Retroviral-mediated transduction of normal and patient cells with Rac2-containing vectors.

(A) Recombinant retroviral vectors constructed as described in “Materials and methods” were based on MIEG3, a modified MSCV vector, and contain either the WT Rac2 sequence (HR2WT) or the D57N mutant cDNA derived from the patient (HR2MU). Also shown is the vector expressing the Flag-tagged WT murine Rac2 (MIEG3FR2). LTR, long terminal repeat; IRES, internal ribosome entry site; EGFP, enhanced green fluorescent protein. (B) Flow analysis of NIH/3T3 cells infected with respective retrovirus vectors and analyzed for expression of GFP; horizontal axis shows GFP expression. (C) Western blot analysis of transduced and sorted NIH/3T3 cells for Rac2 protein. Expression of the Rac2 protein is identified in NIH/3T3 cells infected with HR2MU (patient mutant cDNA, lane 2), HR2WT (WT cDNA, lane 4), MIEG3FR2 (murine Flag-tagged Rac2, lane 5), and the viral producer cell expressing MIEG3FR2 virus (lane 6). No expression is seen in NIH/3T3 cells or NIH/3T3 cells infected with the virus expressing only EGFP (lane 1,3). The size of the murine Rac2 protein is slightly higher because of the Flag tag. The faint cross-hybridizing bands seen in lanes 1 and 3 are caused by cross-hybridization with Rac1.21