[Construction and identification of Rac1-GTPase lentivirus].
OBJECTIVE
To construct lentiviruses carrying dominant negative mutant of Rac1-GTPase (Rac1N17) or the constitutive active mutant of Rac1-GTPase (Rac1L61) and expressing enhanced green fluorescent protein (EGFP) bicistronically.


METHODS
The lentiviral expression plasmid Plenti6/v5-Rac1N17 and Plenti6/v5-Rac1L61 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 Rac1L61 and Rac1N17, which were used to infect the prefrontal cortex neurons (PFCs) from neonatal SD rats. The transfection efficiency and biological activity of different Rac1 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 1x10(6) TU/ml. Analysis of Rac1 biological activity showed that Rac1N17 lentivirus particles infection significantly inhibited epidermal growth factor-stimulated Rac1 activity in the PFCs, while Rac1L61 lentivirus particles enhanced the Rac1 activity. The transfection efficiency of these Rac1 mutant lentivirus particles exceeded 80% in the PFCs. Morphologically, the PFCs exhibited distinct dendritic branches after infection by these lentiviruses.


CONCLUSIONS
The lentiviruses carrying Rac1 dominant negative mutant and constitutive active mutant have been successfully constructed. The lentiviral particles can efficiently infect neonatal rat PFCs and lend important support for the study of Rac1-GTPase.
Wang
Bin
B
Department of Pathophysiology and Key Laboratory of Proteomics of Guangdong Province, Southern Medical University, Guangzhou 510515, China. wangbin1983piero@163.com
Li
Juan
J
Zhang
Lei
L
Zhang
Lin
L
Zhang
Lu
L
D016475:3T3 Cells; D000818:Animals; D000831:Animals, Newborn; D002540:Cerebral Cortex / Q000166:cytology; D020558:GTP Phosphohydrolases / Q000096:biosynthesis* / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009474:Neurons / Q000378:metabolism / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D015398:Signal Transduction; D014162:Transfection; D020830:rac1 GTP-Binding Protein / Q000096:biosynthesis* / Q000235:genetics
D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D020558:GTP Phosphohydrolases; D020830:rac1 GTP-Binding Protein