新的β_2m基因打靶载体构建方法的建立

New strategy of constructing the β_2m gene targeting vector

目的 :采用常规方法 (同源片段的插入方向与Neo基因相同 )构建载体的同时 ,将两条同源臂反向插入Neo基因的两侧 ,构建新型小鼠 β2 m基因替换型打靶载体 β2 m -pPNT ,即增加 3’同源臂的长度 ,探讨同源臂插入位置和长度变化对同源重组率的影响。方法 :设计和合成引物 ,经PCR从小鼠 β2 m -pSV2△HXgpt基因组克隆分别扩增出长度为 0 8kb和 4 2kb的 β2 m基因片段 ,作为 5’和 3’同源臂 ,分别反向插入载体pPNT的Neo基因上游和下游 ,构建小鼠 β2 m基因替换型打靶载体 β2 m -pPNT。结果 :经过PCR、限制性内切酶及DNA序列测定 ,证实此两条同源臂包含小鼠 β2 m的起始区和表达区 ,表明载体构建成功。结论 :PCR技术是构建基因打靶载体的简单而可靠方法。增加 3’同源臂的长度对研究提高胚胎干细胞基因敲除的同源重组率提供新的途径。

AIM: The purpose of this study was to establish a new strategy for constructing the mouse β 2m gene targeting vector in order to increase the homologous recombination frequency in contrast with our previous one, which was successfully constructed in the normal way.METHODS: A 4.2 kb 3' arm and a 0.8 kb 5' arm were amplified by PCR from the mouse β 2m-pSV2△HXgpt genomic clone. They included the start region and the three exons, which were separated into two parts from exons 2 (the main coding block) for the two arms——5' arm and 3' arm.RESULTS: The two fragments, in reverse orientation to the Neo gene, were cloned into pPNT respectively on either side of Neo. They were identified by PCR, restriction analysis and sequence analysis as well. CONCLUSION: The mouse β 2m gene targenting vector has been cloned successfully.