猪α-1,3-半乳糖转移酶基因打靶载体的构建

目的:构建猪α-1,3-半乳糖转移酶(GGTA1)基因的正负筛选打靶载体。方法:以原代猪胚胎成纤维细胞基因组DNA为模板,采用长程PCR方法扩增出GGTA1基因的2条片段;以长约2kb包含部分第9外显子的片段为同源短臂,在XbaⅠ和ClaⅠ位点插入pLoxP质粒正筛选标记neo基因的3'端;以长约5.4kb包括部分第8外显子、全部第8内含子及部分第9外显子的片段为同源长臂,于NotⅠ位点插入该质粒中neo基因的5'端;2.7kb的负筛选标记tk基因位于载体中同源短臂的3'端外侧。结果与结论:酶切、PCR及测序结果表明,同源臂被正确连接至质粒pLoxP,成功构建了猪GGTA1基因正负筛选打靶载体pSL/GT。

Construction of Porcine Alpha-1,3-Galactosyltransferase Gene Knockout Vector

Objective：To construct a porcine α-1,3-galactosyltransferase（GGTA1） gene knockout vector with positive-negative selection markers.Methods：Two GGTA1 genomic fragments were amplified by PCR from genomic DNA of primary porcine fetal fibroblasts.The targeting vector was designed to be able to use positive-negative selection.The short arm is a 2 kb fragment containing most part of exon 9 of GGTA1 gene.It was inserted into the XbaⅠ-ClaⅠ site at 3＇ end of the positive selection marker neo sequence in pLoxP plasmid.The long arm is a 5.4 kb fragment containing part of exon 8,whole intron 8 and part of exon 9 of GGTA1 gene.It was inserted into the NotⅠ site at the 5＇ end of the neo sequence in the plasmid.The 2.7 kb negative selection marker tk was at the 3＇ end outside of the short arm in the vector.Results Conclusion：Restriction analysis,PCR and sequencing results indicated that homologous arms were inserted into pLoxP plasmid correctly.A porcine GGTA1 gene targeting vector pSL /GT with positive-negative marker was constructed.