A method for constructing a flounder testis cell line

A construction method and cell line technology, applied in the field of seawater fish cell culture, can solve problems such as the establishment of cell lines with no gonad tissue

Inactive Publication Date: 2017-11-10
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But so far, there are only flounder fin ray cell lines (Ocean University of China, Tong Shangliang, Aquaculture, 1997,156:327-333) and embryonic cell lines (Yellow Sea Fisheries Research Institute of China Fisheries Research Institute, Chen Songlin, Diseases of Aquatic Organisms, 2004, 60: 241-246), kidney cell line (Yellow Sea Fisheries Research Institute of China Fisheries Research Institute, Wang Na, Journal of Fish Diseases, 2011, 34: 81-85) and the laboratory established by the applicant There are 5 cell lines including the brain cell line and the muscle satellite cell line, but there is no report on the establishment of the gonad tissue cell line of this fish species

Method used

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  • A method for constructing a flounder testis cell line
  • A method for constructing a flounder testis cell line
  • A method for constructing a flounder testis cell line

Examples

Experimental program
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Effect test

Embodiment 1

[0024] The establishment method of flounder testis cell line, the steps are as follows:

[0025] 1) Preparation of cell culture medium: take Hyclone company DF12 culture medium, add fetal bovine serum accounting for 20% of the total volume of cell culture medium to the culture medium, 10ng / ml recombinant human basic fibroblast growth factor (Recombinant HumanFGFb, bFGF) , 15ng / ml recombinant human epidermal growth factor (Recombinant Human EGF, EGF), 100U / mL penicillin, 100μg / mL streptomycin, pH value is 7.2, stored at 4°C for later use.

[0026] 2) Primary culture: under aseptic conditions, the testis tissue of healthy flounder with a body weight of 347.5 g was taken, and placed in a sterile glass plate with 4 mL of Hyclone DF12 culture solution supplemented with 400 U / mL penicillin and 400 μg / mL streptomycin After soaking for 5 minutes, discard the culture medium, rinse the testis tissue twice with 2mL PBS (pH 7.2), suck out the PBS, cut the testis tissue into minced pieces,...

Embodiment 2

[0029] Identification and application of testis cell line of flounder

[0030] 1) Cryopreservation and recovery of cells

[0031] Freezing of cells:

[0032] Select the above-mentioned subcultured testicular cells in the exponential growth phase with a cell density of more than 90% in different generations, and digest them according to the conventional method. After digestion, the cells shrink under the microscope and discard the trypsin solution. Completely, add 2 mL of fresh cell culture medium prepared in step 1) of the above-mentioned embodiment to the original bottle to prepare cell suspension. Transfer the cell suspension to a 15mL centrifuge tube, centrifuge at 2200g for 3min, discard the supernatant. Suspend the cells with 2 mL of pre-cooled cryopreservation solution (Hyclone DF12 cell culture medium containing 10% dimethyl sulfoxide), and transfer to 2 mL cryopreservation tubes, put the cryopreservation tubes into the programmed cooling box for 4 ℃ for 30 minutes, ...

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Abstract

The invention relates to a seawater fish cell culture technology, in particular to a method for constructing a flounder testis cell line. The method of trypsinization of tissue blocks is used for the primary culture of testis tissue, and in the subculture, nutrient-rich medium and mild trypsinization are used to subculture, and then the cell line is constructed. The cell morphology of the established flounder testis cell line was fibroblast-like, and the cell line could provide a large number of testis cells. Not only can it be directly used in the study of functional genes related to sex differentiation of flounder, but it is also expected to become a platform for theoretical research at the molecular and cellular level of flounder. The construction method of the flounder testis cell line of the present invention can be applied to the in vitro culture of testis cells of other fishes.

Description

technical field [0001] The invention relates to a seawater fish cell culture technology, in particular to a method for constructing a flounder testis cell line. Background technique [0002] The construction and cultivation of animal cell lines has long been a powerful tool for research in endocrinology, toxicology, genetics, virology, immunology, and tumor therapy. Up to now, nearly 280 different fish cell lines have been established one after another, while there are only about 100 cell lines of seawater fish and saltwater fish, which are far from satisfying their needs in physiology, virology, toxicology, Application requirements in various fields such as tumor and genetic engineering. [0003] Flounder (Paralichthys olivaceus) belongs to Osteophytes, Subclass Radix, Pleuriformes, Suborder Flounder, Flounderidae, and the genus Flounder. Its meat is fresh and tender, and it is one of the important seawater cultured fish. As with most flatfish, the fact that females are ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071C12R1/91
Inventor 彭丽敏郑媛尤锋吴志昊邹玉霞
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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