小鼠釉基质丝氨酸蛋白酶基因打靶载体的构建及鉴定

Construction and identification of mouse EMSP1 gene targeting vector

目的:对牙釉质丝氨酸蛋白酶(EMSP1)基因进行体外扩增,构建基因打靶载体,利用基因打靶技术建立转基因动物模型,研究EMSP1生理功能和病理学意义。方法:根据EMSP1的DNA序列,采用Oligo6.0软件设计两对引物,以129品系小鼠基因组DNA为模板,分别扩增长为5 000 bp和1 700 bp片段作为同源长短臂。将其分别插入通用型基因敲除载体pSSC-9的正向筛选基因neo两侧,用PCR方法、限制性酶切和DNA序列分析进行鉴定。结果:采用双酶切鉴定,阳性重组克隆分别切出5000 bp和1700 bp片段,表明长短臂已克隆于载体中。DNA序列分析表明,成功构建EMSP1基因打靶载体pSSC-9-EMSP1。结论:成功构建小鼠EMSP1基因打靶载体。

Objective To amplify the enamel matrix serine proteins 1 （EMSP1） in vitro and to establish gene targeting vector and transgenic mouse models to study Methods Two pairs of primers were designed by Oligo the physiological and pathological function of EMSP1. 6.0 software and synthesized according to EMSP1 gene sequence. Two gene fragments of 129 strain mouse EMSP1 were amplified from mouse genomic DNA by PCR-5 000 bp 5＇ arm and 1 700 bp 3＇arm. The two arms were cloned into a universal gene knockout vector-pSSC-9 respectively on either side of the positive selective marker neo. The targeting vector was identified by PCR, restriction endonuclease and sequence analysis. Results The restriction endonuclease identifiation result indicated that 5 000 bp and 1 700 bp fragments were obtained and homogeneous long and short arms were cloned into vector. DNA squence analysis showed that EMSP1 gene targeting vector pSSC-9-EMSP1 was successfully constructed. Conclusion The mouse EMSP1 gene targeting vector is constructed successfully.