Studies On Androgen-regulated Kap147/HAGT-Kpn Hybrid Promoter In Vitro And In Vivo

Gene expression and regulation determine the organism’s metabolism,growth and development.The specific characterization of organisms is dependent on the cell-tissue specific expression of the gene. The promoters are the important regulatory elements that directly regulate gene expression,which is responsible for the temporal and spatial specificity of gene expression.Kidney androgen-regulated protein is a specific expression in epithelial cells of proximal convoluted tubules. There is a3-4-fold stimulation of KAP mRNA after androgen treatment, suggesting KAP mRNA is highly regulated by androgen, Study on androgen-regulated protein promoter activity in different tissues and its regulatory of androgen is very helpful to establishmentof targeting Expression System.We constructed recombinase plasmids pGL3-Kap147/KpnI-Luciferase and pGL3-Kap147-/KpnI-Cre using Luciferase and Cre recombinase as report genes. We demonstrated that report genes were expressed in OK, LLC-MK2that is from epithelial cells of proximal convoluted tubules. Kap147/Kpnl had a activity that was7-8higher after20nM DHT treatment in OK and LLC-MK2cells.suggesting Kap147/Kpn is cell-specific and regulated by androgen.pGL3-Kap147/KpnI-Luciferase plasmid comlex was injected in male mice via intraperitoneal injection. Kidneys,heart,liver,lungs,testis were harvested on24hours after injection.Luciferase gene expression was detected in Kidney by RT-PCR, Immunofluorescence and Bioluminescence imaging. We also detected uciferase gene expression in testis. We further demonstrated HAGT-Kpn-Kap147hybrid promoter is the cell-specific.Based on potential androgen responsive sites in the HAGT-Kpn segment using analysis of conserved sequence of androgen responsive element, HAGT-Kpn fragment is divided into five parts.Luciferase gene recombinant plasmid infused five Truncated Fragments of HAGT-Kpn were constructed successfully and have enhancer effect to147bp Kap promoter at cell level,whichdo not affect the cell specificity of Kap promoter.