利用Red系统快速改构含鼠β-酪蛋白基因的细菌人工染色体  

The Rapid Modification of a Bacterial Artificial Chromosome Containing Mouse β-casein Gene with Red System

作　　者：辛艳丽[1] 贾玉艳[1] 王恒梁[1] 邓继先[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071

出　　处：《中国生物工程杂志》2005年第8期45-50,共6页China Biotechnology

基　　金：国家"863"计划资助项目(2002AA206621)

摘　　要：采用Red系统介导的同源重组方法对含有鼠β-酪蛋白基因的RPCI23-440C1BAC进行快速改构。首先通过PCR方法,获得两端带有鼠β-酪蛋白基因同源序列的tPAm-Zeo同源重组片段,然后将此同源重组片段电击转化至已含有编码Red重组酶质粒的RPCI23-440C1BAC菌中,在λRed重组系统的帮助下,通过同源重组片段两端与RPCI23-440C1中β-酪蛋白同源的序列在菌体内与β-酪蛋白基因发生同源重组,将其置换。最后利用Zeocin抗性基因两侧的FRT位点,通过FLP位点专一性重组将抗性基因剔除。经Southern blot和序列分析鉴定表明,获得了重组正确且无编码两种重组酶质粒的tPAm-RPCI23-440C1BAC克隆。The bacterial artificial chromosome containing mouse β-casein gene is rapidly modified by homologous recombination mediated Red system. First, PCR was used to obtain the tPAm-Zeo and fragments flanking with homologous arms for homologous recombination of mouse beta-casein gene in RPCI23-440C1. The PCR products were electro-transformed into the RPCI23-440C1 BAC packaging strain DH10β expressing Red recombinase. With the help of λ Red recombination system, beta-casein gene was replaced by the tPAm-Zeo in vivo. Finally, due to the existence of the FRTs sequence flanking Zeocin gene, Zeocin genes were deleted by FLPpromoted recombination events. DNA Sequencing showed that tPAm-RPCI23-440C1 BAC clone containing no plasmids coding for the recombinases were obtained.

关 键 词：细菌人工染色体 λ RED重组系统 Β-酪蛋白基因 FLP Southern 同源重组 BAC克隆 鼠 同源序列 

分 类 号：Q786[生物学—分子生物学] Q78