Method for separating spermatogonial stem cells of turbot

A spermatogonial stem cell and separation method technology, which is applied in the separation field of turbot spermatogonial stem cells, can solve the problems of decline, serious inbreeding, frequent disease and yield, etc., and achieves the effects of high purity, high density, and complete morphological structure.

Active Publication Date: 2018-04-17
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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Problems solved by technology

Turbot is an imported species. Due to long-term breeding in China, inbreed

Method used

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  • Method for separating spermatogonial stem cells of turbot

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Embodiment 1

[0019] (1) Collect 2-3 tails of turbot weighing 300-500g at the age of about 1, separate the gonads of the male fish substage in the live body, and put them into 100ml of pre-cooled 0.1M PBS;

[0020] (2) Cut the gonads into about 0.5g portions on crushed ice, and cut them into pulp;

[0021] (3) Add 1 mL of L-15 culture medium containing 1% double-antibody (penicillin / streptomycin) to wash, centrifuge at 200 g for 5 min at 4°C, discard the supernatant, and repeat twice;

[0022] (4) Put into L-15 culture solution containing 1mL 0.2% trypsin, 5% fetal bovine serum, and 0.05% NaseI and incubate at 25°C for 2 hours, and gently blow and beat with a pipette to disperse the gonad cells;

[0023] (5) filter with a nylon mesh with a pore size of 42 μm, remove undissociated cell masses, and collect the filtrate;

[0024] (6) Add 3mL-15 culture medium to wash, blow and mix with a pipette, centrifuge at 200g at 4°C for 5min, discard the supernatant, repeat twice;

[0025] (7) Add 1mL ...

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Abstract

The invention relates to a method for separating spermatogonial stem cells of seawater fishes, in particular to a method for separating spermatogonial stem cells of turbot. The method comprises the steps as follows: pretreated complete gonads of the turbot which is 1-1.5 years old are washed with an L-15 medium containing double antibodies (penicillin/streptomycin) and digested by enzymes, digested digestive fluid is subjected to density gradient centrifugation, and accordingly, the spermatogonial stem cells with high purity and high density are obtained. According to the method for separatingthe spermatogonial stem cells of the seawater fishes, the spermatogonial stem cells separated with the method have complete morphological structure, high purity and large density, and can be used forpreservation of the spermatogonial stem cells and transplantation research of the stem cells. According to the method, a new method and a new idea are provided for preservation of marine fish germplasm resources and cultivation of new varieties.

Description

technical field [0001] The invention relates to a method for separating spermatogonial stem cells of seawater fish, in particular to a method for separating spermatogonial stem cells of turbot. Background technique [0002] Turbot is a marine fish with high economic value in my country. Its meat is tender and nutritious, and it is very popular among consumers. Turbot is an imported species. Due to long-term breeding in China, inbreeding is more serious, resulting in frequent disease and production decline. 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 successfully obtained offspring. If the spermatogonial stem cells of the fish are separated and preserved, they can be used to obtain donor eggs or sperm through a su...

Claims

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Application Information

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IPC IPC(8): C12N5/076
CPCC12N5/061C12N2509/00
Inventor 刘清华李军王文琪杨敬昆徐世宏王学颖
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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