A method for isolating spermatogonial stem cells from turbot

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

Active Publication Date: 2021-04-20
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, inbreeding is more serious, resulting in frequent diseases and reduced production.

Method used

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  • A method for isolating spermatogonial stem cells from 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 solution to wash, blow and mix with a pipette, centrifuge at 200g at 4°C for 5min, discard the supernatant, repeat twice;

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

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Abstract

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. The pretreated intact glands of 1-1.5-year-old turbot were washed with L-15 culture solution containing double-antibody (penicillin / streptomycin), digested with enzymes, and the digested solution was subjected to density gradient centrifugation to obtain high-purity and High density of spermatogonial stem cells. The invention provides a method for separating spermatogonial stem cells from seawater fish. The spermatogonial stem cells obtained by the method are complete in shape and structure, high in purity and high in density, and can be used for preservation of spermatogonial stem cells and research on stem cell transplantation. This method provides a new method and a new idea for the preservation of seawater fish germplasm resources and the cultivation of new species.

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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Patent Type & Authority Patents(China)
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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