[Investigation of Lentiviral Vectors Based Integrin β8 RNAi System in Neonatal Rats' Brain].

Objective: To construct lentiviral vectors expressing pSicoR- shRNA and evaluate its efficiency of RNA interference in neonatal rats' brain.

Methods: Plasmid vectors pSicoR- shRNA and pSicoR-control,as well as lentiviral packaging system and were amplified respectively and plasmid DNA was identified by restriction enzyme digestion. Lentiviral packaging system and expressing vector pSicoR- shRNA/pSicoR-control were co-transfected into packaging cell line 293T. Lentiviral particles expressing -shRNA or control sequence packaged and secreted by 293T were collected,concentrated by PEG-it,and viral titers were assayed by 50% tissue culture infective dose (TCID50). RNAi for integrin in neonatal rats' brain was performed by intraventricular injection of lentivirus expressing -shRNA and rats received lentivirus expressing -shRNA were served as control. Green fluorescent protein (GFP) expression after intraventricular injection of GFP-Lentivirus was observed under fluorescence microscope, mRNA and β8 protein expression were detected by RT-PCR and Western blot respectively,all of which were performed to evaluate the RNAi efficiency and to choose the optimal time for intervention.

Results: Restrictive endonuclease digestion and agarose gel electrophoresis showed plasmids as same as the expected size. Lentiviral titers for LV-control after concentration was 1.0×10 PFU/mL,and for LV- shRNA 5.0×10 PFU/mL.One day after intraventricular injection of lentiviral vectors containing GFP sequence,lenticivirus genome was integrated into host cells and emitted green fluorescence. A relatively strong green fluorescence could be observed in brain slides 2 d,3 d and 5 d after intraventricular injection. Western blot and RT-PCR demonstrated a maximum inhibition happened 3 d after intraventricular injection of LV- shRNA,the inhibitory rate for mRNA and β8 protein were 56% and 51%,respectively.

Conclusion: Lentiviral vectors expressing -shRNA are successfully constructed and lentiviral mediated -RNAi is successfully applied for use.