Intracranial Administration of P Gene siRNA Protects Mice from Lethal Chandipura Virus Encephalitis

Kumar, Satyendra; Arankalle, Vidya A.

For the transfection of siRNA, amaxa nucleofactor II device and supplemented kit V solution was used as suggested by the manufacturer. For transfection using HiPerfect reagent, HEK-293 cells were seeded 12-16 hr before transfection in a six-well plate (1610 5 per well). Lipid-siRNA complexes were prepared by incubating 100 pmol of siRNA with HiPerfect reagent (QIAGEN, Valencia, CA) in the appropriate volume as recommended by the manufacturer. To determine transfection efficiency, P-2 siRNA tagged with Alexa Fluor-647 was used (Qiagen, Valencia, CA). P gene siRNA validation with pAcGFP1N1-CHPV-P Cells were transfected with P gene siRNA (P-1 to P-4) at a concentration of 100 pmol and 2 mg of target expressing plasmid pAcGFP1N1-CHPV-P. 24 hr post transfection, cells were harvested and flow cytometry and real time one step RT-PCR were carried out. For real time one step RT-PCR, RNA was isolated using RNAeasy minikit (QIAGEN, Valencia, CA) as described by Figure 1 . Schematic representation of gene arrangement in CHPV and siRNAs used. P and M genes were targeted for siRNA synthesis. The numbers were assigned arbitrarily and do not indicate location (refer table 1 for correct location of siRNA). Different siRNA used to determine tolerance for mismatches using P-2 siRNA number in parentheses represent the positions at which mismatches were incorporated (For sequence of P-2 siRNA mutants refer