Construction and Identification of shRNA Expression Vector Specific for Small GTPase Rap1

Rap1 shRNA expression vector was constructed to suppress the expression of Rap1 by RNA interference (RNAi) technology and study the interference role of Rap1 shRNA in the mouse liver cells. Based on the mRNA sequence of Rap1,Three target sequences (Rap1 siRNA1,Rap1 siRNA2,Rap1 siRNA3)and one corresponding negative control (HK)were designed. Rap1 siRNA and HK were synthe- sized and cloned into the EGFP reporter plasmid pGenesil-3. The Rap1 siRNA expression vector was successfully constructed and identified by double endonuclease digestion and sequence analysis. Forty Kunming mice weighing 18～22 g were randomly divided into 4 groups:groupⅠ,HK was transfected (n= 10);groupⅡ,Rap1 shRNA1 was transfected (n=10);groupⅢ,Rap1 shRNA2 was transfected (n=10); groupⅣ,Rap1 shRNA3 was transfected (n=10). The intraperitoneal dosing of expression vectors was 2.0～ 2.5 mg/kg at 0,16,24 h. The mouse live cells were collected at 48 h after transfection. RT-PCR and immunofluorescence were used to determine the transfection rate and the mRNA and protein level of Rap1.The transfection efficiency of groupⅠ,Ⅱ,Ⅲ,Ⅳ was more than 60%. Rap1 mRNA and protein expression of groupⅠwas obvious higher than groupⅡ,Ⅲ,Ⅳ (P 0.05). Rap1 mRNA and protein expression of groupⅡwas significantly degrader than groupⅢ,Ⅳ(P0.05).