Figure 1: XTR alleles facilitate Cre-mediated inactivation and subsequent Flp-dependent restoration of endogenous genes.

(a) Cre converts XTR alleles to the TR allele, thereby inactivating gene function. Flp restores gene function by conversion to R. (b) Schematic of the XTR allele. Cre drives irreversible inversion of a double-floxed gene trap consisting of a splice acceptor (SA) enhanced GFP complementary DNA and the polyadenylation transcriptional terminator sequence (pA). Inversion can proceed either through sequential action of Cre on Lox2272 sites then Lox5171 sites (2272 intermediate) or Lox5171 then Lox2272 sites (5171 intermediate). Stable inversion accepts splicing from upstream exons in the host gene, reads out GFP expression and then terminates transcription, leading to functional inactivation of the host gene’s expression. Flp drives deletion of the gene trap (SA-GFP-pA), thereby restoring normal splicing of the host gene. AdCre and AdCre followed by AdFlpO treatment is indicated (c,d). PCR-based detection of Rb (c) and p53 (d) XTR, TR, R and wild-type (+) alleles in MEFs of the indicated genotype. (e) Detection of GFP reporter expression from TR alleles in Rb^TR/TR and p53^TR/TR MEFs by flow cytometry analysis. Representative of ⩾3 cell lines. (f) Immunoblot analysis of Rb and GFP expression in Rb^XTR/XTR MEFs treated sequentially with AdCre and/or AdFlpO as indicated. β-Tubulin is a loading control. (g) Immunoblot analysis of p53 and GFP expression in p53^XTR/XTR MEFs treated sequentially with AdCre and/or AdFlpO as indicated. Hsp90 is a loading control. (h) 3T3 proliferation assay of p53^XTR/XTR MEFs treated sequentially with AdCre (day 3) then AdFlpO (day 21) as indicated. Representative of two p53^XTR/XTR cell lines.