Fig. 2

Tumor suppressor genes knockout by RCAS-gRNA plasmids induce high-grade-gliomas. a Schematic illustration of the RCAS-gRNA plasmids. b In vitro validation of the RCAS-gRNA against Trp53, Pten, Cdkn2a and a non-targeting control (Ctrl). Western blot analysis, using the indicated antibodies, on whole cell extracts from NIH-3T3 TVA-Cas9 fibroblasts and Ntv-a; LSL-Cas9 neural stem cells (NSCs) transduced with pMSCVhygro-CRE (NSC TVA-Cas9). To induce p53 expression, the cells were collected 24 h after exposure to ionizing radiation (10 Gy). c Table summarizing the injections performed in the Ntv-a; Nes-Cre; LSL-Cas9 and Gtv-a; hGFAP-Cre; LSL-Cas9 pups and adult mice. Co-injection of RCAS-PDGFB and the RCAS-gRNA against different tumor suppressor genes accelerate tumor formation, increases the tumor penetrance and the frequency of high-grade gliomas. d Hematoxylin and eosin (H&E) and immunohistochemical stainings (IHCs), using the indicated antibodies, of representative RCAS-PDGFB/gRNA tumors. To note PTEN expression in the normal vasculature but not in the tumor cells of the RCAS-PDGFB + RCAS-Pten-gRNA tumor. Insets show higher magnification images. Scale bars: H&E 100 μm; IHCs 50 μm. e Western blot analysis, using the indicated antibodies, on whole cell extracts from tumorspheres. f IHCs for p53 (left panels) and p21 (right panels) on the indicated tumors 1 h after exposure to 10 Gy IR. Scale bars: 50 μm. g Western blot analysis, using the indicated antibodies, on whole cell extracts from tumorspheres 24 h after exposure to 10 Gy IR. h Quantitative real-time PCR (qPCR) analysis on mRNA extracted from tumorspheres 3 h after exposure to 10 Gy IR. Data presented as mean ± SD (n = 3); A.U. arbitrary unit