牛肌肉生长抑制素基因打靶载体的构建与鉴定

本试验旨在构建用于牛肌肉生长抑制素(MSTN)基因敲除的置换型打靶载体。基于已发布的MSTN基因序列，选取第3外显子约600 bp作为靶位点，在其上、下游设计2条同源臂，分别为4.4和1.4 kb。以pPNTⅢ为骨架载体，在其2个多克隆位点处插入同源臂，构建出置换型打靶载体MSTN-KO-pPNTⅢ。结果显示，经DNA测序及酶切鉴定证实1.4 kb同源短臂和4.4 kb同源长臂均正确插入基础载体中。结果表明，成功构建出牛MSTN-KO-pPNTⅢ打靶载体。

Construction and Identification of Bovine Myostatin Gene Targeting Vector

The objective of this study was to construct a targeting vector for knocking-out bovine myostatin (MSTN) gene. Based on the bovine MSTN gene sequence, we chose third exon about 600 bp as a target site. The homologous arms of bovine MSTN gene were amplified from the genomic DNA, which were about 4.4 and 1.4 kb in length, respectively. The two fragments were cloned into pPNTⅢ vector and located at both sides of the neomycin resistance gene to construct targeting vector MSTN-KO-pPNTⅢ. By sequence analysis and restricted endonucleases digestion, the 1.4 kb short arm and 4.4 kb long arm were detected in the targeting vector. In conclusion, the replacement targeting vector for bovine MSTN gene had been successfully constructed.