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Paralichthys olivaceus muscle satellite cell line establishing method, specific primer for identifying paralichthys olivaceus muscle satellite cell marker gene and application of specific primer

A satellite cell and marker gene technology, applied in artificial cell constructs, animal cells, vertebrate cells, etc., can solve the uncertainty of muscle satellite cell survival and proliferation ability, and is not suitable for fish muscle satellite cell culture and operation. Cumbersome and other problems, to achieve the effect of easy operation, simple operation and simple operation

Pending Publication Date: 2015-12-30
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
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  • Description
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Problems solved by technology

[0003] So far, only non-fish muscle satellite cell isolation and culture methods such as rats, rabbits, pigs, and chickens have been reported, which are cumbersome to operate and easy to contaminate, and are not effective in maintaining the survival and proliferation of muscle satellite cells for a long time. There are uncertainties and are not suitable for fish muscle satellite cell culture

Method used

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  • Paralichthys olivaceus muscle satellite cell line establishing method, specific primer for identifying paralichthys olivaceus muscle satellite cell marker gene and application of specific primer
  • Paralichthys olivaceus muscle satellite cell line establishing method, specific primer for identifying paralichthys olivaceus muscle satellite cell marker gene and application of specific primer
  • Paralichthys olivaceus muscle satellite cell line establishing method, specific primer for identifying paralichthys olivaceus muscle satellite cell marker gene and application of specific primer

Examples

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Embodiment 1

[0028] The establishment method of flounder muscle satellite cell line, the steps are as follows:

[0029] 1) Preparation of cell culture medium: Take the MEM culture medium of Hyclone Company, add 20% of the total volume of fetal bovine serum to the culture medium, 10ng / mL human basic fibroblast growth factor, 100U / mL penicillin, 100μg / mL chain Mycin, pH 7.0-7.4, stored at 4°C, for later use.

[0030] 2) Primary culture: Aseptically take the skeletal muscle tissue near the tail of flounder with a weight of 120 g, and place it in a sterile glass plate with 4 mL of MEM culture solution from Hyclone Company added with 400 U / mL penicillin and 400 μg / mL streptomycin 3- After 5 minutes, suck out the culture medium, wash the muscle tissue twice with 1.0-1.2mL PBS (pH7.2), suck out the PBS, and then cut the muscle tissue into about 1mm 3 Add small pieces of the muscle tissue to 1 mL of the cell culture medium prepared in step 1) above to suspend the small pieces of muscle tissue, an...

Embodiment 2

[0033] Identification and Application of Flounder Muscle Satellite Cell Line

[0034] 1) Cryopreservation and recovery of cells

[0035] Freezing of cells:

[0036]Select the subcultured muscle cells that are in the exponential growth phase and the cell density reaches more than 90%, and digest them according to the conventional method. After digestion, the cell shrinkage is observed under the microscope, and then the trypsin digestion solution is discarded. Continue to observe under the microscope until the cells become round. , add 2 mL of fresh above-mentioned embodiment step 1) to prepare cell culture medium in the original bottle to prepare cell suspension. Transfer the cell suspension to a 15mL centrifuge tube, centrifuge at 2200rpm for 2min, and remove the supernatant. Suspend the cells with 2 mL of pre-cooled cryopreservation solution (i.e., MEM cell culture medium from Hyclone Company containing 10% dimethyl sulfoxide), and transfer to 2 mL cryopreservation tubes, p...

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Abstract

The invention relates to a marine fish cell culture technology, in particular to an aralichthys olivaceus muscle satellite cell line establishing method, a specific primer for identifying a paralichthys olivaceus muscle satellite cell marker gene and application of the specific primer. The aralichthys olivaceus muscle satellite cell line establishing method comprises the following steps: performing primary culture and subculture of aralichthys olivaceus muscular tissues, and then establishing a cell line; after establishing, performing freezing preservation, resuscitation and identification, wherein a cell culture fluid adopted to primary culture and subculture is obtained through adding fetal calf serum and a human basic fibroblast growth factor into the cell culture fluid. The built aralichthys olivaceus muscle satellite cell line is in the format of fusiform or spindle-shaped monocytes, allows continuous passage, can provide a great number of aralichthys olivaceus muscle satellite cells, and not only can be directly used for researches on aralichthys olivaceus muscle development related functional genes to provide rich cell sources for researches on the molecular mechanism of a fish muscle differentiation pathway, but also is expected to provide a research platform for researches and application of muscle character improvement during aralichthys olivaceus culture production.

Description

technical field [0001] The invention relates to seawater fish cell culture technology, in particular to a method for constructing a flounder muscle satellite cell line and specific primers for identifying a flounder muscle satellite cell marker gene and application thereof. Background technique [0002] Muscle satellite cells are a kind of myogenic stem cells originating from the embryonic mesoderm. Their activation, proliferation and differentiation are the important basis for the enlargement and number of skeletal muscle cells and the transformation of muscle fibers. Skeletal muscle is the main component of fish meat. The benefit of aquaculture comes from the rapid growth of fish skeletal muscle, so the growth and normal development of skeletal muscle is very important for the development of fish farming. The in vitro culture of muscle satellite cells is an important means and technology to understand the mechanism of proliferation, differentiation and regeneration of skel...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12Q1/68C12Q1/06C12Q1/04C12N15/11
Inventor 郑媛彭丽敏尤锋谭训刚焦爽
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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