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Pomfret muscle tissue cell line

A technology of muscle tissue and cell lines, applied in the direction of bone/connective tissue cells, animal cells, tissue culture, etc., can solve the problems of occasional difficulties in cell lines, and achieve the effect of tight arrangement of cells and short passage time

Pending Publication Date: 2021-11-12
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the primary culture technology of fish cells is currently a relatively mature technology, there are still great chances and difficulties in obtaining cell lines that are stable and suitable for the construction of experimental models.

Method used

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  • Pomfret muscle tissue cell line
  • Pomfret muscle tissue cell line
  • Pomfret muscle tissue cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Construction of Cell Lines

[0029] The silver pomfrets used in the present invention all come from Xiangshan Gangwan Aquatic Seed Co., Ltd., Ningbo, Zhejiang. The cultivation temperature is 16-32°C, salinity 26-30, pH: 8.21±0.3, dissolved oxygen: 7.35±0.05mg / L. Healthy, vigorous growth, weight 50g.

[0030] Solution preparation

[0031] D-hanks solution: 50ml D-hanks solution + 1ml 100X penicillin-streptomycin double antibody solution (purchased from Gibco), stored at 4°C.

[0032] Basal medium: 49ml L15 medium (Leibovitz’s L15 medium, purchased from Gibco) + 1ml 100X penicillin-streptomycin double antibody solution (purchased from Gibco), stored at 4°C.

[0033] Complete medium: 10ml fetal bovine serum (fetal bovine serum, FBS, purchased from Gibco) + 0.5ml 100X penicillin-streptomycin double antibody solution and 39.5ml L15 medium (Leibovitz's L15 medium, purchased from Gibco), 4 Store at ℃.

[0034] Cell freezing medium: 5ml DMSO (purchased from Invi...

Embodiment 2

[0041] Example 2: Cryopreservation and recovery of silver pomfret muscle cells PaM

[0042] 1) Cryopreservation: Take a bottle of silver pomfret muscle cell line cells in logarithmic growth phase, adopt the classic trypsin digestion method, digest with 2 ml of 0.25% trypsin, and then centrifuge to collect the cell pellet. Gently pipet the cell pellet with 1 ml of the prepared cell freezing solution to make it evenly dispersed in the freezing solution. The cell line cryopreservation followed the gradient cooling principle, and the cryopreservation solution was prepared as 90% FBS+10% DMSO. The cryopreservation tube is programmed to cool down in a gradient cooling box filled with isopropanol, and placed at -80 °C for 24 hours. Isopropanol can ensure that the cells are gradually cooled down in the freezing solution, and finally placed in liquid nitrogen for long-term cryopreservation.

[0043] 2) Recovery: Cell recovery follows the principle of rapid fusion. Take the cryovial o...

Embodiment 3

[0045] Example 3: Karyotype analysis of silver pomfret muscle cells PaM

[0046] 1) Cytogenetic analysis of PaM cells was performed at passage 20. The muscle cells of silver pomfret in the logarithmic growth phase were taken, and the cells were digested with colchicine with a final concentration of 10 μg / ml for 5 h to obtain a cell suspension.

[0047] 2) Centrifuge the collected cell suspension at 1000 rpm for 10 min, gently aspirate and discard the supernatant, add 3 mL of preheated (37°C water bath in advance) hypotonic solution dropwise, and distribute the cells evenly in the suspension. Add 5ml of hypotonic solution dropwise, invert the centrifuge tube to a horizontal level twice, and place it in a 37°C water bath for 30 minutes.

[0048] Add 0.5 ml of pre-cooled Carnot's fixative (freshly mixed methanol: glacial acetic acid = 3:1) into the centrifuge tube, and fix for 10 min.

[0049] 3) Centrifuge the above cell suspension at 2000 rpm for 10 min, aspirate and discard ...

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Abstract

The invention provides a pomfret muscle cell line. The pomfret muscle cell line is a pomfret muscle cell PaM with the preservation number of CCTCC NO: C2021154. The pomfret muscle tissue cell line provided by the invention can be continuously passed to 50 generations and is kept stable so far, and a large number of stable pomfret muscle cells can be provided for nutrition metabolism, functional gene research, breeding research and the like. The provided cell line is excellent in character and is a fibroblast, the cell is fusiform or irregular triangle-shaped, a plurality of irregular bulges extend outwards from a cell body, and the cells are tightly arranged. The cell is short in generation time, can be cryopreserved and recovered, and and can perform various molecular biology experiments such as siRNA interference, plasmid transfection and the like.

Description

technical field [0001] The invention belongs to the technical field of fish cell culture, and in particular relates to a silver pomfret muscle tissue cell line. Background technique [0002] Silver Pomfret (Pampus argenteus) belongs to Perciformes (Perciformes), Pomfretaceae (Stromateidae), and Pomfret (Pampus). It is delicious, nutritious, and widely distributed. It is one of the important seawater economic fishes in my country. In recent years, under the dual requirements of protecting fishery resources and meeting market demand, our team has carried out the artificial breeding of silver pomfret, and achieved major breakthroughs in recent years, gradually realizing industrialization. However, while the artificial breeding of silver pomfret is advancing rapidly, it is also facing prominent problems such as weak basic research and frequent occurrence of diseases. Therefore, research on disease control, breeding of improved varieties, and genetic background of silver pomfret ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077C12R1/91
CPCC12N5/0658C12N2509/00C12N2509/10C12N2500/44C12N2500/34C12N2501/90C12N2501/115C12N2501/11C12N2501/305
Inventor 张友仪王亚军胡佳宝张曼李亚亚孙嘉初蒋欢王冠林朱家杰王想兵
Owner NINGBO UNIV
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