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Updated: Sep 15, 2026
Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction
Published on: September 15, 2014
[Investigation of Lentiviral Vectors Based Integrin β8 RNAi System in Neonatal Rats' Brain]
Bin-Zhi Tang1, Yi Qu2, Feng-Yan Zhao2
1Department of Pediatrics,Sichuan Academy of Medical Sciences & Sichuan Provincial People's Hospital/University of Electronic Science and Technology of China,Chengdu 610072,China.
Objective:
To construct lentiviral vectors expressing pSicoR-β8 shRNA and evaluate its efficiency of RNA interference in neonatal rats' brain.
Methods:
Plasmid vectors pSicoR-β8 shRNA and pSicoR-control,as well as lentiviral packaging system pDM2G,g/p RRE and pRSV Rev were amplified respectively and plasmid DNA was identified by restriction enzyme digestion. Lentiviral packaging system and expressing vector pSicoR-β8 shRNA/pSicoR-control were co-transfected into packaging cell line 293T. Lentiviral particles expressing β8-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 β8 in neonatal rats' brain was performed by intraventricular injection of lentivirus expressing β8-shRNA and rats received lentivirus expressing β8-shRNA were served as control. Green fluorescent protein (GFP) expression after intraventricular injection of GFP-Lentivirus was observed under fluorescence microscope,β8 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 1.0×108 PFU/mL,and for LV-β8 shRNA 5.0×108 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-β8 shRNA,the inhibitory rate for β8 mRNA and β8 protein were 56% and 51%,respectively.
Conclusion:
Lentiviral vectors expressing β8-shRNA are successfully constructed and lentiviral mediated β8-RNAi is successfully applied for in vivo use.
