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Method for establishing stable transgenic flounder embryo cell strain

A technology of embryonic cells and transgenes, applied in the field of marine fish genetics, can solve problems such as less involvement

Active Publication Date: 2017-02-15
OCEAN UNIV OF CHINA
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Problems solved by technology

[0003] However, efficient cell transfection methods in marine fish such as flounder have not yet been reported, and the establishment of stable transgenic cell lines in marine fish cells is rarely involved. One of the major bottlenecks in gene-like function

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  • Method for establishing stable transgenic flounder embryo cell strain
  • Method for establishing stable transgenic flounder embryo cell strain
  • Method for establishing stable transgenic flounder embryo cell strain

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Embodiment Construction

[0023] The applicant found in long-term research that using the promoter whose nucleotide sequence is SEQ ID NO: 1 can enable the high-efficiency expression of foreign genes in the embryonic cells of flounder fish; and carried out the method of transfection and screening Optimization, finally obtained the present invention.

[0024] 1. Construction of exogenous plasmids

[0025] 1) Insert the flounder β-actin gene promoter (SEQ ID NO: 1) before the green fluorescent protein (EGFP) to construct a transgenic plasmid, and design a pair of flounder β-actin promoter genes with PCR primers for restriction endonuclease sites, in which a HindIII and KpnI enzyme cutting site is added to the 5' and 3' primers for PCR. Its primers are as follows:

[0026] 5'-primer:5'-CCCAAGCTTCGCGTCGTGCCCCAGTGT

[0027] 3'-primer: 3'-CGGGGTACCGGCTGAACTGCAGAGAGGAGAGA

[0028] The PCR reaction program was as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 sec, annealing at 68°...

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Abstract

The invention provides a method for establishing a stable transgenic flounder embryo cell strain. The method for establishing stable transgenic flounder embryo cell strain comprises the following steps: transfecting flounder embryo cells by using plasmids which can widely express enhanced green fluorescent protein promoted by a beta-actin promoter in flounders and can express G418 resistant protein; after 24h, observing expression of the EGFP (express enhanced green fluorescent protein) under a fluorescent microscope, and through G418 screening, obtaining the embryo cells with stable genetic expression. By the method, an exogenous gene widely expressed in the flounders and promoted by the beta-actin can be effectively transfected into the flounder embryo cells, and can be genetically expressed in the flounder embryo cells stably, so that a novel method is provided for researching for flounder genes, the problem that difficult cellular-level gene function analysis and research caused by low transient transfection efficiency of the flounder cells is solved, and a novel technical means is provided for a molecular-level genetic breeding work of the flounders, and a transgenic method for stably inheriting a marine fish cell line is provided.

Description

technical field [0001] The invention belongs to the technical field of marine fish genetics, and in particular relates to a method for establishing a stable transgenic flounder embryo cell line. Background technique [0002] Cell transfection refers to the process by which eukaryotic cells acquire new genetic markers due to the incorporation of exogenous DNA or RNA. It is used more and more widely in biological experiments such as studying gene function, regulating gene expression, mutation analysis and protein production. To date, various methods for transfecting cells with exogenous genes have been developed. It mainly includes virus-mediated exogenous gene transfection cell method and non-virus-mediated cell transfection method. The above method has successfully established a variety of stable genetically expressed cell lines in plants and mammals. [0003] However, efficient cell transfection methods in marine fish such as flounder have not yet been reported, and the ...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10
Inventor 汪波张全启王慧贞杨帆齐洁王志刚王旭波于海洋贺艳刘金相
Owner OCEAN UNIV OF CHINA
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