Fig. 6

GLuc monitoring of SCLC induced by adenoviral sgRNA delivery to Cas9 mice. a Experimental scheme for SCLC induction and monitoring with adenoviral vectors (AV) expressing Cre and Trp53/Rb1/Rbl2-targeting sgRNAs. b Validation of sgRNA function by T7 endonuclease I Assay using genomic DNA from uninfected and AV-PRL.Cre infected NIH3T3-Cas9 cells. NTC, no template control. c Western Blot of LSL-Cas9 fibroblasts infected with AV-PR.Cre and AV-PRL.Cre showing downregulation of p53, Rb1 and Rbl2/p130 protein levels. β-actin is shown as loading control. d Immunohistochemistry for GLuc and Cas9 expression in the lung of mice 2 weeks post infection. Scale bar, 50 µm. e Mutation spectrum of Trp53, Rb1 and Rbl2 sgRNA target loci (flanked by dashed lines). f Kaplan–Meier survival plot of LSL-GLuc mice infected with indicated CRISPR-AVs (AV-C.Cre: n = 14; AV-PR.Cre: n = 12; AV-PRL.Cre: n = 8). Shown are median survival and P-values from Log-rank (Mantel-Cox) test. g Temporal development of blood GLuc activity in mice from f following infection with indicated AVs. Shaded area represents the GLuc background activity. h Total fold change in blood-GLuc activity over the course of tumor development. P-values from Tukey’s multiple comparisons test. i Time point when blood-GLuc activity reached its maximum. P-value from an unpaired, two-sided t-test. j Time difference between the time of sacrifice (survival) and the time point when blood-GLuc activity was first elevated, i.e. exceeded the background range. k Correlation between time of sacrifice (survival) and the time point when blood-GLuc activity was first elevated. Shown is the linear regression with 95% confidence interval All error bars indicate SD, all data points represent biological replicates/individual mice