【摘要】 利用DNA的同源重组原理，通过基因打靶技术，在小鼠胚胎干细胞（ES细胞）中将pMCl－neo导入hprt座位，实现了基因组内指定基因的定位致变．通过电穿孔导入质粒pRV4．0线性化DA，分别用G418与6－TG筛选HPRT突变子．经抗性检验及DNA印迹分析，证明得到了一株预期的定位转化细胞，转化效率为1．32×108．载体的非同源序列对定位致变的效率和整合方式没有影响．由于采取了有效的措施，所获HPRT－ES细胞株仍维持了未分化和二倍体状态，保留了胚胎于细胞的特性．更多还原

【Abstract】 hprt locus has been disrupted in murine embryonic stem(ES) cells by gene targeting. Thelinearized targcting vector pRV4.0 was introduccd into ES cclls by electroporation. HPRT- mutants inwhich the hprt gene had been disrupted by insertion of pMCl-neo cassctte into its 8th exon viahomologous recombination was selected with G418 and 6-thioguanine. Three independent ES cell strainswas isolated in a pool of 7.6×107 treated cells. With furthor tests of drug resistance and Southernhybridization it was proved that only C19 was thc HPRT- mutant in which target event did oeurred. Theoverall targeted efficiency was 1.32×10-3. The heterologous sequence of pUC9 DNA flanking the targeting sequence did not affect the targcted efficiency and integration pattem of DNA. As a result of thecareful choice of culture condition and transforming procedure, the HPRT- ES strain C19 still remainedits undiffcrentiating feature and diploid karyotype.更多还原