Separation and culture method for germ cells of gonadal tissue of tegillarca granosa

A technology of germ cells and gonads, which is applied in the field of separation and cultivation of gonad germ cells of clam gonads, to achieve the effect of improving disinfection and sterilization performance and less impurities in sperm or eggs

Active Publication Date: 2020-02-11
ZHEJIANG MARICULTURE RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no reports about the in vitro culture of gonad cells of cockles at home and abroad, and the in vitro culture methods of gonad cells of other shellfish are not suitable for in vitro culture of gonad cells of cockles. Therefore, the study of gonad cells of cockles in vitro The method of cultivation is crucial

Method used

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  • Separation and culture method for germ cells of gonadal tissue of tegillarca granosa
  • Separation and culture method for germ cells of gonadal tissue of tegillarca granosa
  • Separation and culture method for germ cells of gonadal tissue of tegillarca granosa

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Effect test

Embodiment 1

[0046] (1) Clean the surface of the cockles, remove sediment, microalgae and other attachments, temporarily raise them in sand-filtered seawater for 1 day, soak them in penicillin-streptomycin mixed solution 1× for 24 hours, and disinfect them for later use.

[0047] (2) Wipe the body surface of the clam with 70% alcohol cotton, open the shell, wash the tissue of the clam with a sterilized buffer solution, and place it on a sterile ultra-clean operating table. Use sterile ophthalmic scissors to open the gonad, and then use a 20ml sterile syringe (without needle) to draw the gonad tissue.

[0048] (3) Put the gonad tissues into petri dishes containing 10ml pre-cooled disinfection buffer for disinfection under ice bath conditions, replace the disinfection buffer after 20min, shake the culture dish slowly, and centrifuge to remove the disinfection buffer.

[0049] (4) Digest the gonad tissue with a mixture containing 0.05% trypsin and 0.2% collagenase at 23°C for 8 minutes, and g...

Embodiment 2

[0057] Example 2 Fluorescent expression of the functional gene Foxl2 related to gonad development after estrogen induction in gonad cells of clam gonads

[0058] Sperm cells and egg cells cultured in a biochemical incubator at 26°C for 4 days were replaced with the medium that had been added with 7β-estradiol to continue culturing for 48 hours. Experimental groups with multiple concentrations were set up. The concentrations of 7β-estradiol were 60 pg / ml, 300pg / ml, 600pg / ml, then use a sterile Pasteur pipette to blow and absorb the cells, transfer them to a 15ml sterile centrifuge tube, remove the medium after centrifugation, extract RNA, and use real-time quantitative PCR to analyze the Foxl2 gene expression level and Estrogen-induced effects on the expression of this gene. The result is as figure 2 It was shown that the expression level of Foxl2 gene in egg cells was 13 times higher than that in male gonads, indicating that the gene was expressed in both sperm and eggs, and...

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Abstract

The invention discloses a separation and culture method for germ cells of gonadal tissue of tegillarca granosa. The separation and culture method comprises the following steps: firstly, sucking gonadal tissue of tegillarca granosa by a sterile injector under a sterile condition, carrying out disinfection, performing digestive enzymolysis on the gonadal tissue at 23 DEG C with a mixed solution containing 0.05% of pancreatin and 0.2% of collagenase, collecting a cell suspension, carrying out centrifugation, removing a supernatant, suspending precipitated cells with a disinfection buffer solution, controlling the horizontal centrifugation speed at 2600 rpm/m when taking sperm cells from male gonadal tissue, controlling the horizontal centrifugation speed at 1600 rpm/m when taking egg cells from female gonadal tissue, taking the cell suspension, paving a 6-hole plate, carrying inverted adherent culture in a biochemical culture box at 26 DEG C for 24 h, then setting the plate right, adding1 mL of culture medium to each hole, then, replacing a half of culture medium every 2 d, carrying out constant-temperature standing culture for 5-10 d, and performing primary culture. The separation and culture method selects culture conditions of the gonadal tissue of the tegillarca granosa through a large quantity of basic experiments, so that gonadal cells can be separated, purified, cultured and survive in vitro.

Description

technical field [0001] The invention relates to the technical field of shellfish culture, in particular to a method for separating and culturing germ cells of gonad tissue of clams. Background technique [0002] Mud cockles belong to the phylum Molluscs, class Bivalvia, order Dendonta, family Cockles, and the genus Cockles. Chinese traditional cultured shellfish. It is distributed all over the coast of China, and the spawning period from August to October is the peak production season. In addition, artificial breeding is carried out in Hebei, Shandong, Zhejiang, Fujian, and Guangdong, and the output is quite abundant. Clam meat is delicious and can be eaten fresh or soaked in wine, or dried. Clam meat contains a lot of protein and vitamins. The blood of cockles is bright red, and there is a golden-like color line on the edge of the meat. [0003] The gonad tissue of cockles is easy to observe and operate, and it is a good material for micromanipulation and reproductive de...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/07
CPCC12N5/0601C12N2500/12C12N2500/14C12N2500/16C12N2509/00
Inventor 滕爽爽肖国强柴雪良方军邵艳卿任鹏
Owner ZHEJIANG MARICULTURE RES INST
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