牛肌肉生长抑制素基因敲除打靶载体的构建.pdfVIP

中国农业科学 2012,45(2):359-368 Scientia Agricultura Sinica doi: 10.3864/j.issn.0578-1752.2012.02.019 牛肌肉生长抑制素基因敲除打靶载体的构建 赵丽华，梁 浩，云 亭，李荣凤 （内蒙古大学哺乳动物生殖生物学与生物技术教育部重点实验室, 呼和浩特 010021 ） 摘要：【目的】构建两套用于牛肌肉生长抑制素（myostation，MSTN）基因敲除的置换型打靶载体。【方法】 设计并合成含有限制性内切酶酶切位点的引物，利用聚合酶链式反应（polymerase chain reaction，PCR）以牛 耳皮肤组织基因组DNA 为模板分别扩增出两套打靶载体的同源短臂和长臂，再分别插入到两套打靶专用基础载体 pMCS-PLP 和PⅢ中，构建两套用于牛MSTN 基因敲除的置换型打靶载体pPLP-MSTN 和PⅢ-MSTN。【结果】经过PCR、 T 载体连接和DNA 测序，证实载体pPLP-MSTN 包含2.8 kb 同源短臂和4.0 kb 同源长臂，载体PⅢ-MSTN 包含1.3 kb 同源短臂和6.8 kb 同源长臂；经过限制性内切酶酶切鉴定，证实两套载体的同源臂分别正确插入到基础载体内。 【结论】两套牛MSTN 基因敲除打靶载体pPLP-MSTN 和PⅢ-MSTN 构建成功，载体pPLP-MSTN 为不含负筛选标记的 传统基因敲除打靶载体，载体PⅢ-MSTN 为不含负筛选标记的荧光蛋白启动子捕获打靶载体。 关键词：牛; 生长抑制素基因; 基因敲除打靶载体; 胎儿成纤维细胞 Construction of Knock-Out Vectors for Bovine MSTN ZHAO Li-hua, LIANG Hao, YUN Ting, LI Rong-feng (Key Laboratory of Mammalian Reproductive Biology and Biotechnology, Ministry of Education, Inner Mongolia University, Hohhot 010021) Abstract : 【Objective 】The objective of this study is to construct two replacement targeting vectors for knocking-out bovine MSTN gene. 【Method 】Primers with restricted endonucleases locus were designed and synthesized. The short arms and long arms for both vectors were isolated from the genomic DNA of cattle by polymerase chain reaction (PCR). Then, these fragments were cloned into basic targeting vectors pMCS-PLP and PⅢ to construct two replacement targeting vectors (pPLP-MSTN and PⅢ -MSTN), respectively. 【Result 】Two constructed gene targeting vectors were identified by PCR, T-vector ligation and DNA sequencing. The 2.8 kb short arm and the 4.0 kb long arm were detected in the targeting vector pPLP-MSTN, and