基于Cre重组酶体系的小鼠HPRT定点整合打靶载体的构建及其应用

Construction and application of a mouse HPRT targeting vector with Cre recombinase recognition site lox66

目的 :构建带有 Cre重组酶识别位点变异体 lox6 6的针对小鼠 HPRT基因的基因打靶载体 ,并用于小鼠胚胎干细胞基因打靶研究。方法 :利用含 HPRT基因组 DNA片段的质粒 p MP8和人工合成含 lox6 6序列的寡核苷酸 ,构建含 Cre重组酶识别位点变异体 lox6 6的置换型打靶载体 ,经过限制性内切酶酶切鉴定其结构正确后 ,用电穿孔法对小鼠胚胎干细胞 R1株进行基因转染 ,经 G418/ 6 - TG(6 - thioguanine)分组药物筛选 ,进行 p SP- HPRT- lox6 6 - Neo载体的应用研究。结果 :得到了与设计一致的置换型打靶载体 p SP- HPRT- lox6 6 - Neo;将线性化的打靶载体导入 ES细胞后 ,能与靶位点有效地发生同源重组 ,获得了 2 0个靶基因被灭活了的 ES细胞克隆。结论 :此研究成功构建了含 Cre重组酶识别位点变异体 lox6 6针对小鼠 HPRT基因位点的置换型打靶载体 。

Objective: To study the construction and application of a vector with Cre recombinase recognition site lox66 for mouse HPRT gene targeting in embryonic stem(ES) cells. Methods: Using the HPRT genomic DNA fragment and synthesized oligonucleotides, pSP HPRT lox66 Neo was designed and constructed as a replacement vector by common molecular cloning techniques. After the linearized pSP HPRT lox66 Neo DNA was electroporated into ES cells, and transfected cells were cultured in G418/6 TG drug selection medium. The recombination efficiency of this vector was tested. Results: The main components of pSP HPRT lox66 Neo were a positive selection gene Neo, lox66, long and short homologous fragments of mouse HPRT gene and plasmid backbone. Twenty ES cell clones with HPRT gene inactivated were obtained. Conclusion: An effective replacement vector with Cre recombinase recognition site lox66 is constructed and applied to HPRT gene targeting in ES cells.