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In-vitro culture, identification and induced differentiation method of sebastes schlegeli myoblasts

A technique for myoblasts and in vitro culture, applied in the field of cell culture

Active Publication Date: 2020-05-15
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with other vertebrates, there is no relevant report on the culture and induction of myoblasts in seawater fish

Method used

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  • In-vitro culture, identification and induced differentiation method of sebastes schlegeli myoblasts
  • In-vitro culture, identification and induced differentiation method of sebastes schlegeli myoblasts
  • In-vitro culture, identification and induced differentiation method of sebastes schlegeli myoblasts

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The primary culture of the muscle cells of embodiment 1, Xu's flat scorpionfish, concrete method is as follows:

[0039] The tertiary antibody (BI, no.PB180424) is a mixed antibiotic composed of 10000units / mL penicillin, 10mg / mL streptomycin, and 1250units / mL nystatin; 1% of the tertiary antibody means adding 1mL of the tertiary antibody to 99mL of medium. FGFb (Solarbio, no. P00032). Fetal bovine serum (BI, no.04-001-1A). DMEM / F12 (BI, no. 01-172-1ACS). L-15 (Gibco, no. 11415-064). 0.25% trypsin (Gibco, no. 12605-028). Horse serum (BI, no. 04-004-1B).

[0040] 1) Sampling and disinfection of muscle tissue blocks of Scorpius xushii: ①Select juvenile scorpionfish xushii that had just died at the age of 3 to 6 months, and sample the muscle tissue pieces. ②Before sampling, disinfect the surface of the fish with 75% alcohol, wipe the alcohol with toilet paper, and repeat three times to remove bacteria on the surface of the fish as much as possible. ③ When sampling, fi...

Embodiment 2

[0045] The identification method of the subcultured cell species described in Example 1, the present embodiment takes the 30th generation subcultured cells, and the specific method is as follows: 1. The identification of the cell species: by amplifying the 18S rRNA gene of the muscle cell line and the scorpionfish Xu's Compare the homology of 18S rRNA gene in muscle tissue to determine the source of the cell line, design specific primers for 18S rRNA gene, and use the phenol-chloroform method to extract the total DNA of SSCM cells and the total DNA of the muscle tissue of Scorpius xushii. The PCR system is 2.5uL Taq Enzyme buffer (10X), 2uL dNTPs (10mol / L), 0.5uL each primer (10umol / L), 1uL DNA template, 0.2uL taq enzyme, 18.3uL water, the total system is 25uL, after mixing, carry out PCR amplification, amplification The growth conditions were pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 59°C for 30 seconds, extension at 72°C for 1 m...

Embodiment 3

[0059] The method for inducing differentiation of subcultured cells in Example 1: ① 0-24h: set the time for inoculating cells to 0h, and 0-24h is the growth medium, and the growth medium is L-15 medium containing 15% FBS , the cells grow normally at 24h; 24-48h: the growth medium is replaced with a differentiation medium at 24 hours, and the differentiation medium is L-15 medium containing 2% DHS, and cells begin to fuse at 48h; 48-72h : The differentiation medium continued to be cultured, and a large number of cells fused at 72 hours; 72-96h: The differentiation medium was continued to be cultured, and the cell fusion was completed at 96 hours, and almost all of them were multinucleated myotubes; ② For cells cultured in growth medium and differentiation medium Take pictures every 24 hours to record the process from mononuclear muscle cells to fusion into multinucleated muscle fibers; ③ use Hoechst and phalloidin to stain the nucleus and cytoplasm of the cells at 96 hours respe...

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Abstract

The invention relates to an in-vitro culture, identification and induced differentiation method of sebastes schlegeli myoblasts, and belongs to the field of molecular biology. The cultured sebastes schlegeli myoblast can be subjected to subculture, and is used for basic research on the muscle growth of marine scleroderma; the induced differentiation of myoblasts in vitro can be effectively carriedout, and a basis is provided for the molecular mechanism research of fusing monocytes into polycytes in vivo.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and in particular relates to a method for culturing, identifying and inducing differentiation in vitro of scorpionfish myoblasts. Background technique [0002] Xu's flat scorpionfish (Sebastes schlegelii), commonly known as black scorpion, black head, etc., is an important marine economic fish and an excellent species for breeding. It is mainly distributed in the Bohai Sea, the Yellow Sea and the East China Sea. Xu's flat scorpionfish has a wide range of living temperatures and can survive the winter in deep-water sea cages. It is one of the few species suitable for long-term farming in deep-water cages in the north. It has been widely cultured in deep-water cages along the northern coast, but its growth rate is slow. , usually takes 2-3 years to reach commodity specifications. Growth is one of the most valuable economic traits for genetic improvement of Scorpius xushii. The muscle of fish...

Claims

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Application Information

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IPC IPC(8): C12N5/077C12Q1/6869
CPCC12N5/0658C12N2509/10C12N2509/00C12N2501/115
Inventor 贺艳齐洁孔祥福张全启
Owner OCEAN UNIV OF CHINA
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