Method for establishing and identifying cynoglossus semilaevis testis cell line

A semi-smooth tongue sole and identification method technology, applied in artificial cell constructs, microbial-based methods, animal cells, etc., can solve problems such as no reports of semi-smooth tongue sole gonad cell lines, limit research on sex-related gene functions of semi-smooth tongue sole, and achieve chromosome identification methods Intuitive, accurate and reliable molecular marker identification method, appropriate effect of growth and development stage

Inactive Publication Date: 2012-01-04
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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Problems solved by technology

However, the lack of in vitro cultured cell lines derived from the gonads of tongue sole greatly limits the functional studies of sex-related genes in tongue sole, the discovery of new sex-determining genes, the confirmation of sex expression regulatory networks, the relationship between sex genes and sex genes and growth Gene Interrelationships Revealed
So far, although embryonic cell lines, liver cell lines and cardiomyocyte cell lines have been established in tongue sole, there is still no report on the gonad cell line of tongue sole

Method used

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  • Method for establishing and identifying cynoglossus semilaevis testis cell line
  • Method for establishing and identifying cynoglossus semilaevis testis cell line
  • Method for establishing and identifying cynoglossus semilaevis testis cell line

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Embodiment 1

[0032] 1. The construction of the testicular cell line of tongue sole of semi-smooth tongue, the steps are as follows, including: preparation of complete medium for primary cells, primary culture and subculture of testicular cells.

[0033] (1) Preparation of complete cell medium: Dissolve the commercially available GIBCO standard MEM medium into 1000ml, then add 2.38g of Hepes buffer, fully dissolve, stir and mix well, adjust the pH to 7.2-7.5, filter and sterilize, use as Basal medium, stored at 4°C; before use, add 15%-20% of the total volume of fetal calf serum, 0.1% of the total volume of β-mercaptoethanol, 2ng / ml human basic fibroblasts to the basal medium before use factor, 1ng / ml leukemia inhibitory factor, 1nmol sodium pyruvate and 1nmol glutamine, prepared into a complete medium;

[0034] (2) Primary culture of testicular cells: select 2-year-old healthy male half-smooth tongue sole weighing about 250 g (adult fish have a high degree of gonad differentiation, weak ce...

Embodiment 2

[0042] Characterization of cell growth performance:

[0043] In order to monitor the growth of testicular cell line of tongue sole and determine the optimal growth condition of testicular cell line of tongue sole, the 30th passage cells were inoculated in seven 12-well plates, 6 wells per plate, 2×10 per well. 5 Three 12-well plates were cultured in incubators at 10°C, 20°C, 24°C and 30°C, respectively. The other four were cultured at the same 24°C but in complete medium supplemented with 5%, 15%, 20% and 25% fetal bovine serum, respectively. On the 1st, 2nd, 3rd, and 4th days after inoculation, any 3 wells of each 12-well plate were digested with 0.25% trypsin, the cells were suspended, and counted on a cell counting plate. Draw a growth curve, such as figure 2 , 3 shown. It shows that the testis cell line of tongue sole grows and proliferates faster at 24°C and 30°C than at 10°C and 20°C; the growth and proliferation speed in the medium containing 20% ​​fetal bovine ser...

Embodiment 3

[0045] Identification of the source of gonad cell lines in tongue sole females using specific molecular markers.

[0046] Take a bottle (25cm 2 ), DNA was extracted by phenol-chloroform method; CSGC cells were identified by female-specific marker CseF382 (DQ487760). The female-specific fragment CseF382 primers (CseF382N1: 5'-ATTCACTGACCCCCTGAGAGC-3'; CseF382C1: 5'-AACAACTCACACA CGACAAATG-3') have been proven to amplify a specific fragment of 350bp in the genome of the female half-smooth tongue sole, while in the male half-smooth tongue sole There are no specific bands. PCR amplification was carried out using the DNA of the male, female, and CSGC cells of the sex-determined half-smooth tongue sole as templates. The PCR conditions were as follows: 25 μl of the amplification system contained 2 μl of 10×PCR buffer, 0.5 μl of Taq DNA polymerase ( 5 U / μl), 0.5 μl each of primers (10 μM), 1 μl dNTP (2.5 mM), 1 μl template DNA (0.5 mM), and 19.5 μl sterilized ultrapure water. PCR p...

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Abstract

The invention relates to a method for establishing and identifying a cynoglossus semilaevis testis cell line, which comprises the following steps: adopting a MEM (Minimum Essential Medium), adding 20 percent of fetal calf serum, 0.1 percent of B-mercaptoethanol, 2 ng / ml of human alkaline fibroblastic growth factors, 1 ng / ml of leukemia inhibiting factors, 1 nmol of sodium pyrurate and 1 nmol of glutamine and preparing into a complete medium; inoculating a tissue block into a culture bottle, overturning and inversely placing the bottle to culture for 3 h and positively placing the culture bottle after the tissue block is attached onto a wall, so that the tissue block is soaked into the culture medium for culture. A trypsin digestion method is adopted for subculturing cells. One cynoglossus semilaevis testis cell line is established by adopting the above method and is subcultured to fiftieth generations. Meanwhile, the invention also provides a method for identifying a chromosome of the established cell line and the level of a molecular marker and a method for identifying the sensitivity of the cell line to related viruses. The method for establishing the cynoglossus semilaevis testis cell line can be used for culturing other fish gland cells and establishing the cell line, thereby having wider promotion and application prospect.

Description

Technical field: [0001] The invention belongs to marine biological cell culture technology, and relates to a method for culturing gonad cells of tongue sole semismoothus and constructing and identifying cell lines. Background technique: [0002] Semi-smooth tongue sole (Cynoglossus semilaevis) is an economic marine fish in the northern coast of my country, with an annual output value of more than 1 billion yuan. However, there are obvious growth differences between male and female individuals of semi-smooth tongue sole, and the growth rate of female fish is 2-4 times that of male fish. The bottleneck problem of slow growth of male fish needs to be solved urgently in the aquaculture industry. [0003] Genetically, the female of S. half-smooth tongue sole has a heteromorphic W chromosome and belongs to the ZW / ZZ sex determination type, which is relatively rare in fish. Analyzing the sex determination mechanism of tongue sole will not only help to reveal the nature of the grow...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12Q1/04C12Q1/68C12Q1/02C12Q1/70C12R1/91
Inventor 陈松林张博沙珍霞王贤丽王娜杨长庚刘珊珊
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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