牛α(1,3)-半乳糖转移酶基因片段的克隆及一种全新的无启动子打靶载体的构建

Ooning of the Genomic Fragments of Bovine a(1,3)-galactosyltransferase Gene and Construction of a New Kind of Promoterless Targeting Vector

利用La-PCR的方法,从牛的基因组中成功地获得分别约为2.4kb、15.0kb的两段扩增产物。两个片段分别克隆于pCR—XL—TOPO载体。测序结果表明所克隆的两片段均为牛α(1,3)GT基因的基因组序列。其中2.4kb片段位于5～7外显子之间,包括了完整的第五及第六内含子;15.0kb片段位于3～4外显子,包括了完整的第三内含子。15.0kb、2.4kb片段分别作为打靶载体的5’、3’同源臂,使用融合基因策略构建了一种全新的无启动子打靶载体7zf—neo17.4。电击转染牛胎儿成纤维细胞以期敲除牛的α(1,3)—GT基因。

Using the LA-PCR method, we have cloned two fragments from bovine genome which are about 2. 4kb, 15.0kb respectively. The 2.4kb fragment between the fifth exon and the sixthth exon includes the whole fifth intron and the sixth intron. The 15. Okb fragment between the third exon and the fourth exon includes the whole third intron. Sequencing result reveals the two fragments are both the segments of bovine GT gene. The 15. Okb and 2.4kb fragments were used to contruct a new promoterless targeting vector 7zf-neo17.4 . Transfecting the bovine fetal fibroblast cell with the new targeting vector we want to knock out bovine α(1 ,3)-GT gene.