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Construction method for Hexagrammos otakii cell line

A technology of the Otaki Hexaline fish and its construction method, which is applied in the direction of artificial cell constructs, animal cells, vertebrate cells, etc., and can solve the problems that the population of the wild Otaki Hexaline fish cannot meet the supply and the mariculture industry needs to be developed. , to achieve the effect of easy operation and simple process steps

Inactive Publication Date: 2012-06-13
DALIAN OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, the market demand for Otaki hexaline has been increasing day by day, and the population of wild Otaki hexaline is far from meeting the supply, and its mariculture industry needs to be developed

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] ⑴ Preparation of cell culture medium

[0016] Choose DMEM / F12 medium from Hyclone Company, add fetal bovine serum, human basic fibroblast growth factor, type I insulin-like growth factor and chondroitin sulfate to the medium, so that fetal bovine serum accounts for 20% of the total volume , the concentration of human basic fibroblast growth factor is 5ng / ml, the concentration of type I insulin-like growth factor is 40ng / ml, and the concentration of chondroitin sulfate is 20ug / ml;

[0017] Store at 4°C;

[0018] ⑵, primary culture

[0019] Take the fin tissue of Otaki six line fish on the ultra-clean workbench, and soak it in a mixture of penicillin and streptomycin. The concentration of penicillin is 100IU / ml, and the concentration of streptomycin is 100ug / ml. for 30 minutes; then rinse with PBS, cut into 0.8-1.2mm in a small amount of 5% FBS-DMEM / F12 (Ph7.2) 3 block, then use 0.5% hyaluronidase and 0.2% type II collagenase to jointly digest the block fin tissue for ...

Embodiment 2

[0024] ⑴ Preparation of cell culture medium

[0025] Choose the DMEM / F12 medium from Hyclone Company, and add fetal bovine serum, human basic fibroblast growth factor, type I insulin-like growth factor and chondroitin sulfate to the medium, so that the fetal bovine serum accounts for 20% of the total volume. , the concentration of human basic fibroblast growth factor is 5ng / ml, the concentration of type I insulin-like growth factor is 405ng / ml, and the concentration of chondroitin sulfate is 20ug / ml;

[0026] Store at 4°C;

[0027] ⑵, primary culture

[0028] Take the rostral tissue of Otaki six line fish on the ultra-clean workbench, and soak it in a mixture of penicillin and streptomycin. The concentration of penicillin is 100IU / ml, and the concentration of streptomycin is 100ug / ml. for 30 minutes; then rinse with PBS, cut into 0.8-1.2mm in a small amount of 5% FBS-DMEM / F12 (Ph7.2) 3 block, then use 0.5% hyaluronidase and 0.2% type II collagenase to jointly digest the blo...

Embodiment 3

[0033] ⑴ Preparation of cell culture medium

[0034] Choose the DMEM / F12 medium from Hyclone Company, and add fetal bovine serum, human basic fibroblast growth factor, type I insulin-like growth factor and chondroitin sulfate to the medium, so that the fetal bovine serum accounts for 20% of the total volume. , the concentration of human basic fibroblast growth factor is 5ng / ml, the concentration of type I insulin-like growth factor is 405ng / ml, and the concentration of chondroitin sulfate is 20ug / ml;

[0035] Store at 4°C;

[0036] ⑵, primary culture

[0037] Take the kidney tissue of Otaki six line fish on the ultra-clean workbench, rinse once with 5% FBS-DMEM / F12 (Ph7.2) and cut into 0.8-1.2mm 3 block; (Visceral tissue can not be treated with double antibody and joint digestion).

[0038] The above-mentioned tissue blocks were evenly inoculated on 25 cm 2 Add 3 ml of culture medium to the cell culture flask, and incubate at 25°C CO 2 Start the primary culture in the in...

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Abstract

The invention discloses a construction method for a Hexagrammos otakii cell line. The construction method comprises the following steps: 1, selecting a DMEM / F12 medium, adding a fetal calf serum, a human basic fibroblast growth factor, an I type insulin-like growth factor and chondroitin sulfate to prepare a cell culture solution, and storing the cell culture solution in a 4DEG C environment; 2, carrying out double resisting and rinsing processing on tissues of the Hexagrammos otakii on a super-clean workbench, and carrying out primary culture in a culture bottle; and 3, preparing a cell suspension after cells grow into a single layer, and adding the culture solution to subculture. The method of the invention has the advantages of simple step and easy operation; the morphology of the constructed tissue cell line of Hexagrammos otakii fins (HOFs), Hexagrammos otakii lips (HOLs) and Hexagrammos otakii kidneys (HOKs) likes a fiber, the serial passage of the cell line can be realized, and the Hexagrammos otakii cell line can be directly applied to pathogenic characteristic researches, vaccine developments and function gene researches, so needs for application researches of theoretical researches, virus vaccine developments and the like of Hexagrammos otakii are satisfied; and the construction method is also suitable for constructing cell lines of fins, lips and kidneys of other fishes.

Description

technical field [0001] The invention relates to a method for constructing a cell line, in particular to a method for constructing a cell line of Otaki hexaline fish. Background technique [0002] Otaki six line fish Hexagrammos otakii Also known as European-style six-line fish, commonly known as yellow croaker, it is a cold-temperate bottom fish in the coastal waters. It is distributed in the Yellow Sea, Bohai Sea, Japan, North Korea, and the Far East of Russia. Its meat is delicious and tender, and it has "northern grouper" known as. Otaki hexaline has strong adaptability, low temperature resistance, and the biological characteristics of early sexual maturity and fast generation alternation. It inhabits near offshore rocks and islands, and has a small range of migratory activities. Therefore, it is an ideal fish species for reef bay farming. In recent years, the market demand for Hexaline hexalis has been increasing day by day, and the population of Hexaline hexalis in t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
Inventor 李霞郭莹秦艳杰姜志强
Owner DALIAN OCEAN UNIV
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