In-vitro culture, passage and purification method of carp oogonial stem cells

Abstract

The invention provides an in-vitro culture, passage and purification method of carp oogonial stem cells, and belongs to the technical field of cell in-vitro culture. The invention provides an in-vitro culture method of carp oogonial stem cells, which comprises the following steps: selecting female fish, separating ovarian tissues, separating primary oogonial stem cells from the ovarian tissues, carrying out adherent culture, and carrying out subculture and purification on the oogonial stem cells subjected to adherent culture. The invention also provides cryopreservation and resuscitation of the carp oogonial stem cells obtained by passage and purification. According to the method, the carp oogonial stem cells are cultured by utilizing a tissue adherence method, and long-strip-shaped, triangular and irregular polygonal cells can be observed to climb out of small tissue blocks and extend to the periphery in the third day; after cell passage, the growth state of the carp oogonial stem cells is good. The carp oogonial stem cells subjected to in-vitro culture, passage, purification, cryopreservation and recovery have typical germline stem cell characteristics and pluripotency.

Description

Technical Field

[0001] The invention belongs to the technical field of in vitro cell culture, and in particular relates to an in vitro culture, passage and purification method of carp oogonial stem cells. Background Art

[0002] Carp is one of the main freshwater aquaculture species in my country. In order to improve the growth, quality, disease resistance and other traits of carp, fish breeders have adopted group selection, family selection, hybrid breeding, molecular marker-assisted selection and other technologies to cultivate 32 new carp varieties approved by the state. However, since the age of carp's first sexual maturity is generally 2 to 3 years, it takes at least 8 to 12 years to breed a new variety, resulting in a long breeding cycle for new carp varieties. Therefore, how to overcome the long generation interval that restricts the breeding process of new carp varieties is a major technical problem in carp genetic breeding. Fish reproductive stem cell transplantation provides an optional technical approach to solve the above problems. As a special cell type with the ability to self-renew and the potential to produce gametes (sperm and eggs), fish reproductive stem cells play an indispensable role in biological reproduction and the maintenance of genetic diversity. In recent years, with the rapid development of molecular biology technology and cell in vitro culture technology, the in vitro operation of reproductive stem cells has broad application prospects, especially in the protection of endangered fish germplasm resources, the creation of new germplasm, and the breeding of new varieties. Its technical application potential has attracted much attention. By combining germ stem cell culture technology, cell transplantation technology and gene editing technology, the genetic characteristics of target traits of aquatic animals can be quickly and directed at the cellular level, such as enhancing disease resistance, increasing growth rate, etc., greatly shortening the breeding cycle and improving breeding efficiency.

[0003] Oogonial stem cells are a type of reproductive stem cells that exist in the ovary and have the ability to self-renew and differentiate. In the research on the culture and transplantation of fish reproductive stem cells, although the oogonial stem cells of many fish (such as zebrafish, eel, catfish and other fish) have been successfully isolated and cultured in vitro, for most aquatic animals, due to their different living habits and biological characteristics, specific oogonial stem cell isolation and culture techniques still need to be further developed and optimized. However, carp currently does not have a technical system for long-term in vitro culture and preservation of oogonial stem cells, which restricts the use of reproductive stem cell transplantation technology to shorten the breeding cycle of excellent varieties and improve carp breeding efficiency. Summary of the invention

[0004] The invention provides an in vitro culture, passage and purification method for carp oogonial stem cells, establishes an in vitro long-term culture system for carp oogonial stem cells, and the established carp oogonial stem cell system has high purity and good pluripotency.

[0005] The invention provides an in vitro culture method of carp oogonial stem cells, comprising the following steps: soaking ovaries taken from female carp in L-15 basic culture medium and then placing in carp serum, cutting the ovaries into pieces and evenly spreading them in a T25 culture bottle, culturing at 26-30 DEG C in a CO2-free environment, adding L-15 complete culture medium for further culturing after culturing for 4-6 hours, and obtaining primary carp oogonial stem cells in adherent culture;

[0006] The L-15 basal medium comprises the following components: L-15 powder, 5 mol / L HEPES buffer, 50 μmol / L β-mercaptoethanol, and sterilized water;

[0007] The L-15 complete culture medium uses the L-15 basic culture medium as the basic culture medium and also includes the following components: 10-20% by volume of fetal bovine serum, 2-20 ng / mL of basic fibroblast growth factor, 2-20 ng / mL of leukemia inhibitory factor, 0.5-2% by volume of carp serum, 0.5-2% by volume of zebrafish embryo extract, 0.5-2% by volume of L-glutamine, 600-1000 IU / mL of penicillin, 0.6-1 mg / mL of streptomycin and 1-3 μg / mL of amphotericin B.

[0008] The sex of the female carp is identified, and the primer pair used for identification includes CYC-male marker-F with nucleotide sequences as shown in SEQ ID No.1 and CYC-male marker-R with nucleotide sequences as shown in SEQ ID No.2.

[0009] The present invention also provides an adherent primary carp oogonial stem cell line cultured by the in vitro culture method.

[0010] The present invention also provides a method for subculturing the above-mentioned adherent primary carp oogonial stem cell line, comprising the following steps:

[0011] (1) After the confluence of the adherent primary carp oogonial stem cell line reaches more than 80%, the L-15 complete medium is removed and the cells are washed with PBS;

[0012] (2) After washing with PBS, digesting with a mixture of PBS and 0.25% trypsin-EDTA for 4 to 6 minutes, terminating the digestion with L-15 complete medium, aspirating the liquid and centrifuging at 1000 to 1500 rpm for 4 to 6 minutes, removing the supernatant, and adding L-15 complete medium to prepare a cell suspension; (3) allocating the cell suspension collected in step (2) to a new T25 culture flask at a ratio of 1:(2 to 4), adding L-15 complete medium, and culturing and subculturing at 26 to 30° C. and without CO2.

[0013] The present invention also provides a method for purifying the above-mentioned adherent primary carp oogonial stem cell line, comprising the following steps: using the above-mentioned passaging method to pass the above-mentioned adherent primary carp oogonial stem cell line to the third generation, and then performing continuous differential adhesion to obtain purified carp oogonial stem cells.

[0014] Preferably, the continuous differential adhesion comprises 4 to 6 consecutive subcultures for differential adhesion culture.

[0015] Preferably, the continuous differential adhesion comprises digesting the third generation carp oogonial stem cells with a mixture of PBS and 0.25% trypsin-EDTA for 4 to 6 minutes, terminating the digestion with L-15 complete medium, aspirating the liquid and centrifuging at 1000 to 1500 rpm for 4 to 6 minutes, removing the supernatant, adding L-15 complete medium, mixing, and then inoculating into a culture dish, adhering to the wall for 30 to 50 minutes, aspirating the cell suspension and inoculating into a new culture dish, and completing one differential adhesion culture.

[0016] The present invention also provides purified carp oogonial stem cells obtained by the purification method.

[0017] The present invention also provides a freezing solution of the above-mentioned adherent primary carp oogonial stem cell line or the above-mentioned purified carp oogonial stem cell, which comprises a mixture of L-15 complete culture medium, fetal bovine serum and DMSO, or a mixture of fetal bovine serum and DMSO.

[0018] The present invention also provides the use of the above-mentioned adherent primary carp oogonial stem cell line or the carp oogonial stem cells after being frozen and revived in cultivating new carp varieties.

[0019] Beneficial effects: The present invention provides an in vitro culture method of carp oogonial stem cells, comprising selecting female carp, isolating ovarian tissue, isolating primary oogonial stem cells from the ovarian tissue and then performing adherent culture, and performing subculture and purification of the adherently cultured oogonial stem cells. The present invention also provides cryopreservation and recovery of the subcultured and purified oogonial stem cells. The present invention utilizes the tissue adherence method to culture carp oogonial stem cells, and on the third day, it can be observed that long strips, triangular shapes, and irregular polygonal cells crawl out of the small tissue pieces and extend to the surrounding areas; after cell subculture, the carp oogonial stem cells are in good growth condition.

[0020] After passage using the in vitro culture method of the present invention, the third generation carp oogonial stem cells contain a large number of fibroblasts. After differential attachment, some fibroblasts can be removed. After continuous differential attachment of carp oogonial stem cells, the purity of the cells is analyzed based on morphological observation, and the purity of the carp oogonial stem cells of the 14th generation can reach 66.73%. After cryopreservation and resuscitation using the method of the present invention, the resuscitation survival rate is above 90%. Morphological observation of the resuscitated cells shows that the cells are in good condition, the cell outline can be clearly seen, and the cell nucleus is intact.

[0021] The carp oogonial stem cells obtained by the present invention after in vitro culture, passage and purification, cryopreservation and resuscitation have typical germ stem cell characteristics and pluripotency, which overcomes the long generation interval constraint in the process of new carp variety breeding, shortens the cycle of excellent variety breeding and improves carp breeding efficiency, and provides an important technical basis for the genetic breeding of carp. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the result of sex identification of Yellow River carp;

[0023] Figure 2 Cell diagrams of primary culture methods of Yellow River carp oogonial stem cells, where (a) is the tissue attachment method, (b) is the trypsin digestion method, (c) is the chelating agent dissociation method, and (d) is the first generation of Yellow River carp oogonial stem cells; the black circles in the figure contain triangular cells; the white ovals contain long strips of cells; the black arrows indicate irregular cells; the white arrows indicate gonadal tissue blocks; the red arrows indicate cell vacuoles;

[0024] Figure 3 : The growth curve of Yellow River carp oogonial stem cells, wherein (a) is the growth curve of Yellow River carp oogonial stem cells under different temperature conditions, (b) is the growth curve of Yellow River carp oogonial stem cells under different FBS concentrations, (c) is the growth curve of Yellow River carp oogonial stem cells under different culture medium conditions, and (d) is the growth curve of Yellow River carp oogonial stem cells under the conditions of lacking different growth factors;

[0025] Figure 4 The figure shows the cells purified by differential attachment of Yellow River carp oogonial stem cells, where (a) shows the cells before differential attachment, (b) shows the cells after differential attachment, the black arrows indicate fibroblasts, and the white arrows indicate Yellow River carp oogonial stem cells;

[0026] Figure 5 The results of freezing and reviving the oogonial stem cells of Yellow River carp are shown in Figure 1. (a) is the survival rate of cell revival, (b) is the cell state under the condition of freezing solution 1, and (c) is the cell state under the condition of freezing solution 2.

[0027] Figure 6 This is the ALP staining result of Yellow River carp oogonial stem cells;

[0028] Figure 7 This is the RNA-FISH result of Yellow River carp oogonial stem cells;

[0029] Figure 8 This is the immunofluorescence result of Yellow River carp oogonial stem cells;

[0030] Fig. 9 This is the result of RT-PCR of specific marker gene of Yellow River carp oogonial stem cells;

[0031] Fig.10 The results of in vitro differentiation of Yellow River carp oogonial stem cells are shown in Figure 1. (a) is an epithelial cell, (b) is an astrocyte, (c) is a neural cell, and (d) and (e) are embryoid bodies.

[0032] Fig.11 This is the RFP staining result of Yellow River carp oogonial stem cells;

[0033] Fig.12 The figure shows the colonization of Yellow River carp oogonial stem cells transplanted into zebrafish embryos. In the figure, AC represents the colonization of oogonial stem cells in the chimera at 2 days (A), 3 days (B) and 4 days (C) after transplantation of zebrafish recipients.

[0034] Fig.13 The figure shows the colonization of Yellow River carp oogonial stem cells transplanted into crucian carp embryos. In the figure, AD indicates the colonization of oogonial stem cells in the chimera at the stages of 24 hpf (A), 10 dpf (B), 12 dpf (C) and 20 dpf (D) of the crucian carp recipient.

[0035] Fig.14 The results of transplanting oogonial stem cells from Yellow River carp into larval crucian carp 7 to 9 days after fertilization are shown in Figure 5. AC represents the colonization of oogonial stem cells in the chimera at 2 days (A), 3 days (B) and 4 days (C) after transplantation. DETAILED DESCRIPTION

[0036] The invention provides an in vitro culture method of carp oogonial stem cells, comprising the following steps: soaking ovaries taken from female carp in L-15 basic culture medium and then placing in carp serum, cutting the ovaries into pieces and evenly spreading them in a T25 culture bottle, culturing in a CO2-free environment at a temperature of 26-30 DEG C, adding L-15 complete culture medium for further culturing after culturing for 4-6 hours, and obtaining primary carp oogonial stem cells in adherent culture;

[0037] The L-15 basal medium comprises the following components: L-15 powder, 5 mol / L HEPES buffer, 50 μmol / L β-mercaptoethanol, and sterilized water;

[0038] The L-15 complete culture medium is based on the L-15 basal culture medium and also includes the following components: 10-20% by volume of fetal bovine serum, 2-20 ng / mL of basic fibroblast growth factor, 2-20 ng / mL of leukemia inhibitory factor, 0.5-2% by volume of carp serum, 0.5-2% by volume of zebrafish embryo extract, 0.5-2% by volume of L-glutamine, 600-1000 IU / mL of penicillin, 0.6-1 mg / mL of streptomycin, and 1-3 μg / mL of amphotericin B.

[0039] When the selected carp is young and the gonadal differentiation is not obvious, the present invention also needs to perform sex identification on the selected carp. For example, in the embodiment, a 10-month-old carp is used as an example for explanation. At this time, the gonadal differentiation is not obvious, and the selected carp needs to be sex identified. In a specific embodiment of the present invention, the sex identification can be PCR identification, wherein the primer pair for identification can be a primer pair composed of CYC-male marker-F shown in SEQ ID No.1 and CYC-malemarker-R shown in SEQ ID No.2.

[0040] CYC-male marker-F: 5'-CCTTGTTGTTTGTGGTGCTG-3';

[0041] CYC-male marker-R: 5'-ATTGCAAGCTGGACTGGTTT-3'.

[0042] When using the primer pair for sex identification, in a specific embodiment of the present invention, a PCR reaction system is constructed using carp tail fin DNA as a template, including: 0.5 μL of upstream and downstream primers, 1 μL of template DNA, 5 μL of 2×PCRmix, and ddH2O added to 10 μL. In a specific embodiment of the present invention, the PCR reaction conditions are as follows: 95°C for 3 min; 95°C for 30 s, 60°C for 30 s, 72°C for 30 s, 35 cycles; 72°C for 5 min; and stored at 4°C. The present invention performs agarose gel electrophoresis on the PCR amplification product. In a specific embodiment of the present invention, the PCR amplification product is added to the gel well of agarose gel with a concentration of 1.5%, and electrophoresis is performed at 120V for 30 min. If there is only a single 120bp band, it proves to be a female carp. If there are double bands, that is, one is 120bp and the other is 452bp, it is a male carp.

[0043] The present invention takes ovarian tissue from female carp and separates primary oogonial stem cells. When taking ovarian tissue, in a specific embodiment of the present invention, the selected female fish is first anesthetized, such as by using MS-222 for anesthesia, and the concentration of MS-222 can be 100-200 mg / L, such as 100 mg / LMS-222. After the anesthesia, 0.1% potassium permanganate solution is used for disinfection, and the disinfection time can be 20-30 minutes, such as 30 minutes; then the gills are cut and bled, and then 75% alcohol is evenly sprayed on the surface of the fish body, and after disinfection, the fish body is transferred to a clean bench to take out the ovaries.

[0044] In a specific embodiment of the present invention, when separating carp primary oogonial stem cells from the ovary, firstly, the ovary is washed 3 times (3 to 5 times) in a sterile culture dish with PBS containing penicillin-streptomycin-amphotericin B to remove other tissues, wherein the concentration of penicillin-streptomycin-amphotericin B in the PBS can be 5 to 10%, such as 8%. Then, the ovary washed and other tissues are immersed in L-15 basic culture medium, and after immersion, the ovary tissue is placed in a centrifuge tube containing carp serum to break the ovarian tissue, such as cutting it into 1 mm pieces with scissors. 3 Take small pieces and evenly spread them in a T25 culture bottle. The immersion time of the L-15 basic culture medium described in the present invention can be 15 to 30 minutes, such as 20 minutes. The specification of the centrifuge tube described in the present invention can be 1.5mL, and the volume containing carp serum can be 200 to 600μL, such as 200μL. In the specific implementation of the present invention, it is preferred to pre-treat the T25 culture bottle before spreading the block tissue in the T25 culture bottle, such as treating it with 1% gelatin 1h to 2h in advance, and then spreading the block tissue.

[0045] The present invention cultured a T25 culture bottle containing block ovarian tissue, such as cultured in an environment of 26 to 30°C and without CO2 for 4 to 6 hours, and then added L-15 complete culture medium to continue culture, and added L-15 complete culture medium again after 18 to 24 hours, and then replaced the culture medium every 1 to 3 days, and the amount of liquid replacement can be 1 / 2 to 2 / 3 of the original culture medium volume. In a specific embodiment of the present invention, the first time to add L-15 complete culture medium can be after cultured for 4 hours at 30°C and without CO2, and the amount of addition can be 1 to 2.5 mL, such as 2 mL; the second time to add can be after continuing to culture for 24 hours, and the amount of L-15 complete culture medium added can be 1.5 to 3 mL, such as 2 mL. Thereafter, the interval time for replacing the culture medium can be 1 to 3 days, such as 2 days, and the amount of liquid replacement can be 1 / 2 to 2 / 3, such as 1 / 2.

[0046] The L-15 basic culture medium of the present invention comprises the following components: L-15 powder, 5 mol / L HEPES buffer, 50 μmol / L β-mercaptoethanol, and sterile water; the L-15 complete culture medium is obtained by adding fetal bovine serum (FBS), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), carp serum, zebrafish embryo extract, L-glutamine (LG), penicillin, streptomycin and amphotericin B to the L-15 basic culture medium. In a specific embodiment of the present invention, the volume percentage of FBS can be 10-20%, such as 15%; the final concentration of bFGF can be 2-20 ng / mL, such as 2 ng / mL. L; the final concentration of LIF can be 2-20 ng / mL, such as 2 ng / mL; the volume percentage of carp serum can be 0.5-2%, such as 1% carp serum; the volume percentage of zebrafish embryo extract can be 0.5-2%, such as 1%; the volume percentage of L-glutamine can be 0.5-2%, such as 1%; the content of penicillin can be 600-1000 IU / mL, such as 800 IU / mL; the content of streptomycin can be 0.6-1 mg / mL, such as 0.8 mg / mL; the content of amphotericin B can be 1-3 μg / mL, such as 2 μg / mL.

[0047] The present invention also provides an adherent primary carp oogonial stem cell line cultured by the in vitro culture method.

[0048] By using the in vitro culture method of the present invention, i.e., using the tissue adhesion method to culture carp oogonial stem cells, on the third day, long strips, triangles, and irregular polygonal cells can be observed crawling out of the small tissue pieces and extending to the surrounding areas; after cell passaging, the carp oogonial stem cells are in good growth condition.

[0049] The present invention also provides a method for subculturing the above-mentioned adherent primary carp oogonial stem cell line, comprising the following steps:

[0050] (1) after the confluence of the adherent primary carp oogonial stem cell line reaches more than 80%, the L-15 complete medium is removed and the cells are washed with PBS;

[0051] (2) After washing with PBS, digesting with a mixture of PBS and 0.25% trypsin-EDTA for 4 to 6 minutes, terminating the digestion with L-15 complete medium, aspirating the liquid and centrifuging at 1000 to 1500 rpm for 4 to 6 minutes, removing the supernatant, and adding L-15 complete medium to prepare a cell suspension;

[0052] (3) The cell suspension collected in step (2) is distributed into a new T25 culture flask at a ratio of 1:(2-4), and after adding L-15 complete medium, culture and subculture are performed at 26-30°C in the absence of CO2.

[0053] When the subculture is performed in the present invention, it is necessary to use cells that are attached to the wall of carp oogonial stem cells and have a cell confluence of more than 80%. After removing the culture medium, wash with PBS and discard the waste liquid, and then digest with pancreatic enzymes. In a specific embodiment of the present invention, 1 to 2 mL of PBS is added for 2 to 4 washes, such as adding 1 mL of PBS for washing, and repeating 3 times; when digesting, in a specific embodiment, a mixture of PBS and 0.25% pancreatin-EDTA is added, and the volume ratio of PBS and 0.25% pancreatin-EDTA in the mixture is 1:1. In an embodiment of the present invention, when digesting, 1 to 2 mL of PBS and 1 to 2 mL of 0.25% pancreatin-EDTA mixture are added in sequence, and digestion is 4 to 6 minutes, wherein the mixture is 1 mL of PBS and 1 mL of 0.25% pancreatin-EDTA, and the digestion time is 5 minutes. After the digestion operation, in a specific embodiment of the present invention, L-15 complete medium is used to terminate the digestion, wherein the amount of L-15 complete medium added can be 1 to 2 mL, such as 1 mL of L-15 complete medium to terminate the digestion, and the liquid is aspirated and placed in a new centrifuge tube.

[0054] The present invention centrifuges the centrifuge tube to remove the supernatant, wherein the centrifuge speed in a specific embodiment can be 1000-1500 rpm, such as 1200 rpm; the centrifuge time can be 4-6 min, such as 5 min.

[0055] The present invention redistributes the cell sediment obtained by centrifugation into a new T25 culture bottle. In a specific embodiment, the distribution is performed according to a ratio of 1: (2-4) such as 1: 3, and L-15 complete medium is added to the T25 culture bottle. The amount of L-15 complete medium added can be 3-6 mL / T25 culture bottle, such as 4 mL / T25 culture bottle, and then culture is performed under CO2-free conditions of 26-30° C., such as 30° C. After passage to the 10th generation, the carp oogonial stem cells are passaged once every 4-5 days.

[0056] The present invention also provides a method for purifying the above-mentioned adherent primary carp oogonial stem cell line, comprising the following steps: using the above-mentioned passaging method to pass the above-mentioned adherent primary carp oogonial stem cell line to the third generation, and then performing continuous differential adhesion to obtain purified carp oogonial stem cells.

[0057] In a specific embodiment of the present invention, the differential attachment method is used to purify carp oogonial stem cells, specifically comprising digesting the third generation of Yellow River carp oogonial stem cells with a mixture of PBS and 0.25% trypsin-EDTA for 4 to 6 minutes, terminating the digestion with L-15 complete medium, aspirating the liquid and centrifuging at 1000 to 1500 rpm for 4 to 6 minutes, removing the supernatant, adding L-15 complete medium and mixing, and then inoculating it into a culture dish for 30 to 50 minutes, aspirating the cell suspension and inoculating it into a new culture dish, completing a differential attachment culture, removing part of the fibroblasts; and performing differential attachment for 4 to 6 consecutive passages. In the embodiment of the present invention, a mixture of PBS and 0.25% trypsin-EDTA in a volume ratio of 1:1 is used for digestion for 4 to 6 minutes, such as 5 minutes, and the digested cells are transferred to a centrifuge tube containing L-15 complete medium. In one embodiment of the present invention, the centrifugation speed is 1200 rpm, the time is 4 to 6 minutes, such as 5 minutes, the supernatant is removed and L-15 complete medium is added to obtain a cell suspension which is inoculated in a culture dish. After 30 to 50 minutes, such as 40 minutes, the cell suspension is transferred to a new culture dish to remove some fibroblasts.

[0058] The present invention also provides purified carp oogonial stem cells obtained by the purification method.

[0059] The present invention also provides a freezing solution of the above-mentioned adherent primary carp oogonial stem cell line or the above-mentioned purified carp oogonial stem cell, which comprises a mixture of L-15 complete culture medium, fetal bovine serum and DMSO, or a mixture of fetal bovine serum and DMSO.

[0060] In the specific embodiment of the present invention, two groups of cryopreservation solutions are provided. Cryopreservation solution 1 is a mixture of L-15 complete medium, FBS and DMSO, and the volume ratio of L-15 complete medium, FBS and DMSO is 7:2:1, which is prepared and used immediately; cryopreservation solution 2 is a mixture of FBS and DMSO, and the volume ratio of FBS and DMSO is 10:1, which is prepared and used immediately. After freezing cells for a period of time using the above two groups of cryopreservation solutions, the cells are revived and their resuscitation survival rates are detected. It is found that the resuscitation survival rate of cryopreservation solution 1 is slightly higher than that of cryopreservation solution 2, but the difference is not significant (P>0.05).

[0061] The present invention also provides a freezing method matched with the freezing solution, which specifically comprises the following steps:

[0062] (1) When the cell confluence of carp oogonial stem cells reaches 80-90%, remove the original culture medium and wash gently with PBS to remove the waste liquid;

[0063] (2) Mix PBS and 0.25% trypsin-EDTA for digestion, then terminate the digestion with L-15 complete medium and transfer the liquid to a centrifuge tube;

[0064] (3) After centrifugation, discard the supernatant and add freezing solution and transfer to a dedicated cryotube; first keep it at -80°C overnight and then transfer it to a liquid nitrogen tank for long-term storage.

[0065] In one embodiment, the number of PBS light washes in step (1) of the present invention is 3 to 5 times, such as 3 times; in one embodiment, the reagent used for digestion in step (2) is a mixture of PBS and 0.25% trypsin-EDTA, preferably PBS and 0.25% trypsin-EDTA with a volume ratio of 1:1; the digestion time can be 4 to 6 minutes, such as 5 minutes. Then, the digestion is terminated with L-15 complete medium, and the liquid is transferred to a new centrifuge tube. In one embodiment of the present invention, the centrifugation parameters in step (3) of the present invention can be 1000 to 1500 rpm, such as 1200 rpm; the time can be 4 to 6 minutes, such as 5 minutes.

[0066] The present invention can revive frozen cells, and the revival method specifically includes the following steps:

[0067] (1) Take out the frozen carp oogonial stem cells, immediately place them in a 37°C water bath, and gently shake them until they are completely thawed;

[0068] (2) transferring the completely thawed cells to 5-15 mL of L-15 complete medium, such as 10 mL, and centrifuging at room temperature at 1000-1500 rpm, such as 1200 rpm, for 3-5 min, such as 5 min;

[0069] (3) Discard the supernatant and add 3-10 mL, such as 5 mL, of L-15 complete medium and mix well. Take 1 mL of the cell suspension for cell counting. Transfer the remaining cell suspension to a T25 cell flask;

[0070] (4) Take 10 μL of the mixed cell suspension and mix it with 10 μL of trypan blue reagent. Use a blood cell counting plate to calculate the cell recovery survival rate based on the ratio of live cells to total cells. Incubate at 30°C without CO2 for 18 to 28 hours. After 24 hours, replace with fresh L-15 complete medium.

[0071] The resuscitation survival rate of the carp oogonial stem cells of the present invention is above 90%. Morphological observation of the resuscitated cells shows that the cells are in good condition, the cell outlines can be clearly seen, and the cell nuclei are intact.

[0072] The present invention also provides the use of the above-mentioned adherent primary carp oogonial stem cell system, the above-mentioned purified carp oogonial stem cells or the carp oogonial stem cells frozen and revived by the above-mentioned freezing solution in breeding new carp varieties.

[0073] In order to further illustrate the present invention, the in vitro culture, passage and purification methods of carp oogonial stem cells provided by the present invention are described in detail below in combination with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0074] The culture medium used in the embodiment of the present invention is formulated as follows:

[0075] DMEM complete medium: obtained by adding fetal bovine serum (FBS), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), carp serum, zebrafish embryo extract, L-glutamine (LG), penicillin, streptomycin and amphotericin B to DMEM basal medium (Gibco, 11995065). In a specific embodiment of the present invention, the volume percentage of FBS can be 10-20%, such as 15%; the final concentration of bFGF can be 2-20 ng / mL, such as 2 ng / mL; the final concentration of LIF can be 10-20 ng / mL, such as 10 ng / mL. The final concentration can be 2-20 ng / mL, such as 2 ng / mL; the volume percentage of carp serum can be 0.5-2%, such as 1% carp serum; the volume percentage of zebrafish embryo extract can be 0.5-2%, such as 1%; the volume percentage of L-glutamine can be 0.5-2%, such as 1%; the content of penicillin can be 600-1000 IU / mL, such as 800 IU / mL; the content of streptomycin can be 0.6-1 mg / mL, such as 0.8 mg / mL; the content of amphotericin B can be 1-3 μg / mL, such as 2 μg / mL.

[0076] DMEM-F12 complete medium: obtained by adding fetal bovine serum (FBS), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), carp serum, zebrafish embryo extract, L-glutamine (LG), penicillin, streptomycin and amphotericin B to DMEM-F12 basal medium (Gibco, 31330095). In a specific embodiment of the present invention, the volume percentage of FBS can be 10-20%, such as 15%; the final concentration of bFGF can be 2-20 ng / mL, such as 2 ng / mL; The final concentration of LIF can be 2-20 ng / mL, such as 2 ng / mL; the volume percentage of carp serum can be 0.5-2%, such as 1% carp serum; the volume percentage of zebrafish embryo extract can be 0.5-2%, such as 1%; the volume percentage of L-glutamine can be 0.5-2%, such as 1%; the content of penicillin can be 600-1000 IU / mL, such as 800 IU / mL; the content of streptomycin can be 0.6-1 mg / mL, such as 0.8 mg / mL; the content of amphotericin B can be 1-3 μg / mL, such as 2 μg / mL.

[0077] M199 complete medium: obtained by adding fetal bovine serum (FBS), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), carp serum, zebrafish embryo extract, L-glutamine, penicillin, streptomycin and amphotericin B to M199 basal medium (Gibco, 11150059). In a specific embodiment of the present invention, the volume percentage of FBS can be 10-20%, such as 15%; the final concentration of bFGF can be 2-20 ng / mL, such as 2 ng / mL; the final concentration of LIF can be 10-20 ng / mL, such as 10 ng / mL. The concentration can be 2-20 ng / mL, such as 2 ng / mL; the volume percentage of carp serum can be 0.5-2%, such as 1% carp serum; the volume percentage of zebrafish embryo extract can be 0.5-2%, such as 1%; the volume percentage of L-glutamine can be 0.5-2%, such as 1%; the content of penicillin can be 600-1000 IU / mL, such as 800 IU / mL; the content of streptomycin can be 0.6-1 mg / mL, such as 0.8 mg / mL; the content of amphotericin B can be 1-3 μg / mL, such as 2 μg / mL.

[0078] Example 1

[0079] The gonad differentiation of Yellow River carp is not obvious when it is 10 months old, and the sex of Yellow River carp cannot be distinguished by naked eyes. The present invention designs Yellow River carp sex identification primers using the specific gene sequences of male and female fish in the carp genome to identify the sex of experimental fish.

[0080] (1) DNA extraction: Take a small piece of fresh Yellow River carp fin and place it in DNA lysis buffer. Digest it at 55°C for 3 h, then inactivate it at 98°C for 10 min, and store it at -20°C.

[0081] (2) PCR amplification: Using the DNA of the caudal fin of Yellow River carp as a template, PCR amplification was performed using the carp sex identification specific primers CYC-malemarker-F / R. The PCR reaction system was: 0.5 μL of each upstream and downstream primer, 1 μL of template DNA, 5 μL of PCRmix, and ddH2O to 10 μL. The PCR reaction conditions were as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, 35 cycles; extension at 72°C for another 5 min; storage at 4°C.

[0082] (3) Agarose gel electrophoresis detection: Take the PCR amplification product and add it to the wells of agarose gel with a concentration of 1.5%, and perform electrophoresis for 30 minutes (voltage is 120V). Figure 1 As shown, female fish have a single band with a fragment size of 120 bp; male fish have double bands with fragment sizes of 120 bp and 452 bp respectively.

[0083] Example 2

[0084] Adherent culture of primary cells:

[0085] (1) According to the results of sex identification in Example 1, female Yellow River carp were selected and anesthetized at a concentration of 100 mg / L MS-222.

[0086] (2) Disinfect with 0.1% potassium permanganate solution for 30 minutes, cut the gills and bleed, then spray the fish body surface evenly with 75% alcohol. After disinfection, transfer to the clean bench and remove the ovaries.

[0087] (3) Wash the cells three times in a sterile culture dish with PBS containing 8% penicillin-streptomycin-amphotericin B to remove other tissues.

[0088] (4) Soak in L-15 basal medium for 20 min, take out and place in a 1.5 mL centrifuge tube containing 200 μL carp serum, and cut the gonad into 1 mm pieces with scissors. 3 Take small pieces and spread them evenly in a T25 culture flask.

[0089] (5) Incubate at 30°C without CO2 for 4 h. Add 2 mL of L-15 complete medium.

[0090] (6) After 24 hours, carefully add 2 mL of L-15 complete medium to the T25 cell culture flask and continue culturing.

[0091] (7) The culture medium was replaced every two days with the replacement volume being 1 / 2 of the original culture medium volume.

[0092] When the Yellow River carp oogonial stem cells were cultured using the tissue adherence method, on the third day, long strips, triangles, and irregular polygonal cells could be observed crawling out of the tissue blocks and extending to the surrounding areas ( Figure 2 Middle a). After cell passage, the growth of Yellow River carp oogonial stem cells was good ( Figure 2 (d)

[0093] Comparative Example 1

[0094] In order to explore which primary culture method is more conducive to the in vitro isolation and culture of Yellow River carp oogonial stem cells, trypsin digestion and chelating agent dissociation methods were set up to isolate Yellow River carp oogonial stem cells for culture. Trypsin digestion method: 10-month-old female Yellow River carp was anesthetized at a concentration of 100 mg / L MS-222, disinfected with 0.1% potassium permanganate solution for 30 minutes, and the gills were cut to bleed. Then, it was treated with 75% alcohol and transferred to the clean bench to take the ovaries. Wash 3 times in a sterile culture dish with PBS containing 8% penicillin-streptomycin-amphotericin B triple antibodies to remove other tissues. Soak in a cell culture dish containing penicillin-streptomycin-amphotericin B triple antibodies for 10 minutes. Soak in a cell culture dish containing 1mL 0.25% trypsin and 50μL carp serum, and cut the ovaries into 1mm 3 Small pieces, digest for 2 hours. Add 2mL complete medium to stop digestion, transfer to a new enzyme-free tube, centrifuge at 3000rpm for 5 minutes, and discard the supernatant. Add 4mL L-15 complete medium, gently beat to mix, and then inoculate into a T25 cell culture flask and place in a 30℃ CO2-free incubator for culture. The chelating agent dissociation method is the same as the trypsin digestion method, except that 1mM EDTA is added on the basis of 0.25% trypsin digestion.

[0095] The results showed that the method of isolating oogonial stem cells by trypsin digestion could obtain a large number of single cells suspended in the culture medium. On the 7th day of isolation, triangular and irregular polygonal cells could be observed growing on the wall ( Figure 2 b), but there are still a large number of white suspension cells that do not adhere to the wall. The cells obtained by this method are mainly fibroblast-like cells, with a small proportion of oogonial stem cells, and it is difficult to pass them on. The cell state after passaging is not good. The cells cultured by the trypsin adhesion method are difficult to continue to pass on after the 4th generation. Using the chelating agent dissociation oogonial stem cell culture method, 1mM EDTA is added on the basis of 0.25% trypsin digestion, and single suspension cells can also be separated. On the 5th day, it can be observed that the cells separated from the ovaries are spindle-shaped, with vacuolar structures in the cytoplasm and slender protrusions extending from both ends ( Figure 2 (c) The cells obtained by this method are difficult to continue subculture after the third generation.

[0096] Example 3

[0097] 1. Cell subculture:

[0098] (1) When the Yellow River carp oogonial stem cells adhere to the wall and the cell confluence reaches more than 80%, remove the culture medium.

[0099] (2) Add 1 mL of PBS for washing, repeat 3 times and discard the waste liquid.

[0100] (3) Add 1 mL of PBS and 1 mL of 0.25% trypsin-EDTA mixture in sequence and digest for 5 min. Then, add 1 mL of L-15 complete medium to terminate the digestion and aspirate the liquid into a 15 mL centrifuge tube.

[0101] (4) Centrifuge at 1200 rpm for 5 min and remove the supernatant.

[0102] (5) Add L-15 complete medium and distribute the cells to a new T25 culture flask at a ratio of 1:3. Add L-15 complete medium to make up to 4 mL, and then culture at 30°C under CO2-free conditions.

[0103] (6) After the 10th generation, Yellow River carp oogonial stem cells were subcultured approximately every 4 to 5 days.

[0104] 2. In order to explore the optimal growth conditions of Yellow River carp oogonial stem cells, the cell proliferation of Yellow River carp oogonial stem cells was measured under different temperatures, serum concentrations, culture media, and growth factor deficiency.

[0105] (1) The 14th generation Yellow River carp oogonial stem cells were digested to prepare a cell suspension.

[0106] (2) Count the number of Yellow River carp oogonial stem cells using a blood cell plate, inoculate 5000 cells / well into a 96-well plate, and ensure that each well contains 100 μL L-15 complete culture medium.

[0107] (3) Seven additional gradients (160,000, 80,000, 40,000, 20,000, 10,000, 5,000, and 2,500) were set up with three replicates. After the Yellow River carp oogonial stem cells adhered to the wall for 4 h, 10 μL of CCK-8 reagent was added. After standing for 2.5 h, the OD value (450 nm) was measured with an ELISA reader, and the cell standard curve was calculated.

[0108] (4) Experiments were designed based on different temperatures (26°C, 28°C, and 30°C), different serum concentrations (5%, 10%, 15%, and 20%), different basal culture media (L-15, DMEM, DMEM-F12, and M199), and different cell growth factor deficiencies (bFGF, LIF, carp serum, zebrafish embryo extract, and L-glutamine). Each group had only one variable, and all other conditions were the same, with no CO2.

[0109] (5) After cell inoculation, CCK-8 reagent was added at a fixed point every day, and the OD (450 nm) value was measured after standing for 2.5 h. The effects of different culture media on cell proliferation were detected until the 6th day.

[0110] (6) The cell culture medium was changed every 2 days.

[0111] The results are as follows Figure 3 As shown, the culture temperature of 30℃ is more conducive to the proliferation of Yellow River carp oogonial stem cells. On the 6th day, the number of cells at 30℃ was significantly higher than that at 26℃ and 28℃ (P<0.01); the number of oogonial stem cells at 15% FBS concentration was significantly higher than that at other serum concentrations on the 6th day (P<0.01); among L-15, DMEM, DMEM-F12 and M199 basal culture media, L-15 medium is more conducive to the culture of Yellow River carp oogonial stem cells. On the 6th day, the number of cells was significantly higher than that of other culture media (P<0.01), and the number of cells was more than twice that of other culture media; it was found that in oogonial stem cells, cell proliferation was significantly inhibited in the culture medium lacking carp serum and zebrafish embryo extract (P<0.01); the proliferation of Yellow River carp oogonial stem cells was determined under different culture media, serum concentrations, growth factor deficiency and other conditions. The results showed that the optimal culture temperature of Yellow River carp oogonial stem cells was 30°C, the optimal serum concentration was 15%, and the optimal culture medium was L-15. The lack of carp serum and zebrafish embryo extract had the greatest impact on the proliferation of oogonial stem cells, and the cells hardly proliferated ( Figure 3 ).

[0112] Example 4

[0113] The present invention uses the differential adhesion method to purify the Yellow River carp oogonial stem cells:

[0114] (1) When the cells reached the third generation, differential adhesion purification of Yellow River carp oogonial stem cells was performed, and the cells were digested for 5 min with a mixture of PBS and 0.25% trypsin-EDTA in a volume ratio of 1:1. The digestion was terminated with L-15 complete medium and the cells were transferred to a centrifuge tube containing L-15 complete medium.

[0115] (2) Centrifuge at 1200 rpm for 5 min, remove the supernatant, add L-15 complete medium, mix well, and inoculate into a culture dish. After culturing for 40 min, aspirate the cell suspension and transfer it to a new culture dish, and remove some fibroblasts.

[0116] (3) Perform differential attachment for 4 to 6 consecutive passages.

[0117] The third generation of Yellow River carp oogonial stem cells contains a large number of fibroblasts, and some of the fibroblasts can be removed after differential adhesion. After continuous differential adhesion of Yellow River carp oogonial stem cells, the cell purity was analyzed based on its morphological observation ( Figure 4 ), the purity of the 14th generation Yellow River carp oogonial stem cells could reach 66.73% (Table 1).

[0118] Table 1 Purity of Yellow River carp oogonial stem cells

[0119]

[0120] Example 5

[0121] Cell cryopreservation and cell recovery

[0122] In order to optimize the freezing conditions of Yellow River carp oogonial stem cells, two groups of freezing solutions were set up. The preparation method of freezing solution 1 was to mix L-15 complete medium, FBS and DMSO in a ratio of 7:2:1, and it was prepared and used immediately; the preparation method of freezing solution 2 was to mix FBS and DMSO in a ratio of 10:1, and it was prepared and used immediately. After the cells were frozen for a period of time, the cells were revived, and their survival rate was detected to optimize the freezing conditions of Yellow River carp oogonial stem cells.

[0123] 1. The freezing method is as follows:

[0124] (1) When the cell confluence of the 19th generation Yellow River carp oogonial stem cells reached 80% to 90%, the original culture medium was removed and the cells were gently washed with PBS for 3 times to remove the waste liquid.

[0125] (2) Mix 1 mL of PBS and 1 mL of 0.25% trypsin-EDTA for 5 min of digestion, then terminate the digestion with 1 mL of L-15 complete medium and transfer the liquid to a 15 mL centrifuge tube.

[0126] (3) After centrifugation at 1200 rpm for 5 min, discard the supernatant, add freezing solution and transfer to a cell-specific freezing tube.

[0127] (4) First store at -80°C overnight and then transfer to a liquid nitrogen tank for long-term storage.

[0128] 2. The resuscitation method is as follows:

[0129] (1) Take out the 19th generation frozen Yellow River carp oogonial stem cells, immediately place them in a 37°C water bath, and gently shake them until they are completely thawed.

[0130] (2) Transfer the completely thawed cells to a 15 mL centrifuge tube containing 10 mL of L-15 complete medium and centrifuge at 1200 rpm for 5 min at room temperature.

[0131] (3) Discard the supernatant and add 5 mL of L-15 complete medium and mix well. Take 1 mL of the cell suspension for cell counting. Transfer the remaining cell suspension to a T25 cell flask.

[0132] (4) Take 10 μL of the mixed cell suspension and 10 μL of trypan blue reagent, add it to the blood cell counting plate, and calculate the cell recovery survival rate by the ratio of live cells to total cells. Incubate at 30℃ without CO2. After 24 hours, replace with fresh L-15 complete medium.

[0133] The results showed that the survival rate of Yellow River carp oogonial stem cells was above 90% ( Figure 5 The results in Figure a) show that the cell freezing method of the present invention is scientific, and the recovery survival rate of cryopreservation solution 1 is slightly higher than that of cryopreservation solution 2, but the difference is not significant (P>0.05). The morphological observation of the recovered cells showed that the cells were in good condition, the cell outlines could be clearly seen, and the cell nuclei were intact ( Figure 5 b and c).

[0134] Example 6

[0135] 1. Characterization of Yellow River Carp Oocyte Stem Cells

[0136] The Yellow River carp oogonial stem cells cultured by the present invention have typical characteristics of reproductive stem cells and pluripotency. Alkaline phosphatase (ALP) is a phenotypic marker of reproductive stem cells and can express a strong fluorescent signal. After alkaline phosphatase staining (see Figure 6 ), and observed that the Yellow River carp oogonial stem cells were strongly positive.

[0137] The vasa gene is a marker gene for germline stem cells. The characterization of Yellow River carp oogonial stem cells by cellular RNA in situ hybridization revealed that most cells were positive for the vasa gene ( Figure 7 ). The localization of vasa-a and vasa-b genes in the Yellow River carp oogonial stem cells is consistent. DAPI staining was used to determine that the nuclear structure of the Yellow River carp oogonial stem cells was intact. The localization fusion of the nucleus and vasa gene revealed that the vasa gene was more highly expressed in the cytoplasm of the Yellow River carp oogonial stem cells. Through the analysis of vasa gene expression in reproductive stem cells, it was found that the purity of the 21st generation oogonial stem cells could reach 90.74%.

[0138] Further immunofluorescence analysis of vasa gene expression at the protein level revealed that vasa was positive in almost all Yellow River carp oogonial stem cells ( Figure 8 ).

[0139] 2. Detection of specific marker genes related to reproductive stem cells

[0140] 2.1 The steps for extracting total RNA from cells are as follows:

[0141] (1) The 15th generation oogonial stem cells were cultured at 5×10 4 After the cell coverage rate reached 80% to 90%, the cells were collected in a 2 mL centrifuge tube filled with TRIzol and temporarily stored in a -80°C refrigerator.

[0142] (2) Take out the TRIzol-treated cell samples from -80°C and thaw them on ice.

[0143] (3) Precool the centrifuge to 4°C, add 200 μL of chloroform to the sample, shake vigorously for 15 to 20 seconds, and then leave at room temperature for 10 minutes.

[0144] (4) Centrifuge at 12,000 rpm for 10 min at 4°C.

[0145] (5) Gently pipette the upper transparent liquid into a new enzyme-free 1.5 mL centrifuge tube (be careful to avoid aspirating the protein in the middle layer).

[0146] (6) Add an equal amount of isopropanol to the supernatant, shake gently until the mixture is clear, place at -20°C for 30 min, then at room temperature for 10 min, and then centrifuge at 4°C, 12,000 rpm for 10 min.

[0147] (7) After centrifugation, a white precipitate appeared. Slowly aspirate the supernatant and add 1 mL of pre-cooled 75% ethanol. Flick the precipitate and centrifuge at 4°C, 12,000 rpm for 10 min.

[0148] (8) Discard the supernatant and centrifuge at 12,000 rpm for 3 min at 4°C. After aspirating the supernatant, let it stand for 5 min to allow the alcohol to evaporate. When the white precipitate becomes transparent, immediately add 20 μL of enzyme-free water and stir to fully dissolve the RNA.

[0149] (9) The RNA concentration was measured using a micro-volume UV photometer and stored at -80°C until use.

[0150] 2.2 RT-PCR steps are as follows:

[0151] (1) Remove genomic DNA: Press PrimeScript TM Add the genomic DNA-free system solution of the RT reagent Kit with gDNA Eraser to the enzyme-free tube, incubate at 42°C for 2 minutes, and store at 4°C.

[0152] Genomic DNA removal system: RNA 1μg, 5×gDNAEraser 1μL, 5×gDNA Eraser Buffer 2μL, ddH2O to make up to 10μL.

[0153] (2) Reverse transcription: Incubate the prepared reverse transcription system on a PCR instrument at 37°C for 15 min, then at 85°C for 5 s, and store at 4°C. Store cDNA at -20°C.

[0154] Reverse transcription system: Remove genomic DNA system 10μL, PrimeScript RT Enzyme Mix Ⅰ 1μL, RT PrimerMix 1μL, 5× PrimeScript Buffer 4μL and ddH2O 4μL.

[0155] 2.3 PCR amplification: The above cDNA was diluted 10 times as a template and the following primers were used for PCR amplification. The PCR primer sequences of vasa, nanos1, nanos3, foxl2a, klf4-2, and β-actin genes are shown in Table 2. The PCR reaction system is: 0.5 μL of upstream and downstream primers, 1 μL of template DNA, 5 μL of 2×PCRmix, and ddH2O to 10 μL. The PCR reaction conditions are: 95°C, 3 min; 35 cycles in total (95°C, 30 s; 60°C, 30 s; 72°C, 30 s); 72°C, 5 min; and stored at 4°C.

[0156] Table 2 RT-PCR primer sequences

[0157]

[0158]

[0159] RT-PCR results Fig. 9 As shown, among the germ stem cell-specific marker genes, the Yellow River carp oogonial stem cells can express vasa, foxl2a, nanos1, nanos3, and klf4-2, and the Yellow River carp oogonial stem cells can highly express foxl2a.

[0160] 3. Verification of the pluripotency of Yellow River carp oogonial stem cells

[0161] To identify the multifunctionality of Yellow River carp oogonial stem cells, retinoic acid and 11-KT were used to induce differentiation of Yellow River carp oogonial stem cells in vitro. 4 Cells were inoculated into 6-well plates and cultured with L-15 complete medium. 24 hours after inoculation, cells were cultured with L-15 complete medium containing 10 μM retinoic acid, which was replaced every two days for 14 days, and the differentiation state of cells was observed under a microscope.

[0162] On the second day after the addition of the induction agent, epithelial cells ( Fig.10 Middle a). When Yellow River carp oogonial stem cells were induced to the 7th day, fibroblasts and neural cells could be observed ( Fig.10b and c). When the Yellow River carp oogonial stem cells were induced to the 14th day, many clustered embryoid bodies appeared on the cell layer ( Fig.10 Middle d); When the Yellow River carp oogonial stem cells were induced to the 30th day, the number of embryoid bodies increased significantly ( Fig.10 (e)

[0163] Example 7

[0164] The ability of purified 20th generation Yellow River carp oogonial stem cells to induce differentiation in vivo was analyzed using zebrafish embryos. After staining the Yellow River carp oogonial stem cells with PKH26, the cells were transplanted into zebrafish blastocyst embryos treated with dnd1-MO using microinjection technology. The steps of receptor preparation, donor cell labeling, and microinjection are as follows:

[0165] 1. Receptor Preparation

[0166] (1) Wild zebrafish that had been cultured for more than 3 months and reached sexual maturity were randomly selected and placed in zebrafish spawning boxes at a ratio of 1:1 female to male at around 4:00 p.m. on the first day. The male and female fish were separated by a transparent partition in the spawning box.

[0167] (2) The next day, under light conditions, the baffle was removed and the fertilized eggs were taken out for microinjection after the zebrafish mated.

[0168] (3) Inject 50 μM dnd1-MO into zebrafish embryos at the zygote stage, and wait for the embryos to develop to the blastocyst stage for cell transplantation.

[0169] 2. PKH26 staining of donor cells

[0170] (1) Select the 20th generation Yellow River carp oogonial stem cells with good growth status. When the cell confluence reaches 80%, digest them with 1 mL PBS and 1 mL 0.25% trypsin-EDTA at room temperature for 5 min. Take 2×10 7 The cells were used for subsequent experiments.

[0171] (2) Add 3 mL of serum-free culture medium and centrifuge at 400 g for 5 min. Discard the supernatant and leave less than 25 μL of residual liquid. Add 1 mL of diluent C and pipette gently to mix to prepare a 2× cell suspension.

[0172] (3) Before staining, add 4 μL PKH26 ethanol solution to 1 mL diluent C and mix thoroughly to make 2× staining solution.

[0173] (4) Add the 2× cell suspension to the 2× staining solution, mix quickly, incubate at 26°C for 3 min, and gently invert the centrifuge tube to mix.

[0174] (5) Add 2 mL of FBS to terminate the staining reaction and incubate for 1 min.

[0175] (6) Centrifuge at 400 g for 5 min at 20°C to 25°C, discard the supernatant, resuspend the cells in 10 mL of fresh L-15 complete medium, transfer to a new centrifuge tube, and repeat the above steps three times. After discarding the supernatant for the last time, resuspend the cells in L-15 complete medium to a concentration of 2 × 10 4 ~3×10 4 The cell suspension of 100 cells / μL was used for subsequent injection.

[0176] 3. Microinjection

[0177] (1) Prepare microinjection transplantation needles. Use a needle puller to pull glass needles and grind them to a suitable caliber for transplantation into zebrafish embryos at the blastocyst stage. Before injection, zebrafish embryos are transferred to a culture dish containing 2% agar.

[0178] (2) Under a stereo microscope, approximately 100 to 300 Yellow River carp oogonial stem cells were transplanted into blastocyst-stage zebrafish embryos using an oil pressure injector.

[0179] (3) The mortality rate was high within 1 to 2 days after transplantation. The eggs were cultured in a 28.5℃ incubator, and dead eggs were picked out and the water was changed 2 to 3 times a day. The embryos were observed regularly, and the chimeras were fixed in a methylcellulose culture dish. The fluorescent Yellow River carp oogonial stem cells were tracked under a fluorescence microscope.

[0180] Donor cells were successfully stained with PKH26 and expressed red fluorescence ( Fig.11 ). The results of cell transplantation showed that ( Fig.12 ), 2 to 4 days after transplantation, it can be observed that the Yellow River carp oogonial stem cells with red fluorescence in the injected zebrafish recipients are colonized in the reproductive-related parts of the zebrafish and migrate to the genital ridge, indicating that the established Yellow River carp oogonial stem cells are pluripotent in the zebrafish.

[0181] Example 7

[0182] Blastocyst stage crucian carp was used to analyze the ability of purified 30th generation Yellow River carp oogonial stem cells to induce differentiation in vivo. After staining the Yellow River carp oogonial stem cells with PKH26, the cells were transplanted into blastocyst stage crucian carp embryos treated with dnd1-MO using microinjection technology.

[0183] 1. Preparation of Carassius auratus Receptor

[0184] (1) Select female fish (carp fish) with a swollen abdomen and obvious ovarian outlines. The abdomen feels soft and elastic when you press it lightly, or eggs flow out of the abdomen when you press it lightly. Select male fish (carp fish) that have milky white semen flowing out of the abdomen when you press it lightly and that will scatter when they enter the water.

[0185] (2) The oxytocin for female fish was a mixture of 4 μg / mL luteinizing hormone-releasing hormone A2, 1.5 mg / mL domperidone, and 150 mg / mL chorionic gonadotropin, with the solvent being glucose solution. The mixture was injected into the abdominal cavity at a 45° angle from the base of the pectoral fin of the crucian carp. Each fish was injected with 1 mL per kilogram. The oxytocin dose for male fish was halved.

[0186] (3) After the injection of the oxytocin, the next day, when artificial egg and semen collection is performed, the crucian carp is anesthetized with anesthetics, and the female fish's abdomen is squeezed to collect the fish eggs. When the fish eggs are collected, the male fish's abdomen is quickly squeezed to collect the semen.

[0187] (4) Place an appropriate amount of fish eggs in a glass culture dish, add a drop of semen, and gently stir with a feather to ensure full fertilization.

[0188] (5) Inject 50 μM dnd1-MO into crucian carp embryos at the zygote stage, and wait until the embryos reach the blastocyst stage for cell transplantation.

[0189] 2. PKH26 staining of donor cells

[0190] (1) The 30th generation of Yellow River carp oogonial stem cells with good growth status were selected. When the cell confluence reached 80%, they were digested with 1 mL PBS and 1 mL 0.25% trypsin-EDTA at room temperature for 5 min. 2 × 10 7 The cells were used for subsequent experiments.

[0191] (2) Add 3 mL of serum-free culture medium and centrifuge at 400 g for 5 min. Discard the supernatant and leave less than 25 μL of residual liquid. Add 1 mL of diluent C and pipette gently to mix to prepare a 2× cell suspension.

[0192] (3) Before staining, add 4 μL PKH26 ethanol solution to 1 mL diluent C and mix thoroughly to make 2× staining solution.

[0193] (4) Add the 2× cell suspension to the 2× staining solution, mix quickly, incubate at 26°C for 3 min, and gently invert the centrifuge tube to mix.

[0194] (5) Add 2 mL of FBS to terminate the staining reaction and incubate for 1 min.

[0195] (6) Centrifuge at 400 g for 5 min at 20°C to 25°C, discard the supernatant, resuspend the cells in 10 mL of fresh L-15 complete medium, transfer to a new centrifuge tube, and repeat the above steps three times. After discarding the supernatant for the last time, resuspend the cells in L-15 complete medium to a concentration of 2 × 10 4 ~3×10 4 The cell suspension of 100 cells / μL was used for subsequent injection.

[0196] 3. Microinjection

[0197] (1) Prepare microinjection transplantation needles. Use a needle puller to pull glass needles and grind them to a suitable caliber for transplantation into crucian carp embryos at the blastocyst stage. Before injection, the crucian carp embryos were transferred to a culture dish containing 2% agar.

[0198] (2) Under a stereo microscope, approximately 100 to 300 Yellow River carp oogonial stem cells were transplanted into blastocyst-stage crucian carp embryos using a hydraulic injection apparatus.

[0199] (3) The mortality rate is high within 1 to 2 days after transplantation. Culture in a fish tank at 28.5℃, pick out dead eggs and change water 2 to 3 times a day. Observe the embryos regularly, fix the embryos in a methylcellulose culture dish, and track the fluorescent Yellow River carp oogonial stem cells under a fluorescence microscope.

[0200] The results are as follows Fig.13 As shown in the figure, 24 hours after fertilization, it can be observed that the cells with red fluorescence in the injected crucian carp embryo recipients are transplanted into the crucian carp embryos. 10 to 20 days after fertilization, it can be observed that the cells with fluorescence are colonized in the gonad-related parts.

[0201] Example 8

[0202] The present invention further uses fry stage crucian carp to analyze the induction differentiation ability of the 30th generation Yellow River carp oogonial stem cells in vivo, after staining the oogonial stem cells with PKH26, the cells are transplanted into dnd1-MO treated crucian carp fry 7 to 9 days after fertilization using microinjection technology.

[0203] 1. Preparation of Carassius auratus Receptor

[0204] (1) Select female fish with a swollen abdomen and obvious ovarian outlines. Their abdomen feels soft and elastic when you press it lightly, or they produce eggs when you press it lightly. Select male fish with milky white semen that oozes out when you press it lightly and that scatters when they enter the water.

[0205] (2) The oxytocin for female fish was a mixture of 4 μg / mL luteinizing hormone-releasing hormone A2, 1.5 mg / mL domperidone, and 150 mg / mL chorionic gonadotropin, with the solvent being glucose solution. The mixture was injected into the abdominal cavity at a 45° angle from the base of the pectoral fin of the crucian carp. Each fish was injected with 1 mL per kilogram. The oxytocin dose for male fish was halved.

[0206] (3) After the injection of the oxytocin, the next day, when artificial egg and semen collection is performed, the crucian carp is anesthetized with anesthetics, and the female fish's abdomen is squeezed to collect the fish eggs. When the fish eggs are collected, the male fish's abdomen is quickly squeezed to collect the semen.

[0207] (4) Place an appropriate amount of fish eggs in a glass culture dish, add a drop of semen, and gently stir with a feather to ensure full fertilization.

[0208] (5) Inject 50 μM dnd1-MO into crucian carp embryos at the zygote stage. Place the crucian carp embryos in a 28.5°C fish tank and culture them. Pick out dead eggs on the 2nd to 4th day, change the water, and wait until they reach the larval stage (7 to 9 days) for cell transplantation.

[0209] 2. PKH26 staining of donor cells

[0210] (1) The 30th generation of Yellow River carp oogonial stem cells with good growth status were selected. When the cell confluence reached 80%, they were digested with 1 mL PBS and 1 mL 0.25% trypsin-EDTA at room temperature for 5 min. 2 × 10 7 The cells were used for subsequent experiments.

[0211] (2) Add 3 mL of serum-free culture medium and centrifuge at 400 g for 5 min. Discard the supernatant and leave less than 25 μL of residual liquid. Add 1 mL of diluent C and pipette gently to mix to prepare a 2× cell suspension.

[0212] (3) Before staining, add 4 μL PKH26 ethanol solution to 1 mL diluent C and mix thoroughly to make 2× staining solution.

[0213] (4) Add the 2× cell suspension to the 2× staining solution, mix quickly, incubate at 26°C for 3 min, and gently invert the centrifuge tube to mix.

[0214] (5) Add 2 mL of FBS to terminate the staining reaction and incubate for 1 min.

[0215] (6) Centrifuge at 400 g for 5 min at 20-25°C, discard the supernatant, resuspend in 10 mL of fresh L-15 complete medium, transfer to a new centrifuge tube, centrifuge at 400 g for 5 min at 20-25°C, repeat 2-3 times. After discarding the supernatant for the last time, resuspend the cells in L-15 complete medium to a concentration of 5 × 10 4 ~1×10 5 The cell suspension of 100 cells / μL was used for subsequent injection.

[0216] 3. Microinjection

[0217] (1) Prepare microinjection transplantation needles. Use a needle puller to pull glass needles and grind them to a suitable caliber for transplantation into zebrafish embryos at the larval stage. Before injection, anesthetize the larvae.

[0218] (2) Under a stereo microscope, approximately 1×10 3 ~2×10 3 The Yellow River carp oogonial stem cells were transplanted into the fry by hydraulic injection.

[0219] (3) Observe the larvae regularly. Anesthetize the transplanted larvae and fix them in an agarose plate. Track the fluorescent oogonial stem cells under a fluorescence microscope.

[0220] The results are as follows Fig.14 As shown in the figure, on the second day after transplantation, red fluorescent cells can be observed in the injected larvae recipients colonized in the genital ridge of crucian carp. On the 13th day after transplantation, red fluorescent cells can be observed colonized in the gonad-related areas.

[0221] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for culturing carp oogonial stem cells in vitro, characterized in that: The following steps are involved: The ovaries taken from female carp are soaked in L-15 basal medium and placed in carp serum, the ovaries are cut into pieces and evenly spread in T25 cell culture bottles, and cultured at 26-30° C. in a CO2-free environment, and after culturing for 4-6 hours, L-15 complete medium is added to continue culturing to obtain primary carp oogonial stem cells cultured by adherent culture; The L-15 basal medium comprises the following components: L-15 powder, 5 mol / L HEPES buffer, 50 μmol / L β-mercaptoethanol, and sterilized water; The L-15 complete culture medium is based on the L-15 basic culture medium and also includes the following components: 10-20% by volume of fetal bovine serum, 2-20 ng / mL of basic fibroblast growth factor, 2-20 ng / mL of leukemia inhibitory factor, 0.5-2% by volume of carp serum, 0.5-2% by volume of zebrafish embryo extract, 0.5-2% by volume of L-glutamine, 600-1000 IU / mL of penicillin, 0.6-1 mg / mL of streptomycin and 1-3 μg / mL of amphotericin B.

2. The in vitro culture method according to claim 1, characterized in that: When the gonadal differentiation of female carp is not obvious, it is necessary to identify the sex of the selected carp.

3. The in vitro culture method according to claim 2, characterized in that: The gender identification includes identification by PCR amplification, wherein the primer pair used for PCR amplification includes CYC-male marker-F as shown in SEQ ID No.1 and CYC-male marker-R as shown in SEQ ID No.

2.

4. The adherent primary carp oogonial stem cell line obtained by culturing using the in vitro culture method according to any one of claims 1 to 3.

5. The method for subculturing the adherent primary carp oogonial stem cell line according to claim 4, characterized in that: The following steps are involved: (1) After the confluence of the adherent primary carp oogonial stem cell line reaches more than 80%, the L-15 complete medium is removed and the cells are washed with PBS; (2) After washing with PBS, digesting with a mixture of PBS and 0.25% trypsin-EDTA for 4 to 6 minutes, terminating the digestion with L-15 complete medium, aspirating the liquid and centrifuging at 1000 to 1500 rpm for 4 to 6 minutes, removing the supernatant, and adding L-15 complete medium to prepare a cell suspension; (3) The cell suspension collected in step (2) is distributed into a new T25 culture flask at a ratio of 1:(2-4), and after adding L-15 complete medium, culture and subculture are performed at 26-30°C in the absence of CO2.

6. The method for purifying the adherent primary carp oogonial stem cell line according to claim 4, characterized in that: The following steps are involved: The adherent primary carp oogonial stem cell line is passaged to the third generation using the passage method described in claim 5, and then subjected to continuous differential attachment to obtain purified carp oogonial stem cells.

7. The purification method according to claim 6, characterized in that: The continuous differential adhesion comprises 4 to 6 consecutive passages for differential adhesion culture.

8. The purification method according to claim 7, characterized in that: The continuous differential adhesion comprises digesting the third generation carp oogonial stem cells with a mixed solution of PBS and 0.25% trypsin-EDTA for 4 to 6 minutes, terminating the digestion with an L-15 complete culture medium, aspirating the liquid and centrifuging at 1000 to 1500 rpm for 4 to 6 minutes, removing the supernatant, adding the L-15 complete culture medium and mixing well, then inoculating the mixture into a culture dish, adhering to the wall for 30 to 50 minutes, aspirating the cell suspension and inoculating it into a new culture dish, and completing one differential adhesion culture.

9. A method for cryopreservation of the adherent primary carp oogonial stem cell line according to claim 4 or the carp oogonial stem cells purified by the method according to any one of claims 6 to 8, characterized in that: The freezing solution used for the freezing includes a mixture of L-15 complete culture medium, fetal bovine serum and DMSO, or a mixture of fetal bovine serum and DMSO.

10. Use of the adherent primary carp oogonial stem cells according to claim 4, the carp oogonial stem cells purified by the purification method according to any one of claims 6 to 8, or the carp oogonial stem cells frozen and revived by the freezing method according to claim 9 in cultivating new carp varieties.

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