Application Of Kap147/Kpn Hybrid Promoter In Conditional Gene Targeting And RNA Interference

Gene targeting and RNA interference are two useful tools for gene function analysis.In order to precisely study gene function in specific tissues and specific developmental period,tissue specific or inducible promoter guided gene knockout or silence are often used.Expression of kidney androgen regulated protein(KAP)is specific in kidney proximal tubule cell-specific and androgen-regulated in vivo.In previous studies,the minimal Kap promoter(Kap147)and a 1.4 kb hetergenerous enhancer to Kap promoter,named hAGT-Kpn,between exons 2 and 3 of human angiotensinogen(hAGT)gene were identified by gene trunction and in vitro transfection experiment.In order to verify the function of Kap147/Kpn chimeric promoter in vivo,in this thesis,transgenic mice with ere recombinase(Cre)as the reporter gene were constructed,their expression characteristics were studied.Also,the posibility of using Kap147/Kpn chimeric promoter to direct cell-specific RNA interference was tested.Three independent transgenic mice,Cre-F0-1,Cre-F0-2 and Cre-F0-3,were obtained.Unfortunately,none of them showed kidney-specific Cre expression.Instead,we found tissue-specific Cre gene transcription and translation in the testis of the offspring of Cre-F0-2 by reverse transcription PCR,fluorescence quantitative PCR and immunohistochemistry.Immunohistochemistry also showed that Cre expression was targeted in the seminiferous tubules near epididymis.After crossing the offspring of Cre-F0-2 with a RFP/EGFP Cre reporter mice,we observed testis-specific gene recombination in their offsprings,which indicated that the offsprings of Cre-F0-2 are useful testis-specific gene targeting transgenic tool mice.Kap147/Kpn chimeric promoter directed Cre expression in the testis of transgenic mice Cre-FO-2 was regulated by androgen.Fluorescence quantitative PCR assay showed that the amount of Cre mRNA in adult mice was 20-30 times higher than that in newborn or senile mice.The transcription of Cre gene was similar to that of endogenerous Kap gene.Compared to uninjected normal mice,injection testosterone to female and male mice resulted in 1.5-2 fold increasment of Cre mRNA,while injection cyproterone acetate resulted in 30%-70%decreasment of Cre mRNA.Kap147/Kpn chimeric promoter can be used for kidney proximal tubule cell-specific RNA interference.Recombinant green fluorescent protein(EGFP)siRNA plasmids harboring Kap147/Kpn chimeric promoter and three different transcription stop signals were successfully constructed.Cell transfection experiments showed that the transcription and expression of EGFP was only reduced about 40%in LLC-MK2 cells(kidney proximal tubule derived epithelial cells),but not in Cos-7 cells(kidney derived fibroblast-like cells)and HeLa cells(cervical cancer derived epithelial cell).