Investigation of Lentiviral Vectors Based Integrin β8 RNAi System in Neonatal Rats’ Brain
Abstract
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.
Keywords: Integrin β8, Lentiviral vectors, RNAi in vivo interference
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LIU J, ZENG L, KENNEDY RM. Et al. ^Pix plays a dual role in cerebral vascular stability and angiogenesis, and interacts with integrin av38. Dev Biol,2012,363(1);95-105.
SIZEMORE RJ. SEEGER-ARMBRUSTER S, HUGHES SM. et al. Viral vector-based tools advance knowledge of basal ganglia anatomy and physiology. J Neurophysiol,2016, 115(4):2124-2146.
CHERY J. RNA therapeutics; RNAi and antisense mechanisms and clinical applications. Postdoc J,2016,4(7) : 35-50.
DANG Y, LOEWEN R. PARIKH НА, et al. Gene transfer to the outflow tract. Exp Eye Res«2017 Л58:73-84[2017-05- 15]. https;//doi. org,/10. 1016/j. exer. 2016. 04. 023.
CHEN MH, LIU YH, XU H. et al. Lentiviral vector- mediated p27kip 1 expression facilitates recovery after spinal cord injury. Mol Neurobiol,2016,53(9) :6043-6056.
COUNSELL JR, ASGARIAN Z, MENGJ, etal. Lentiviral vectors can be used for full-length dystrophin gene therapy. Sci Rep,2017,7(1):79.
MA Lt SHEN F. JUN K. et al. Integrin £8 deletion enhances vascular dysplasia and hemorrhage in the brain of adult Alkl heterozygous mice. Transl Stroke Res, 2016, 7(6):488-496.
ARNOLD TD, NIAUDET C, PANG MF, et al. Excessive vascular sprouting underlies cerebral hemorrhage in mice lacking aV|38-TGF|3 signaling in the brain. Development, 2014.141(23):4489-4499.
ZHENG YQ. LIU JX, LI XZ, et al. RNA interference- mediated downregulation of Beclinl attenuates cerebral ischemic injury in rats. Acta Pharmacologica Sinica.2009,30(7):919-927.
ZHU Z, ZENG XH, TURECEK J. et al. RNA interference of GluNl inhibits neuronal rhythmogenesis in the adult inferior olive. J Mol Neurosci.2015,55(2);416-429.
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