Extended Data Fig. 2: Rb^XTR allows Cre-dependent inactivation and FlpO-dependent reactivation of Rb1.

a, Top, Rb^XTR (expressed): XTR gene trap cassette consists of a splice acceptor (SA), GFP complementary DNA ‘GeneTrap’, and the polyadenylation transcriptional terminator sequence (pA). Stable inversion is achieved by the use of two pairs of mutually incompatible mutant loxP sites (Lox2272 and Lox5171) arranged in the ‘double-floxed’ configuration. In the germline and in normal somatic cells, RB expression is normal in Rb^XTR/XTR mice¹⁰. Middle, Rb^TR (trapped): inhalation of Cre-expressing adenoviral or lentiviral vectors induces the permanent conversion of the Rb^XTR allele to the Rb^TR allele that inactivates Rb1 gene expression. Transcripts are spliced from the upstream exon to the GFP reporter gene and downstream transcription is terminated to functionally inactivate gene function. RB expression is inactivated only in the tumour cells. Bottom, Rb^R (restored): the Rosa26^FlpO-ERT2 allele enables tamoxifen-dependent conversion of trapped Rb^TR to its restored Rb^R allelic state via excision of the gene trap. b, Western blot analysis of 3 KP and 3 KP;Rb^TR/TR tumour-derived cell lines. c, Western blot analysis of 2 KP;Rb^TR/TR tumour-derived cell lines treated with Adeno-Cre as a control, or Adeno-FlpO to restore RB expression. d, Quantitative RT–PCR analysis of 3 KP;Rb^TR/TR tumour-derived cell lines treated with Adeno-FlpO to restore RB expression. Data are normalized to Adeno-Cre-treated cells (control). The log₂-normalized fold change in expression ± s.d. is shown. n = 3 technical replicates for each cell line. e, Volcano plot of differentially expressed genes from RNA sequencing data obtained from KP (n = 4) versus KP;Rb^TR/TR (n = 4) cell lines. The RB signature, defined by genes for which the change in expression is ≥2-fold and the P value adjusted for multiple testing is ≤ 0.05, is boxed. Statistical significance was determined by two-sided Wald’s test using Benjamini–Hochberg correction using DESeq2. f, Kaplan–Meier survival analysis of patients with lung adenocarcinoma whose tumours exhibit a high (n = 114 patients) or low (n = 189 patients) Rb signature. Significance was determined by two-sided log-rank test. P = 0.0175.

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