Establishment Of A Stable GFP-V12Rac1 NIH3T3 Cell Line

Rac1 activity often enriches at the leading edge of migrating cells. Such a mode of Rac1 activity distribution is important for cell motility. Nevertheless, mechanisms underlying this localization remain to be elucidated. To study these mechanisms, we need to establish a cell line stably expressing GFP-V12Rac1 first. Plasmid and lentiviral expression vectors for GFP or GFP-V12Rac1 (constitutively active Rac1 fused with an N-terminal GFP tag) were constructed. NIH3T3 cells were infected with lentiviral vectors, and cells stably expressing genes of interest were selected by flow cytometry and tested. Percentages of cells positive and levels of expression for genes of interest were analyzed by flow cytometry after multiple passages. A 7-AAD and APC Annexin V double staining assay was used to assess survival of NIH3T3 cells expressing GFP-V12Rac1 after overnight serum starvation. Cell spreading and ruffling assays were used to confirm that exogenous GFP-V12Rac1 was of normal function. A Boyden chamber assay was used to test the intrinsic and chemotactic motility of established cell lines. Finally, localization of GFP-V12Rac1 in migrating cells under a wound healing model was observed using the established cell lines. According to the results, most cells of the established cell lines stably expressed genes of interest after multiple passages; exogenous GFP-V12Rac1 didn't affect survival of the cell lines after a 12-hour serum starvation; exogenous GFP-V12Rac1 promoted cell spreading and ruffling; established cell lines retained the ability of chemotaxis. To conclude, lentiviral infection plus flow cytometry selection can be used to establish stable NIH3T3 cell lines; exogenous GFP-V12Rac1 was of normal function; the established cell lines can be used to observe the localization of activated Rac1 in migrating cells.