Rac1 and Tumor Angiogenesis mice, a lentiviral vector for conditional, Cre-lox regulated, stable RNA interference (RNAi) was employed (pSico-Rac1). This vector allows tissue-specific activation of Rac1 short hairpin RNA (shRNA) in Cre-expressing cells in vivo. As a positive control (for inhibiting angiogenesis), pSico-Flk-1 was employed for the depletion of Flk-1 (VEGFR2). As a negative control pSico-Con, targeting firefly luciferase was also used. The efficiency of the system was first tested in vitro. The pSico plasmid contains a GFP-reporter that is excised after Cre- mediated recombination and shRNA expression. Wild-type primary endothelial cells infected with pSico-Rac1 lentiviruses were analyzed for GFP-expression and showed high-efficiency transduction. GFP-positive cells were then isolated by FACS and transfected with a Cre expression-plasmid (pTURBO-Cre). Near complete recombination, with a subsequent loss of GFP expression, was observed for all transduced cells (P,0.01, Figure 2A). Moreover, a significant reduction of both Rac1 and Flk-1 mRNA and protein levels was achieved in pSico-Rac1 or pSico-Flk-1 transduced cells, respectively. As expected, no significant changes in either mRNA or protein levels of Rac1 or Flk-1 were observed in pSico-Con infected cells (Figure 2B). To study the effect of pSico-Rac1 on tumor growth, wild-type/ Tie1-Cre+ and b3-null/Tie1-Cre+ mice were injected subcutane- ously with murine B16F0 melanoma cells and pSico-Con, pSico- Rac1 or pSico-Flk-1 lentiviral suspensions injected intratumorally on days 5 and 10 after tumor cell inoculation. In line with our previous findings [12,27], tumor size in pSico-Con treated b3- null/Tie1-Cre+ mice was greater than similarly treated wild-type/ Tie1-Cre+ mice. Results also showed that although pSico-Flk-1 treatment reduced significantly B16F0 melanoma size (P,0.05) in both genotypes, when compared with pSico-Con controls, pSico- Rac1-treatment did not affect tumor size in wild-type/Tie1-Cre+ mice, but significantly reduced tumor size in b3-null/Tie1-Cre+ mice (Figure 3A). To determine the efficacy of the lentiviruses in vivo, double- immunostaining for PECAM-1 and GFP was performed in sections of tumors grown in wild-type/Tie1-Cre2 or wild-type/ Tie1-Cre+ mice treated with either pSico-Rac1 or pSico-Flk-1 Figure 2. Cre-regulated depletion of Rac1. A. Wild-type primary endothelial cells were infected with pSico-Rac1 lentivirus. High-efficiency transduction was achieved as indicated by uniform GFP expression in infected cells (left panel). Cells sorted for GFP positivity and transfected with a Cre-recombinase expression plasmid (pTURBO-Cre) showed a significant loss of GFP (right panel). Scale bar: 10 mm. B. One week after pTURBO-Cre transfection, levels of mRNA, detected by semi-quantitative RT-PCR (upper panels), and protein, detected by Western blotting (lower panels), showed successful Rac1- and Flk-1-depletion in pSico-Rac1 and pSico-Flk-1 infected cells, respectively. Actin RT-PCR and Western blotting for Hsc-70 were carried out to ensure equal RNA and protein loading, respectively. Bar graphs represent densitometric readouts of mRNA of relative Actin or Hsc-70 protein levels, respectively. *P,0.01. N = 3 independent experiments. doi:10.1371/journal.pone.0009766.g002 PLoS ONE | www.plosone.org 3 March 2010 | Volume 5 | Issue 3 | e9766