Abstract:

Objective    To construct lentiviruses carrying dominant negative mutant of RhoA-GTPase (RhoAN19) or the constitutive active mutant of RhoA-GTPase (RhoAL63) and expressing enhanced green fluorescent protein (EGFP) bicistronically.    Methods    The lentiviral expression plasmid Plenti6/v5- RhoAN19 and Plenti6/v5- RhoAL63 were constructed and identified by restriction enzyme digestion and DNA sequence analysis. The two plasmids were packaged using the ViraPowerTM lentiviral expression system to produce replication-incompetent lentiviruses RhoAL63 and RhoAN19, which were used to infect the prefrontal cortex neurons (PFCs) from neonatal SD rats. The transfection efficiency and biological activity of different RhoA mutants were evaluated and the morphology of the transfected PFCs was observed.    Results    The results of DNA sequencing and restriction enzyme analysis demonstrated correct plasmid construction. The packaged lentiviral titer was 1×106 TU/ml. Analysis of RhoA biological activity showed that RhoAN19 lentivirus particles infection significantly inhibited lysophospatidic acid stimulated RhoA activity in the PFCs, while RhoAL63 lentivirus particles enhanced the RhoA activity. The transfection efficiency of these RhoA mutant lentivirus particles exceeded 80% in the PFCs. Morphologically, the PFCs exhibited distinct dendritic branches after infection by these lentiviruses.    Conclusions    The lentiviruses carrying RhoA dominant negative mutant and constitutive active mutant have been successfully constructed. The lentiviral particles can efficiently infect neonatal rat PFCs. Thus providing important support for the study of RhoA signaling.

Key words: RhoA-GTPase, Lentivirus, Green fluorescent protein, Biological activity