Figure 1
A novel RMCE DV3 system for generating conditional KI mice. (A) pRMCE-DV3 targeting vectors are made by multi-site Gateway cloning in which three pENTR vectors are recombined with a pRMCE-DV3 destination vector. The pENTR vectors contain either a floxed stop (LSL) cassette (triple repeat of SV40 polyA), a cDNA of the gene of interest (GOI) or an eGFP/luciferase reporter (eGFP/Luc). The conditional cassette within the pRMCE-DV3-GOI targeting vector is flanked by a wild type FRT (FRT wt) and a mutated FRT (FRT mut) site and is targeted to ROSALUC mESCs by FLPe-mediated RMCE. Correct integration of the cassette in the Rosa26 (R26) locus restores expression of the neomycin resistance gene (NeoR). Upon Cre-recombinase mediated removal of the floxed stop cassette, the GOI and the eGFP/Luc reporter are expressed from the endogenous R26 promoter. IRES: independent ribosomal entry site; ccdB: control of cell death B; PGK: ATG: translation initiation codon; PGK: phosphoglycerate kinase-1. (B) X-gal staining on parental and Cre-excised RMCE-DV3-LacZ mESCs. Scale bar: 100 µm. (C) Correlation between beta-galactosidase and luciferase activity upon Cre-excision of RMCE-DV3-LacZ mESCs.
