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小鼠胚胎干细胞hprt基因的定位致变

Targeted Disruption of hprt Gene in Murine Embryonic Stem Cells

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【作者】 刘爱民尚克刚

【Author】 Liu Aimin; Shang Kegang(Department of Biology, Peking University, Beijing 100871)

【机构】 北京大学生物学系!北京100871

【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.

【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.

【关键词】 ES细胞系基因打靶hprt基因
【Key words】 ES cell lineGene targetinghprt gene
  • 【分类号】Q344
  • 【被引频次】11
  • 【下载频次】70
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