Application Of MRFP1 Reporter Gene And Gal4-UAS Bipartite Expression System In The Mouse

Many promising tools have been developed to address genetics questions in different model organisms. Several examples include utilization of reporter genes, GAL4/UAS tissue specific expression system, and the newly developed RNAi technology. Transplantation of these techniques from its originated organism to other model systems, especially the mouse, is an attractive challenge.We tested a series of makers such as agouti, Tyrosinase, EGFP and mRFP1 in transgenie mouse and found that mRFP1 could be used as a transgenic marker in mouse. mRFP1 has high pentrance, is easy to be distinguished under daylight, and has obvious dosage dependency, that helps to identify homozygots or heterozygots. Furthermore, when driven by a ubiquitous promoter, mRFP1 expression could be detected in almost all tissues, which makes it a possible maker for tissue or cell transplantion experiments.We set up several different tissue specific high expressed GAL4 transgenic mouse lines include ubiquitously expressed, muscle and skin specific GAL4 lines, by a series transgenic mice, we found the UAS cassette containning CMV mini promoter could be induced with highest efficiency, and the hsp70 mini promoter had the lowest leaky expression. By comparing full length GAL4, G5, G53, VP16, which are different at the activate domain, we found GV , a fusion protein of GAL4 (1-238) and VP16 had the highest induce efficiency. So we made GV and 5xUAS-CMV transgenic mice and detected a better inducible expression.Small double strands RNAs can induce nonspecific inhibition of gene expression in mammalian systems. By cotransfecting two plasmid, which transcript the RNA fragments in opposite directions to form a double strands RNA, we found it activate IFN signal pathway by activating PKR. The activation of IFN pathway induce general protein synthesis inhibition. Using siRNA droved by U6 promoter, we successfully reduced the PKR expression level, and found it could inhibit the activation of IFN pathway activiated by double strands RNA. It provides a possible tool to reduce the nonspecific effect of RNAi in mammalian system.