【作者】 贾玉艳； 辛艳丽； 陈宏； 邓继先；

【Author】 JIA Yu-yan, XIN Yan-li,CHEN Hong, DENG Ji-xian(1 Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China; 2.College of Animal Scienceand Technology, Northwest Sci-Tech University of Agriculture and Forestry, Yangling, Shaanxi 712100, China; 3Institute of Biotechnology, Xuzhou Normal University, Xuzhou, Jiangsu 221116, China)

【机构】 军事医学科学院生物工程研究所5室； 西北农林科技大学动物科技学院；

【摘要】 对细菌人工染色体(bacterial artificial chromosome,BAC)的修饰是研究基因在体内和体外表达和功能的一个有用的方法。以往技术的局限是对BAC的修饰需要多步的克隆把目的序列插入到穿梭载体上。本研究用Red系统将带Zeocin抗性的天然t-PA的编码序列置换掉一个BAC上的β-酪蛋白第2～8外显子。构建了两个带Zeocin抗性的天然t-PA的载体,一个含有牛生长激素polyA信号(bovine growthhormone polyadenylation signal,BGHpA),另一个没有BGHpA。用PCR方法获得两端含有β-酪蛋白同源臂的天然t-PA+Zeocin和天然t-PA+BGHpA+Zeocin的DNA片段,然后电击转化至大肠杆菌DH10β中,在λRed重组系统的作用下,在细菌体内与BAC发生同源重组,置换了BAC中的β-酪蛋白的第2～8外显子。对BAC进行了定位外源基因插入,为转染细胞和制备转基因动物生物反应器提供了一个有效的载体。更多还原

【Abstract】 Modification of bacterial artificial chromosome (BAC) has been a useful method for studying gene expression and function in vitro and in vivo. One of the original limits of BAC modification technique required multiple cloning steps to target foreign sequences into the shuttle vector. Using Red system, we have substituted for PGR fragments containing native t-PA and zeocin resistance gene to the 2nd to 8th extrons of β-casein. We constructed two vectors with native t-PA and zeocin resistance gene, one contains BGHpA, another not. PCR fragments of two vectors were electro-transferred into E.coli DH10 β. With the role of Red recombinant system, the 2nd to 8th extrons of & -casern were in vivo replaced by native t-PA and zeocin resistance gene. Modification of BAG provided a effective vector for transfection of cells and preparation of transgenic animal bioreactors.更多还原