含FLP“交换盒”结构的打靶载体构建及ES细胞打靶研究

利用小鼠HPRT基因组DNA片段和人工合成的含有FLP重组酶识别位点变异体FRT和F3RT序列的寡核苷酸 ,构建了针对小鼠HPRT基因位点的置换型打靶载体pSP HPRT Fneo F₃。经过限制酶酶切及部分测序鉴定其结构正确后 ,将线性化了的打靶载体以电穿孔法导入ES细胞内 ,经G418和 6 -TG双药筛选和分子鉴定 ,得到了 2个在HPRT位点整合有FLP重组酶“交换盒”F Neo F3结构的双交换重组ES细胞克隆 ,为建立基于FLP重组酶介导的盒式交换的高效、定点转基因体系创造了条件.

Construction of a Flp "Exchange Cassette" Contained Vector and Gene Targeting in Mouse ES Cell

Using the HPRT genomic DNA fragment and synthesized oligonucliotides, pSP-HPRT-F-Neo-F3 was designed and constructed as a replacement gene targeting vector by usual molecular cloning techniques. Structure of pSP-HPRT-F-Neo-F3 was identified by restrictive digestion analysis and partly sequencing. Then linearized pSP-HPRT-F-Neo-F3 DNA was electroporated into ES cells, and transfected cells were screened by being cultured in medium containing 200 micrograms/mL G418 and 2 micrograms/mL 6-GT. Twenty-four double drug resistant clones were picked up and analyzed, among them, two clones were proved to have taken place the required recombination by PCR and southern blotting analysis.