猪MSTN基因双筛选标记打靶载体的构建及其功能鉴定

Construction and functional analysis of a pig myostatin gene targeting vector containing double-selection markers

【目的】利用正负筛选策略,构建猪肌肉生长抑素(Myostatin,MSTN)基因的双筛选标记打靶载体。【方法】以猪胎儿成纤维细胞DNA为模板,PCR扩增MSTN基因同源长、短臂;采用PCR和Overlap PCR扩增打靶载体正筛选标记嘌呤霉素和绿色荧光蛋白基因及负筛选标记单纯疱疹病毒胸苷激酶基因。以pUC57载体为骨架载体,在其多克隆位点连接一段包括Frt序列在内的酶切位点的多克隆位点序列,在2个Frt序列之间连接正筛选标记嘌呤霉素基因和绿色荧光蛋白基因,在Frt序列两侧分别连接5.7和1.9kb的MSTN基因同源长、短臂;在同源短臂后连接负筛选标记单纯疱疹病毒胸苷激酶基因,将目的片段与载体定向连接克隆。用脂质体法将打靶载体转染猪肾细胞(PK15细胞),用嘌呤霉素和丙氧鸟苷进行正负筛选,验证正负筛选标记基因的功能。【结果】成功克隆了猪MSTN基因同源长、短臂及正筛选嘌呤霉素和绿色荧光蛋白基因及负筛选单纯疱疹病毒胸苷激酶基因,构建了猪MSTN基因双筛选标记打靶载体,打靶载体长14kb。在打靶载体转染的PK15细胞中,正负筛选标记基因均有生物学活性。【结论】成功构建了猪MSTN基因双筛选标记打靶载体。

【Objective】 A pig myostatin（MSTN） gene targeting vector containing double-selection markers was constructed based on the positive-negative selection strategy.【Method】 The MSTN gene homologous long arm and short arm were cloned in the genome of pig fetus fibroblasts cells by PCR method.The posistive selection cassette puromycin（Puro） and enhanced green fluorescent protein（EGFP） genes and the negative selection cassette thymidine kinase（TK） gene were cloned by PCR and Overlap PCR.A set of cassettes and plasmids was used for creating the targeting vector.A positive selection marker cassette（PGK-Puro-EGFP） including the eukaryotic promoter to permit consecutive selection for recombination and allow positive selection of puromycin resistant transfectants in mammalian cells was flanked by two Frt sequences.EF1α-herpes simplex virus thymidine kinase（TK） minigene was used as the negative selection marker.The homologous long arm with a length of 5.7 kb amplified from pig embryonic fibroblast cell genomic DNA was placed before the first Frt sequence.The homologous short arm with a length of 1.9 kb was cloned between the positive and negative selection cassettes.The plasmid was transfected into PK15 cells with X-tremeGene HP DNA transfection reagent to evaluate the positive and negative selection markers function.【Result】 The homologous long arm and short arm together with the Puro and EGFP and TK gene were successfully cloned.The presence of the correct inserts in the targeting vector with the full length of 14 kb was verified by DNA sequencing.Meanwhile,the PK15 cells conferred resistance to puromycin（Puro） and ganciclovir（GAC）,which suggested the positive and negative selection markers could expressed in the cells.【Conclusion】 A pig myostatin（MSTN） gene targeting vector containing double-selection markers was constructed.