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Development of a positive-negative selection procedure for gene targeting in fish cells

文献类型: 外文期刊

作者: Chen, SL 1 ; Hong, YH 2 ; Schartl, M 3 ;

作者机构: 1.Univ Wurzburg, Bioctr, Physiol Chem 1, D-97074 Wurzburg, Germany

2.Chinese Acad Fisheries Sci, Yellow Sea Fisheries Res Inst, Qingdao 266071, Peoples R China

3.Chinese Acad Sci, Inst Hydrobiol, Wuhan 430072, Peoples R China

关键词: Fish;Gene targeting;Homologous recombination vector;Medaka oryzias-latipes;Embryonic stem-cells;Germ-line chimeras;Homologous recombination;Melanoma;Xiphophorus;Establishment;Zebrafish;Cultures;Expression

期刊名称:AQUACULTURE ( 影响因子:4.242; 五年影响因子:4.723 )

ISSN:

年卷期:

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收录情况: SCI

摘要: The gene targeting technique is a powerful tool for analyzing functions of cloned genes and for generating transgenic animals with site-directed integration of foreign genes. In order to develop this technique in fish, positive-negative selection (PNS) and homologous recombination vectors were constructed, and their expression was examined in fish cells. A vector (pNK) for PNS consists of the neomycin resistance gene (neo) as a positive selectable marker gene and the herpes simplex virus (HSV) thymidine kinase (tk) gene as a negative selectable marker gene. Positive selection with geneticin (G418) of epithelioma papulosum of carp (EPC) cells transfected with linearized pNK vector yielded 350 colonies, while double selection of transfected EPC cells with G418 and gancyclovir (Gc) resulted in nearly complete cell death, demonstrating that the PNS procedure is effective in fish cells. Homologous recombination vectors consist of the Xiphophorus melanoma receptor kinase (X mrk(Y)) gene as homologous sequence in addition to the neo and tk genes. Conditions for homologous recombination vector transfection and drug selection were established. After verification of the feasibility of expression of homologous recombination vectors in EPC cells, the first gene targeting experiments were attempted in the Xiphophorus melanoma cell line, PSM. Positive-negative selection of the targeting vector-transfectants led to a low enrichment in this particular cell line. The reasons for the low enrichment in PSM cells were discussed.

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