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Establishing method of myosatellite cell system of bastard halibut during embryo period

The technology of muscle satellite cells and cell lines is applied in the field of establishing muscle satellite cell lines in the embryonic stage of flounder to achieve the effects of overcoming cell dysfunction, avoiding pollution sources and simple operation.

Active Publication Date: 2018-11-06
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

Method used

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  • Establishing method of myosatellite cell system of bastard halibut during embryo period
  • Establishing method of myosatellite cell system of bastard halibut during embryo period
  • Establishing method of myosatellite cell system of bastard halibut during embryo period

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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 marine fish embryo cell culture technology, in particular to an establishing method of a myosatellite cell system of bastard halibut during an embryonic period. The establishing method comprises the following steps: carrying out in-vitro separation on an embryo during a bastard halibut kirschner capsule formation period, removing an egg membrane, dispersing cells, and carrying out primary culture and secondary culture, thus establishing the myosatellite cell system during the embryonic period. The form of the myosatellite cell system, established by the invention, ofthe bastard halibut is fusiform or spindle-shaped mononuclear cells; myofiber can be differentiated to form by culturing for 5 days or above within a single generation or carrying out induction of anexogenous factor (horse serum). The myosatellite cell system is capable of supplying a large amount of myosatellite cells, and GFD (Green Fluorescent Protein) transfection and cynoglossus semilaevisspleen and kidney necrosis virus challenge assay detection verify that the myosatellite cells can be normally transfected or infected, so that the myosatellite cell system not only can be directly used for researching functional genes related to muscle growth and development of fishes and providing research materials for exploring a molecular mechanism for induction, proliferation and differentiation of muscle cells of the fishes, but also is capable of providing a platform for improved research and application of muscle characters in bastard halibut breeding.

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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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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