA production promoting effect as an active ingredient. And a method for stabilizing fetal colonization is disclosed. Furthermore, JP-T-2009-517078 discloses a method of treating a cell population with a predetermined amount of nicotinamide in order to enhance cell engraftment in bone marrow transplantation (BMT) or the like. However, each of these methods is a method for promoting the survival of isolated cells in vertebrates other than fish as described above, and is applied to the method for inducing differentiation of isolated germ cells of fish into germline. In such a case, it cannot be applied to the improvement of the engraftment ability of germ cells after transplantation into the host gonad.

  In fish, there are several methods for improving the engraftment ability of isolated germ cells into the host gonad in relation to the method of transplanting isolated germ cells into a host individual and inducing differentiation into the germ line. Reporting and disclosure has been made. For example, regarding “fish germ cell characteristics and surrogate parent fish techniques using the same,” the appropriate combination of isolated germ cell donor and host fish includes the physical distance of the egg, in addition to the genetic distance between the donor and host, It has been reported that biochemical and physiological properties need to be similar between the two ("Chemistry and Biology" Vol. 48, No. 10, P680-687, 2010). In addition, regarding "application of marine xenotransplantation technology for spermatogonia to marine fish-optimization of host size and tracking of donor germ cells after transplantation", it is reported that younger hosts are preferable. (Abstracts of Annual Meeting of the Fisheries Science Society of Japan, Vol.2008, Spring, P196, 2008).

  When transplanting isolated germ cells in fish to a host fish, the method of improving the ability of germ cells to engraft the host gonads is to engraft the transplanted germ cells without removing or forming the primordial germ cells of the host. A method for improving the above is disclosed. For example, in zebrafish, a method of removing host primordial germ cells by injecting dnd (dead end) antisense morpholino oligonucleotides (“BIOLOGY OF REPRODUCTION” 78, 159-166,2008), and In a method for inducing differentiation of a separated primordial germ cell into a germ cell line by transplanting a separated primordial germ cell derived from a donor fish into an early embryo of a heterologous host fish, a triploid fish is used as a host, and before and after hatching of the fish A method for efficiently growing or breeding fish by specifically forming donor-derived eggs and / or sperm without forming host-derived eggs and / or sperm by intraperitoneal transplantation (patented) No. 4581083).

  In addition, a method for concentrating germ cells to be transplanted is disclosed as a method for improving the engraftment ability of germ cells to the host gonads when transplanting isolated germ cells to a host fish. For example, germ cells to be introduced (spermatogonia) are visualized with Green Fluorescent Protein (GFP) or EGFP (Enhanced Green Fluorescent Protein), and germ cells emitting fluorescence and other non-fluorescent cells A method of purifying somatic cells by separating them with a cell sorter (flow cytometer) (PNAS Vol. 103, No. 8, P2725-2729, 2006; Japanese Patent No. 4300287) is disclosed. Furthermore, in a method for inducing differentiation into a germ cell line by transplanting isolated fish germ cells into a host fish, as a method for increasing the transplant efficiency, fish-derived isolated germ cells used for transplantation and fish testis are trypsinized. After dissociation by treatment, the dissociated cells were cultured in a culture vessel for a short period of time until the germ cells loosely adhere to the culture vessel, and then transplanted by preparing by separating and collecting the cultured germ cells A method for improving the engraftment ability of germ cells to the gonads of host fish is disclosed (Japanese Patent Laid-Open No. 2011-200169).

  As described above, in the method of using a host fish, transplanting a separated germ cell of a fish different from the host fish to the host fish, inducing differentiation into a germ cell line, and proliferating the fish, etc., the transplanted germ cell Various methods have been disclosed as methods for improving the engraftment ability (engraftment rate) in the host gonad, but when the method is used for surrogate parent fish farming, etc., it is more efficient, Development of a method that can be easily performed at the surrogate parent fish farming site is desired.

  On the other hand, in the fish culture technology, several papers have been reported that examined "water temperature and gonad development" as a study of the effects of the aquaculture environment on cultured fish. For example, for "the development of the gonads of a rainbow trout one-year fish bred in an outdoor pond at 12 ° C in summer and 17 ° C in winter", the testes are spermatogonia, primary and secondary spermatocytes from May to August. However, sperm cells and germ cells in various stages of maturity accounted for, but testicular development was less affected by water temperature ("Aquaculture Research Bulletin" No.12, P9-16, 1987). Regarding the “effect of water temperature on maturity”, natural experimental seawater has a temperature of 20 ° C. or below, and two experimental zones of 20 ° C. and natural water temperature (14-11 ° C.) are maintained from mid-November to mid-January 52 As a result of examining the effect of the water temperature on the gonad maturation, the high water temperature of 20 ° C. suppressed the maturation of the egg cell, but the high water temperature of 20 ° C. had no effect on the sperm cell maturation. ("Fisheries breeding" Vol.37, No.4, P267-274, 1989) has been reported.

In addition, regarding “blue-tailed bluefin tuna breeding using a 70 m ³ aquarium”, for the breeding of the 2009 class group (60 fish), warm seawater of 23 ° C is added during the low water temperature period, and the average water temperature is 16.0 ° C. in, together with the fact that it is possible to greatly suppress the meantime of mortality, the number of germ cells is increased in the male and female gonads, clear that developed toward the gonadal sexual maturation of bluefin tuna with 70m ³ aquarium ("Fisheries breeding" Vol.59, No.3, P473-481, 2011) has been reported. These reports have been researched and reported on “water temperature and gonad development” in fish culture techniques, but in particular, they were not researched and reported on surrogate parent fish culture. It is not reported about the influence of the breeding temperature of the host and the engraftment ability (engraftment rate) of the transplanted germ cells to the host gonad when transplanting the cells into host fish and inducing differentiation into germline .

JP-A-8-27011. JP 2000-500327 Gazette. JP-T 2009-517078. JP 2011-200169A. Japanese Patent No. 4300287. Japanese Patent No. 4581083.

“Chemistry and Biology” Vol.48, No.10, P680-687, 2010. Abstracts of Annual Meeting of the Fisheries Science Society of Japan, Vol.2008, Spring, P196, 2008. “BIOLOGY OF REPRODUCTION” 78, 159-166,2008. PNAS Vol.103, No.8, P2725-2729, 2006. “Aquaculture Research Bulletin” No. 12, P9-16, 1987. "Fisheries breeding" Vol.37, No.4, P267-274, 1989. "Fisheries breeding" Vol.59, No.3, P473-481, 2011.

  An object of the present invention is to use a host fish, and in a surrogate parent fish farming or the like for transplanting a germ cell of a different lineage or heterogeneous fish from the host fish into the host fish to induce differentiation into a germline. To provide a method for improving the efficiency of engraftment of germ cells into the host gonad (hereinafter referred to as "engraftment rate") and increasing the efficiency of transplantation in the induction of germ line differentiation by transplantation of isolated germ cells. It is in.

  The present inventors use a host fish, and transplanted isolation in a surrogate parent fish farming or the like that induces differentiation into a germ line by transplanting a separated germ cell of a fish of a different or different species from the host fish to the host fish. In the intensive study of methods for improving the engraftment of germ cells into the host gonad, the temperature at which the individual host fish is raised affects the engraftment of the transplanted isolated germ cells into the host. As a result, it has been found that the engraftment rate of the transplanted isolated germ cells to the host gonad can be improved by adjusting the breeding temperature of these hosts, and the present invention has been completed.

  That is, the present invention relates to a method for inducing differentiation of a separated germ cell into a germ cell line, which comprises transplanting a fish-derived isolated germ cell into a host fish individual by intraperitoneal transplantation of the host fish before and after hatching. The isolated germ cells transplanted into the peritoneal cavity of the host fish at a temperature that is lower than the host breeding water temperature before transplantation and does not adversely affect the survival and development of the host fish individual It consists of a method for inducing the differentiation of isolated germ cells into the germ line by improving the engraftment ability of the isolated germ cells into the gonads of the host fish by breeding for the period required for migration and engraftment. In the present invention, examples of the isolated germ cells include fish primordial germ cells, spermatogonia, and oocyte cells from which the isolated germ cells are derived.

  Usually, a water temperature suitable for the breeding of the host fish is selected for the breeding of the host fish. However, in the present invention, the host fish is raised at the normal water temperature before transplanting the isolated germ cells into the host fish. And after transplantation, the isolated germ cells transplanted into the peritoneal cavity of the host fish are transferred to the host gonad at a temperature lower than the normal water temperature and not adversely affecting the survival and development of the individual host fish. The period required for engraftment, that is, the low water temperature affects the growth of isolated germ cells transplanted into the peritoneal cavity of the host fish, and the ability of the isolated germ cells to move to the host gonad is imparted. During the period when cells are transferred to the host gonad and engrafted in the host gonad, breeding at the low water temperature can improve the survival rate of isolated germ cells in the host fish gonad. .

  In the present invention, the temperature that is lower than the host breeding water temperature before transplantation and that does not adversely affect the survival and development of the individual host fish is 2 to 4 ° C. lower than the host breeding water temperature before transplantation. Preferably, the period required for the isolated germ cells transplanted into the abdominal cavity of the main fish to migrate and engraft the host gonads is preferably at least 3 weeks after transplantation. In the present invention, the mechanism by which the engraftment rate of the isolated germ cells to the gonad of the host fish is improved by rearing the host fish at a water temperature lower than the normal water temperature after transplanting the isolated germ cells to the host fish is described in detail. Although it is not clear, the effect of breeding the host fish at low water temperature on the growth of the host gonads (growing the host fish at a low water temperature slows the growth of the host gonads), or isolated germ cells Effect on the gonad's ability to move to the host gonad (the ability to raise isolated germ cells in the abdominal cavity by raising the host fish at a low water temperature) improves the survival rate of the isolated germ cells to the host gonad Presumed to be.

  In the present invention, an individual host fish that has undergone transplantation is bred at a temperature lower than the pre-transplant host breeding water temperature. However, in the breeding of host fish at the low water temperature, the low water temperature is transplanted into the peritoneal cavity of the host fish. Affects the growth of the isolated germ cells, activates the ability of the isolated germ cells to move to the host gonad, and imparts the ability of the isolated germ cells to move to the host gonad. The animals are reared for a period of time during which migration takes place and engraftment on the host gonads takes place. For example, when nibs are used as the host fish, the isolated germ cells are transplanted into nibs that have been bred at a breeding water temperature of 24 ° C. before transplanting the isolated germ cells, and then reared at a water temperature of 21 to 20 ° C. for 3 weeks or more. The When Nibe is used as the host fish, the isolated germ cells derived from the fish can be Nibe, Onibe, or Tuna-derived isolated germ cells. In the case of using a horse mackerel as a host fish, the isolated germ cell is transplanted to a horse mackerel bred at a breeding water temperature of 22 ° C. before transplanting the isolated germ cells, and then reared at a water temperature of 20 ° C. for 3 weeks or more. When a maji is used as a host fish, a isolated germ cell derived from Japanese kingfish, yellowtail, or amberjack can be used as the isolated germ cell derived from fish.

  Specifically, the present invention relates to [1] a germ cell line of isolated germ cells comprising transplanting a fish-derived isolated germ cell into a host fish individual by transplanting the host fish into the abdominal cavity of the host fish before and after hatching. In the method of inducing differentiation, transplanted host fish individuals are transplanted into the peritoneal cavity of the host fish at a temperature lower than the host breeding water temperature before transplantation and at a temperature that does not adversely affect the survival and development of the host fish individuals. To the germline of an isolated germ cell that has improved the engraftment ability of the isolated germ cell to the host fish gonad, which is maintained for a period of time necessary for the isolated germ cell to move to the host gonad and engraft [2] The temperature that is lower than the host breeding water temperature before transplantation and that does not adversely affect the survival and development of the individual host fish is 2 to 4 ° C. lower than the host breeding water temperature before transplantation. It is characterized by [1] The method for inducing differentiation of a separated germ cell into a germ cell line which has improved the engraftment ability of the isolated germ cell into the gonad of the host fish according to [1], or [3] a separated germ cell transplanted into the abdominal cavity of the host fish The period of time required for transfer to the host gonad and engraftment is at least 3 weeks after transplantation, and the survival of the isolated germ cell according to [1] or [2] above to the host fish gonad It consists of a method for inducing differentiation of isolated germ cells with improved potency into germ line.

  In addition, the present invention uses [4] nibs as a host, transplants the isolated germ cells to the nibs that have been bred at a breeding water temperature of 24 ° C. before transplantation, and then breeds them at a temperature of 21 to 20 ° C. for 3 weeks or more. [1] The method for inducing differentiation of a separated germ cell into a germ cell line which has improved the engraftment ability of the isolated germ cell to the host fish gonad, [5] The method for inducing differentiation of an isolated germ cell into a germ line, which has improved engraftment ability of the isolated germ cell in the gonad of a host fish according to the above [4], [6] The horse mackerel is used as a host, and after transplanting the isolated germ cells to the horse mackerel that has been bred at a breeding water temperature of 22 ° C. before transplantation, it is bred at a temperature of 20 ° C. for 3 weeks or more. ] Of the isolated germ cell host fish gonad [6] The method for inducing differentiation of isolated germ cells having improved engraftment ability into germ line, and [7] The isolated germ cells derived from fish are kingfish, yellowtail, or amberjack. The method for inducing differentiation of a separated germ cell into a germ line improved in the engraftment ability of the isolated germ cell into the gonad of a host fish.

  The present invention provides a method for increasing the efficiency of transplantation of isolated germ cells by improving the engraftment rate of the transplanted germ cells into the host gonad in a method of inducing differentiation into germ line by transplanting isolated germ cells. . The method for inducing differentiation into a germ line of the present invention is effective for, for example, surrogate parent fish culture in which a separated germ cell of a fish of a different or different species from the host fish is transplanted into the host fish to induce differentiation into the germ line. It can be effectively applied to surrogate parent fish culture in which large-sized fish species that are difficult to cultivate parent fish are produced into easily cultivated fish species. For example, using host fish selected from salmonids, crocodiles, and mackereles as host fish, and using different types of fish, such as tuna, kingfish, yellowtail and amberjack, to produce juvenile fish efficiently And can be used for aquaculture. Since the improvement of the engraftment rate in the host gonads in the induction of germ line differentiation by transplanting the isolated germ cells of the present invention can be performed by adjusting the water temperature in the breeding of the host fish, this method is used for surrogate parent fish farming, etc. When used for the above, it can be simply performed at the farming site, and can be provided as a simple and efficient surrogate parent fish culture technique.

In the test of the method for inducing differentiation into a germ line by transplanting isolated germ cells in the examples of the present invention, nibe is used as the host fish, and the breeding temperature of the host fish is 24 ° C. before transplanting and 21 ° C. after transplanting. It is a graph which shows the survival number (%) of the isolation | separation germ cell to the host gonad after rearing at water temperature for 3 weeks. FIG. 1-a shows a case where nibe is used as a donor of a separated germ cell (germ cell-derived fish) and nibe is used as a host fish. FIG. 1-b shows a rice plant as a donor of a separated germ cell (germ cell-derived fish). Fig. 1-c shows tuna as a separated germ cell donor (germ cell-derived fish), and a separated germ cell into the host gonad when nibe is used as a host fish. The number of surviving fish (%).

  The present invention relates to a method for inducing differentiation of a separated germ cell into a germ line by transplanting a fish-derived isolated germ cell into a host fish individual by transplanting the host fish before and after hatching into the abdominal cavity. The isolated germ cells transplanted into the peritoneal cavity of the host fish at a temperature lower than the host breeding water temperature prior to transplantation and not adversely affecting the survival and development of the host fish It consists of a method for inducing differentiation of a separated germ cell into a germ cell line, which has been reared for a period required for migration and engraftment, thereby improving the ability of the isolated germ cell to engraft the host fish gonad.

  The present invention relates to a method for inducing differentiation of a separated germ cell into a germ line by transplanting a fish-derived isolated germ cell into a host fish individual by transplanting the host fish before and after hatching into the abdominal cavity. The host fish individual is reared at a temperature lower than the host breeding water temperature before transplantation, and the isolated germ cells derived from the fish are transplanted into the host fish individual by transplantation into the abdominal cavity of the host fish before and after hatching. The method itself can be performed using the method specifically disclosed in the specification of the earlier patent application of the present inventor (Japanese Patent No. 4300287).

  In the present invention, the breeding of the host fish is carried out at a water temperature usually employed in the host fish before transplanting the isolated germ cells into the host fish, and after the transplantation, for example, 3 to 4 ° C. lower than the normal water temperature. Raised at water temperature. That is, the breeding of the host fish is usually performed by selecting a water temperature suitable for the breeding of the host fish, but in the present invention, the normal fish is transplanted before the transplantation of the isolated germ cells into the host fish. And is maintained at a temperature that is lower than the normal water temperature after transplantation and that does not adversely affect the survival and development of the individual host fish. The host fish is kept at a low water temperature for a period of time required for the isolated germ cells transplanted into the abdominal cavity of the host fish to move to the host gonad and engraft, and the period is about 3 weeks. This period is required, and the effect is exhibited by breeding at a low temperature within the period.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

  [Separation of test cell germ cells (shrimp, giant crab, bluefin tuna) and transplantation to host fish (shrimp) gonads]

<Preparation of Leibovitz L-15 / FBS culture solution>
Leibovitz's L-15 medium powder (Invitrogen Corporation, Carlsbad, Calif.) 1. 374 g, HEPES (Sigma-Aldrich, St. Louis, MO) 0.598 g in 80 ml of Deionized distilled water (Invitrogen Corporation, 15230-162) Dissolved. Subsequently, the pH was adjusted to 7.8 using 10 N NaOH and 1 N NaOH, and the volume was adjusted to 89.7 ml. Then, using a sterile filter for syringes (Dismic-25cs, Advantec, Tokyo) having a pore size of 0.2 μm. Filter sterilized. Further, 2.5 ml of salmon serum, 10 ml of fetal bovine serum (FBS) was added, and finally antibiotics were added to give concentrations of 50 U / ml penicillin, 50 μg / ml streptomycin, and 50 μg / ml ampicillin.

<Isolation of testis and exfoliation of connective tissue>
After anesthesia of the test fish (nibe, giant crab) in ice-cold seawater or after catching the test fish (tuna), dissect with surgical scissors and tweezers and surgically test the testis Extracted. The isolated testis was temporarily stored in a 48-well plate containing 200 μl of L-15 / FBS 10% (pH 7.8) culture medium. Then, the isolated testis was transferred into a sterile petri dish filled with cooled PBS (-), and the blood vessels attached to the testis and the testicle were removed with tweezers under a stereomicroscope (SZX-10: Olympus, Tokyo). And peeled off.

<Testis dispersion>
150 mg (1-20 tails) of testis from which blood vessels and connective tissue have been exfoliated are put together on a one-hole blood reaction plate, and testicular debris is collected using Weckels scissors (MB-41, NAPOX, Natsume Seisakusho Co., Ltd.). It was in a state. Subsequently, testis pieces were transferred into 1 ml of 0.855 Unit trypsin / PBS (+) solution and incubated at 25 ° C. for about 2 hours. During the incubation, pipetting was performed every 30 minutes in order to promote the dispersion of testicular debris. After the enzyme treatment, the whole cell suspension was transferred to a 15 ml tube and centrifuged at 4 ° C. and 200 × g for 5 minutes using a swing rotor to precipitate testis cells to form a pellet. Subsequently, the supernatant was discarded while taking care not to suck the pellet, and 3 ml of L-15 / FBS 10% (pH 7.8) culture solution was added. In addition, in order to remove an enzyme solution completely, the rinse by the same centrifugation operation was performed twice. After removal of the enzyme solution, 2 ml of L-15 / FBS 10% (pH 7.8) culture solution was added and stirred by pipetting, and the resulting testis cell suspension was subjected to a nylon mesh (NBC Inc, Tokyo, 42 μm). ), The testicular piece produced by incomplete dispersion was removed.

<Preparation of germ cells for transplantation (fluorescent labeling)>
After dissociating the testis of the test fish (nibe, sea bream, bluefin tuna) by trypsin treatment, the obtained cells were labeled with a red fluorescent dye (PKH26, Sigma) according to a conventional method, and then resuspended in the L-15 / FBS medium. Stored on ice in a turbid state.

<Transplantation of isolated germ cells into the host gonad>
The testis of the test fish was dissociated by trypsin treatment, and the collected germ cells were transplanted into the peritoneal cavity of a 10-12 day old (total length: about 4 mm) nibe host. The germ cell transplantation method and the engraftment confirmation method itself can be applied to the spermatogonia transplant method in the marine larvae previously developed by the present inventors (Takeuchi Y, Higuchi K, Yatabe T, Miwa M, Yoshizaki G. Development of spermatogonial cell transplantation in Nibe croaker, Nibea mitsukurii (Perciformes, Sciaenidae). Biology of Reproduction, 81, 1055-1063 (2009) .; Chub mackerel gonads support colonization, survival, and proliferation of intraperitoneally transplanted xenogenic germ cells. Yazawa R, Takeuchi Y, Higuchi K, Yatabe T, Kabeya N, Yoshizaki G. Biology of Reproduction, 82, 896-904 (2010) .; Higuchi K, Takeuchi Y, Miwa M, Yamamoto Y, Tsunemoto K , Yoshizaki G. Colonization, proliferation and survival of intraperitoneally transplanted yellowtail Seriola quinqueradiata spermatogonia in nibe croaker Nibea mitsukurii recipient. Fisheries Science, 77, 69-77 (2011)). Three weeks after transplantation, it was confirmed that spermatogonia derived from the test fish were engrafted in the gonads of the Nibe host. Cells that fluoresce indicate germ cells. The germ cells of the test fish engrafted in the gonad of the nibe are confirmed.

<Management of breeding temperature (water temperature) of host fish (Nibe)>
In this example, the nibs used as host fish were 24 ° C., which is normally used as the breeding temperature (water temperature) for nibs before germ cell transplantation, and 21 ° C., 3 ° C. lower than the temperature after transplantation. Coordinated and managed. In the case of [Donor: Nibe-Host: Nibe], 3 lots in the case of [Donor: Nibe-Host: Nibe], 3 lots in the case of [Donor: Onibe-Host: Nibe], and [Donor: Tuna-Host: Nibe] One lot was tested.

<Result>
The results are shown in Table 1. Moreover, the test lot (Lot) average value of the number of engraftment (%) to the host gonads in the host breeding temperature 24 ° C. group and the 21 ° C. group after transplantation is shown in FIG. 1 for each germ cell donor. In the figure, FIG. 1-a shows a case where nibe is used as a donor of isolated germ cells and nibe is used as a host fish (24 ° C. group average = 17.8, 21 ° C. group average = 49.1). FIG. 1-c shows the isolated germ cells when Onibe is used as a donor of isolated germ cells and Nibe is used as a host fish (24 ° C. group average = 23.6, 21 ° C. group average = 57.0). Shows the number (%) of engrafted germ cells in the host gonads when tuna is used as the donor and nibe is used as the host fish (24 ° C. group = 16, 21 ° C. group = 50). For any germ cell donor, improvement in the engraftment rate to the host gonads was shown in the host breeding temperature group at 21 ° C. (low temperature breeding temperature) after transplantation.

  [Isolation of test-germ germ cells (Populus, yellowtail, amberjack) and transplantation to the gonad of host fish (Maji)]

<Transplantation of test fish germ cells to the mackerel host gonad>
In the same manner as in Example 1, germ cells isolated from Japanese flounder, yellowtail, or amberjack were transplanted into the gonad of host horse mackerel.

<Management of breeding temperature (water temperature) of host fish (Maji)>
In this example, the horse mackerel used as the host fish is 22 ° C., which is normally used as the breeding temperature (water temperature) of the horse mackerel (spawning temperature of the horse mackerel) before the germ cell transplantation. 2 ° C lower, adjusted to 20 ° C and controlled. In the case of [donor: hiramasa-host: maji], 1 lot for [donor: hiramasa-host: maji], 2 lots for [donor: campathi-host: maji], and [donor: yellow-host: maji] 5 lots were tested.

<Result>
The results are shown in Table 2. Table 2 shows the number of engrafted tails (%) in the host gonads at 20 ° C. after the transplantation for each germ cell donor. In the case of using Japanese horse mackerel as a donor of isolated germ cells, and when horse mackerel is used as a host fish, an engraftment rate of 50% is obtained. When a horse mackerel or yellowtail is used as a donor of isolated germ cells, horse mackerel is used as a host fish. Obtained a survival rate of 75-100%. For any germ cell donor, an improvement in the engraftment rate on the host gonads was shown in the host breeding temperature group at 20 ° C. (low temperature breeding temperature) after transplantation.

  The present invention provides a method for increasing the efficiency of transplantation of isolated germ cells by improving the engraftment rate of the transplanted germ cells into the host gonad in a method of inducing differentiation into germ line by transplanting isolated germ cells. . The method for inducing differentiation into a germ line of the present invention is effective for, for example, surrogate parent fish culture in which a separated germ cell of a fish of a different or different species from the host fish is transplanted into the host fish to induce differentiation into the germ line. It can be effectively applied to surrogate parent fish culture in which large-sized fish species that are difficult to cultivate parent fish are produced into easily cultivated fish species. The improvement of the engraftment rate to the host gonads in the induction of germ line differentiation by transplanting the isolated germ cells of the present invention can be achieved by adjusting the water temperature in the breeding of the host fish. It provides a simple and efficient surrogate fish farming technology that can be performed in a simple manner.

Claims (5)

  In a method for inducing differentiation of a separated germ cell into a germ cell line by transplanting a fish-derived isolated germ cell into a host fish individual by intraperitoneal transplantation of the host fish before and after hatching, the host fish is treated with the host fish. After breeding at a water temperature suitable for breeding, the isolated germ cells are transplanted into the host fish, and the individual host fish is transplanted into the peritoneal cavity of the host fish at a temperature 2 to 4 ° C. lower than the host breeding water temperature before the transplantation. Improving the engraftment ability of isolated germ cells to the host fish gonads, which is maintained for at least 3 weeks after transplantation, which is the period required for the isolated germ cells transplanted into the host gonad to migrate and engraft For inducing differentiation of isolated germ cells into germ line.   2. The method according to claim 1, wherein a nib is used as a host, and the isolated germ cells are transplanted to a nib that has been bred at a breeding water temperature of 24 ° C. before transplantation, and then reared at a temperature of 21 to 20 ° C. for 3 weeks or more. A method for inducing differentiation of a separated germ cell into a germ line, which has improved the engraftment ability of the isolated germ cell into a host fish gonad.   The isolated reproductive cell having improved engraftment ability of the isolated reproductive cell to the gonad of the host fish according to claim 4, wherein the isolated reproductive cell derived from fish is a isolated reproductive cell derived from a nib, a giant crab or a tuna. A method for inducing differentiation of cells into germline.   2. The isolation according to claim 1, characterized in that a horse mackerel is used as a host, and the isolated germ cells are transplanted to a horse mackerel bred at a breeding water temperature of 22 ° C. before transplantation and then reared at a temperature of 20 ° C. for 3 weeks or more. A method for inducing differentiation of a separated germ cell into a germ line, which has improved the engraftment ability of the germ cell into the host fish gonad.   The isolated reproductive cell having improved engraftment ability of the isolated reproductive cell to the host fish gonad according to claim 6, wherein the isolated reproductive cell derived from fish is a isolated reproductive cell derived from Japanese kingfish, yellowtail or amberjack. A method for inducing differentiation of cells into germline.