Inhibition Of SV40T Antigen In COS-1 Cell Line By RNA Interference And Foundation Of Transgenic Mice

Since the discovery that synthetic double—stranded 21 — nt small interfering RNA is enough to trigger gene — specific silencing,double — strand RNA — mediated interference(RNAi) has recently emerged as a powerful reverse genetic tool to silence gene expression in multiple organisms including plants,Caenorhabditis elegans, Drosophila and mammalian cells.However, we expect that a approach should be adaptable to situations forwhich delivery of in vitro—synthesized siRNAs may not be practical,such as primary cell cultures,studies in intact animals,and gene therapy.In addition,to date, the most powerful applications of genetic manipulation are realized only with the creation of stable mutants.Simian Virus 40 T antigen is a potent oncoprotein that can induce several types of tumors.SV40T antigen acts as a potent growth stimulator by inactiving p53 and Rb tumor suppressor genes, leading to uncontrolled cellular proliferation and tumor formation.Here, a vector—based siRNA expression system is reported that can induce robust inhibition of endogenous gene stably.Three SV40T sequence—specific siRNAs were designed by us according to the principle of siRNA. No 100% homologous mRNA of other genes was found in Blast.We constructed the three vector that could interfere Simian Virus 40 T antigen expression.We collected the COS-1 cells transfected the siRNA vectors to make some experiments.The result of the check-up including RT-PCR,FACS,FCM and Western—blot showed that a interfering system was founded and useful for RNAi research.Based on the confirmed interfering system, we established a transgenic model that expressed aim gene.Our report demonstrates the value of this transgenic model in the studying the physiological function of the SV40 T antigen of interesting a specific manner.