Myostatin基因打靶载体的构建及猪胎儿成纤维细胞Myostatin基因的敲除

目的:用缺口修复等技术构建Myostatin(肌肉生长抑制素,MSTN)基因打靶载体,并对大白猪胎儿成纤维体细胞进行转染,获得基因敲除细胞。方法与结果:首先构建用于MSTN基因同源长臂(LA)的抓捕载体,然后在大肠杆菌内利用Red同源重组系统介导的缺口修复,从含大白猪MSTN基因座的细菌人工染色体上亚克隆9.9 kb的LA到抓捕载体上,经过部分序列测定,同源性为100%;通过PCR获得1.4 kb的同源短臂(SA);将LA和SA连入载体pLOXP,构建含有neo和tk正负筛选标记基因的MSTN基因打靶载体pLOXP-MSTN-KO;将线性化的pLOXP-MSTN-KO通过电转染整合到大白猪胎儿成纤维细胞基因组中,利用G418和丙氧鸟苷进行药物筛选,获得抗性细胞克隆890个,通过PCR和DNA测序鉴定获得基因敲除的细胞克隆4个。结论:构建了有效的MSTN基因打靶载体,通过转染获得基因敲除细胞,为利用体细胞核移植制备MSTN基因敲除猪奠定了基础。

Construction of a Myostatin Gene-Targeting Vector and Myostatin Gene Knockout of Porcine Fetal Fibroblasts Cells

Objective： To knock out porcine Myostatin（MSTN） gene of porcine fetal fibroblasts cells correctly,a gene targeting vector was constructed via the gap-repair method and other means.Methods Results： The gaprepair vector was constructed,linearized and transformed into Escherichia coli.Mediated by Red homologous recombination system,the 9.9 kb homologous long arm（LA） was subcloned into the vector from bacterial artificial chromosome which harbors the MSTN gene locus of large white pig.Partial sequencing showed that the homology reached to 100% compared with the reported MSTN genomic sequence.Homologous short arm（SA） about 1.4 kb was acquired by PCR.The LA and SA were inserted into vector pLOXP,generating MSTN gene targeting vector pLOXP-MSTN-KO which contained neo and tk positive-negative-selection markers.The pLOXP-MSTN-KO was linearized and electroporated into the porcine fetal fibroblasts cells.After G418 and GANC selection,890 drug resistant cell clones were screened,and from which totally 4 positive clones were confirmed by PCR and DNA sequencing.Conclusion： The constructed gene targeting vector could efficiently target of MSTN locus,and the gene knockout cell clones will be used to produce MSTN knockout pig by means of somatic cell nuclear transplantation.