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A method for establishing a satellite cell line of flounder embryonic muscle

A technology of muscle satellite cells and cell lines, which is applied in the field of establishment of muscle satellite cell lines in the embryonic stage of flounder, to achieve the effects of avoiding pollution sources, increasing repeatability, and avoiding the possibility of pollution

Active Publication Date: 2022-03-04
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the establishment of embryonic cell lines at the embryonic body stage of fish has not been reported yet.
Regarding muscle muscle satellite cell lines, it has been reported that they are also isolated from juvenile or adult muscle tissue, such as rainbow trout (Jean et al., 2010) and flounder (Peng et al., 2016), that is, from fish embryos Muscle satellite cells isolated from

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  • A method for establishing a satellite cell line of flounder embryonic muscle
  • A method for establishing a satellite cell line of flounder embryonic muscle
  • A method for establishing a satellite cell line of flounder embryonic muscle

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037]The establishment method of flounder embryonic muscle satellite cell line, the steps are as follows:

[0038] 1) Prepare complete cell culture medium

[0039] DF12 complete medium contains 20% fetal bovine serum, 10ng / mL human basic fibroblast growth factor, 15ng / mL epidermal growth factor, 100U / mL penicillin, 100μg / mL streptomycin, 100μmol / L NEAA (non-essential amino acid ), the pH value is 7.2-7.4, and it is stored at 4°C for later use.

[0040] 2) Primary culture

[0041] Observing the fertilized eggs of flounder under a stereoscope, take about 50 floating embryos of normally developed Kelvin's bursa formation stage and place them in a petri dish, wash them with sterilized seawater 5 times, 2 minutes each time. Then transfer to another new sterilized petri dish, rinse with sterilized sea water 3 times, and then wash 3 times with PBS containing double antibodies (400 U / mL penicillin, 400 μg / mL streptomycin). Gently crush the outer membrane of the embryo with a steri...

Embodiment 2

[0045] Identification of flounder muscle satellite cell lines: In this example, the source species, cell lineage, growth and genetic stability were identified according to the requirements of cell line identification.

[0046] 1) Cryopreservation and recovery of cells

[0047] Freezing of cells:

[0048] Select the subcultured cells in the above-mentioned embodiment that are in the exponential growth phase and have a cell confluency of more than 80%, and digest them according to conventional methods. Observe under an inverted microscope. When the cells shrink and become about 70% round, discard the trypsin, add 2 mL of complete medium to stop digestion, blow the cells from the bottom of the bottle with a pipette tip, and distribute the cells in a single cell suspension; Transfer the cell suspension to a cryopreservation tube, centrifuge at 2200rpm for 2min; discard the medium, add 2mL of pre-frozen stock solution (serum containing 10% dimethyl sulfoxide) to suspend the cells,...

Embodiment 3

[0061] Application of flounder muscle satellite cell line: In this example, the induced differentiation of this cell line was analyzed and identified for the first time, so as to clarify the characteristics of its muscle satellite cell. At the same time, the spleen and kidney necrosis virus infection experiment of tongue sole of seawater fish was also carried out, which provided materials and basic parameters for the cell line in the research and application of fish virus isolation, identification and vaccine preparation.

[0062] 1) Identification of muscle satellite cell differentiation

[0063] In order to verify that the obtained cell line is a muscle satellite cell line, the marker protein (myosin) expressed in muscle satellite cell differentiation was analyzed. Take the 31st passage cells of this cell line, digest them with 0.25% trypsin, suspend them with 2 mL of complete cell culture medium, and inoculate them in 25 cm 2 In a culture bottle, culture in an incubator at...

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Abstract

The invention relates to a seawater fish embryonic cell culture technology, in particular to a method for establishing a muscle satellite cell line in the embryonic stage of flounder. The embryos of the flounder bursa formation stage were separated in vitro, the egg membrane was removed, the cells were dispersed, and the embryonic muscle satellite cell line was established after primary culture and subculture. The morphology of the flounder embryo muscle satellite cell line established in the present invention is spindle-shaped or spindle-shaped mononuclear cells. In a single generation, if it is cultured for more than 5 days or induced by exogenous factors (horse serum), it can differentiate and form muscle fibers. The cell line can provide a large number of muscle satellite cells, which can be normally transfected or infected by GFP transfection and semi-smooth tongue sole spleen-kidney necrosis virus challenge experiments. Therefore, it can not only be directly used in the research of functional genes related to fish muscle growth and development, provide research materials for exploring the molecular mechanism of induction, proliferation and differentiation of muscle cells, but also provide research materials for the improvement and application of muscle traits in flounder breeding. platform.

Description

technical field [0001] The invention relates to a seawater fish embryonic cell culture technology, in particular to a method for establishing a muscle satellite cell line in the embryonic stage of flounder. Background technique [0002] Animal cell lines and cell culture have become indispensable tools and carriers for studies in physiology, immunology, virology, developmental biology, gene function, and toxicology. The continuous development of the fish farming industry requires research on fish growth and development, disease outbreaks, etc. The development of fish toxicology research is inseparable from the fish cell line platform, so its cultivation has attracted much attention. Since the first fish cell line was established in 1962, more and more fish cell lines have been successfully established and applied. Embryo cells are different from adult tissues in that they have a low degree of differentiation, strong division ability, high diversity, and a wider range of ap...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/077
CPCC12N5/0659C12N2509/00C12N2501/11C12N2501/115C12N2500/32C12N2500/44
Inventor 聂苗苗谭训刚尤锋吴志昊焦爽肖鹏娄雅楠梁冬冬
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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