The Basic Study Of Vector And Target Site On The Therapy Of Marfan Syndrome By RNA Interference

Objective: In order to study the mutant FBN1 gene mRNA-based therapy for Marfan syndrome by RNA interference, we emphasize the experiment on the efficiency of RNAi pBSHHl vector through delivering siRNA to effectively suppress specific gene into mammalian cell, and by the study of the positional effect of RNAi to make sure whether C1037del is an effective RNAi target site.Methods: Plasmids are constructed by using standard techniques with successful annealing. The C1037del is as the target site to construct hairpined RNAi pBSHHl vector pMi under the control of the HI promoter. And so do the pEi and pGi. The reporter piasmids with EGFP fused gene are constructed by cloning into the C1037del sequence. And so does the EX sequence. For advanced study in cultured human HT1080 cell, with the performance of Lipofectamine 2000, the RNAi pBSHHl vectors are co-transfected with EGFP reporter piasmids by paired groups. The green fluorescent positive cells are recorded and analysised by Flow Cytometry.Results: The enzyme map and sequencing results suggest the pGi, pEi, pMi, and the other four EGFP reporter piasmids pEX-Nl (Cl), piVIU-i\l (Cl) are all correct. And by the Flow Cytometry and Picture, the fused EGFP reporter piasmids can express themselves well. The pGi show the most powerful RNAi gene silencing inhibitory, pEi with moderate but sequence-specific inhibitory, while pMi with no noteable RNAi effect.Conclusion: RNAi is an effective and specific gene silencing technique. pBSHHl can be employed as an effective RNAi vector to deliver siRNA into human cells. And the unpredictale RNAi positional effect could disadvantage the C1037del target site.