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Method for separating and transplanting sebastes schlegelii spermatogonial stem cells

A spermatogonial stem cell and dissociation technology, which is applied in the field of separation of spermatogonial stem cells of marine fish, can solve the problems of frequent diseases, serious inbreeding, and yield decline, and achieves good repeatability, simple transplantation method, and little damage to larvae. Effect

Inactive Publication Date: 2020-03-31
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

Xu's flat scorpion belongs to the Scorpidae family and the genus Flat Scorpion. It is an oviparous fish. Due to the long-term breeding in China, inbreeding is more serious, resulting in frequent diseases and reduced production.

Method used

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  • Method for separating and transplanting sebastes schlegelii spermatogonial stem cells
  • Method for separating and transplanting sebastes schlegelii spermatogonial stem cells

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Experimental program
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Embodiment

[0020] 1. The process of isolating spermatogonial stem cells:

[0021] 1) Take 300-400g gonads of the 5-month-old-1-year-old male fish, P. xushii, put them into a centrifuge tube, and cut them into pulp;

[0022] 2) Prepare 500mL of digestion solution, 25mL of FBS, 0.02g of collagenase H, 0.0165 of neutralase II, and 475mL of L-15. Add 250mL of digestive solution to the above-mentioned crushed gonads, mix and incubate at 25°C for 2.5-3h, and gently blow the cells with a pipette to disperse during the incubation;

[0023] 3) Filter the obtained cell dispersion with a 42um pore size nylon sieve, collect the filtrate, add 1mL LL-15 medium to the filtrate for washing, centrifuge at 200g for 5min, and discard the supernatant;

[0024] 4) Add 200uL L-15 medium to the above centrifuged pellet to resuspend the cells, and then stain with trypan blue to observe the state of the cells (see figure 1 ),Depend on figure 1 Observation shows that the cell structure is clear and free of imp...

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Abstract

The invention relates to a method for separating and transplanting live-bearer spermatogonial stem cells, in particular to a method for separating and transplanting sebastes schlegelii spermatogonialstem cells. The method comprises the following steps: digesting and centrifuging the complete gonad of male sebastes schlegeli at the age of 5 months to 1 month or at the early gonad development stageby using a digestive enzyme to obtain spermatogonial stem cells; and then transplanting the spermatogonial stem cells to the abdominal cavity of the fry through microinjection. According to the method for separating and transplanting live-bearer spermatogonial stem cells, the spermatogonial stem cells separated and transplanted by the method are subjected to fluorescence labeling and thus the migration and proliferation processesof donor germ cells can be observed in receptor fish. Therefore, a novel method is provided for preservation and protection of marine fish germplasm resources and cultivation of new varieties.

Description

technical field [0001] The invention relates to the separation of spermatogonial stem cells of seawater fish, in particular to a method for separating and transplanting the spermatogonial stem cells of ovoviviparous fish Scorpius scorpionoides. Background technique [0002] Xu's flat scorpionfish is a marine fish with high economic value in northern my country. Its meat is tender and nutritious, and it is very popular among consumers. Xu's flat scorpion belongs to the Scorpidae family and the genus Flat Scorpion. It is an oviparous fish. Due to the long-generation breeding in China, inbreeding is more serious, resulting in frequent diseases and reduced production. Spermatogonial stem cells in the testis belong to reproductive stem cells, which can be self-renewal and differentiated totipotent, and can differentiate to form germ cells at various stages until mature gametes. Japanese scholars have successfully transplanted spermatogonial stem cells in rainbow trout and succes...

Claims

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Application Information

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IPC IPC(8): C12N5/076A01K67/027
CPCA01K67/0271A01K2207/12A01K2227/40C12N5/061C12N2509/00
Inventor 刘清华李军王学颖周莉
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI