CHEN Li-xiang
Shanghai Public Health Clinical Center, Fudan University, Shanghai 201508, ChinaZHOU Xiao-jing
The first affiliated hospital of bengbu medical college, Anhui Bengbu 233004, ChinaLIU Wen-wen
Affiliated hospital of Chifeng University, Neimenggu Chifeng 024000, ChinaZHOU Wen-jiang
Shanghai Public Health Clinical Center, Fudan University, Shanghai 201508, ChinaREN Xiao-nan
Shanghai Public Health Clinical Center, Fudan University, Shanghai 201508, ChinaYU Shi-yan
Shanghai Public Health Clinical Center, Fudan University, Shanghai 201508, ChinaZHOU Xiao-hui
Shanghai Public Health Clinical Center, Fudan University, Shanghai 201508, China;Key Laboratory of Medical Molecular Virology, Ministry of Education and Health, Shanghai Medical College, Fudan University, Shanghai 200032, ChinaObjective To establish uPA inducible expression system using recombinant retroviral system for the further construction of inducible uPA-SCID animal model. Methods The Inducible expression system need to construct two plasmids:pLNHXO1O2-Alb-GLUC-FMN2A -rtTA and pLNHXO5O6-TRE2-uPA-IRES-ZsGreen respectively. Both plasmids were based on retroviral vector pLNHX, Albumin promoter gene (Alb) and rtTA gene or uPA gene and ZsGreen were obtained by PCR reaction and inserted into pLNHX. The Gaussia enzyme fluorescent element (GLUC) was used to monitor rtTA expression in pLNHXO1O2-Alb-GLUC-FMN2A-rtTA,and the ZsGreen for uPA expression monitoring in pLNHXO5O6-TRE2-uPA-IRES-ZsGreen.The correct constructed plasmids were transfected into packaging cell line GP2-293 to gain recombinant viral particles.NIH/3T3 cells were infected with these viral particles and selected with G418.Gene expression in the surviving cells was confirmed by the PCR method. Results The recombinant retroviral vectors harbouring target genes were successfully cloned.The rtTA gene in pLNHXO1O2-Alb-GLUC-FMN2A-rtTA was expressed, and uPA can be induced to express in pLNHXO5O6-TRE2-uPA-IRES-ZsGreen by doxycycline (Dox) when the plasmid transfected into the HepG-Tet-on cell. The constructed recombinant two retroviral vectors were transfected into GP2-293 packaging cells respectively to gain infectious viral particles.Then,NIH/3T3 cells were infected with these viral particles and single-cell clones which stably expressed the transgenes were successfully established. Conclusion This study primarily established uPA inducible expression system, it laid a foundation for the murine model of inducible liver damage, and provided a novel technical platform for further building the liver humanised murine models for viral hepatitis studying.
