小鼠p16^(INK4a)基因外显子1α胚胎干细胞条件打靶研究

Conditional targeting of p16^(INK4a) exon 1αin mouse embryonic stem cells

目的 研究打靶载体的结构与打靶效率的关系 ,探讨小鼠 p16 INK4 a基因在活体水平上抑制肿瘤发生和发展的功能。方法 利用筛选基因组文库得到的小鼠 p16 INK4 a基因组 DNA片段 ,设计并构建了针对小鼠 p16 INK4 a基因外显子 1α的条件打靶载体 ,其短臂为 2 .0 kb Eco R / Xba 片段 ,长臂为 5 .9kb Spe / Not 片段 ,上游交叉位点 (locus of crossing- over,lox P)位于外显子 1α起始密码上游 2 4 0 bp处 ,下游lox P位于外显子 1α起始密码下游 16 33bp处 ,经重组酶 (Cre)介导后可将外显子 1α和选择标记 Neo基因同时删除。结果 将此条件打靶载体通过电穿孔转导小鼠胚胎干细胞 ,获得 2 4个药物抗性克隆 ,其中 1个经 Southern杂交证实为正确同源重组克隆。结论 在同源臂两侧各用一个

Objective: To study the relationship between targeting vector structure and homologous recombination rate and investigate whether the mouse p16INK4a plays a role in tumor suppression. Methods: A conditional targeting vector with 2. 0 kb EcoR I /Xba I fragment as short arm and 5. 9 kb Spe I /Not I fragment as long arm was built. Of the 2 direct locus crossing-over(loxPs) in the vector, one was inserted at 240 bp upstream of the initiate code of p16INK4a exon 1α and the other at 1633 bp downstream of the initiate code. Both exon 1α and the selection marker Neo will be deleted in targeted cells when mediated by Cre. After linearlization and purification, the targeting vector was introduced into ES cells through electroporation. Results: Twenty-four G418- and gancyclovir-resistant ES cell colonies were picked out and one of them was confirmed as positive by Southern hybridization. Conclusion: Targeting vectors with 2 TK genes flanking the homologous arms are likely to produce good result of homologous recombination.