Gene Targeting System Based On The Recombinant Enzyme In Mouse Es Cells In The Hprt Gene Locus Transfer

Transgenic mice have been widely used in life science study. However, the Iransgenic mice producing ways still have some defect By using microinjection and viral vector infection, transgenic mice can be produced comparatively efficiently, but the expression level of transgene is unpredictable and an accurate analysis of the effect of transgene is quite difficult since the copy number and integration locus are very changeable. The transgene can be introduced to a defined site of mice genome by gene targeting, but the efficiency is very lower. To facilitate the construction of transgenic mice, we planed to develop an efficient, she-specific transgenic mice producing system. By homologous recombination based gene targeting, an integration tag, consisting of site-specific recombinase recognition she and selectable gene, should be place to HPRT locus of mouse genome to construct a transgenic tool mouse. Then, the eggs derived from the tool mouse are used in microinjection. In this procedure, recombinant DNA containing site-specific recombinase recognition sequence is introduced into the egg with she-specific recombinase expression vector. In the injected egg, recombinant DNA should integrate into HPRT locus at higher efficiency through recombinase mediated she-specific integration reaction.In this study, oligonucleotides containing Flp recombinase mutant recognition she F3RT, Cre recombinase mutant recognition she Iox66 and Iox71 respectively, were synthesized Using these synthesized oligonucleotides, universal exchange plasmid pF-Neo-F3 and universal integration plasmid pEGFP-lox66, pEGFP-lox71 were constructed. Replacement gene targeting vector pSP-HPRT-F-Neo-F3 and pSP-HPRT-lox66-Neo were constructed with mouse HPRT genomic DNA fragment Then, Rl ES cell at tog phase were electroporated to introduce in linearized targeting vector pSP-F-Neo-F3 and pSP-HPRT-lox66-Neo. Through G418 and 6-TG screening, double resistant clones were obtained and further analyzed by PCR and genomic DNA Southern blotting. Two out of twenty-four pSP-HPRT-F-Neo-F3 drug resistant clones were identified to have taken the desired double exchange recombination.To examine the feasibility of she-specific integration strategy based on Flp recombinase mediated cassette exchange at mouse HPRT locus, exchange plasmid pF-HPRT-F3, Flp recombinase expression plasmid pCMV-Flp and pEF-Flp were constructed. Plasmid pF-HPRT-F3 and pCMV-Flp, pEF-Flp were coelectroporated into one of two obtained double exchange recombinant ES cell clones Fl 8. After HAT medium selection, HAT resistant cloneswere obtained and forty-eight clones were analyzed by G418 resistant test and Southern blot Among them, six clones were proved to have taken desired RMCE recombination. The result demonstrated that the strategy of using Flp remombinase mediated cassette exchange to introduce transgene into mouse HPRT locus is practicable at least in mouse ES cell level. All those have laid foundation for the construction of transgenic tool mouse and the establishment of recombinase based, efficient she-specific transgenic mice producing system.