RMCE as described by Seibler and co-workers [36]. A: The Rosa26 allele is targeted with a cassette encoding the ΔATG-zsGreen gene, a Hygromycin resistance (Hyg^R) marker and the FLPe recombinase. Constitutive PGK and CAGGS promoters control the expression of Hyg^R and FLPe, respectively, whereas expression of zsGreen is controlled by the endogenous Rosa26 gene promoter prior to the F3 recombination site. A FRT recombination site flanks the 3′ end of the targeted locus. B: Introduction of doxycycline-inducible shRNA expression constructs via FLPe recombinase-mediated cassette exchange is achieved by co-transfection of the targeted ESCs with a FLPe expression plasmid and an RMCE exchange cassette encoding an F3 site, Δ5′ Neomycin resistance (Neo^R) marker, the H1-tet promoter, an shRNA of choice, the enhanced tet-repressor itetR controlled by the CAGGS promoter and an FRT site. C: The original RMCE exchange vector is ideally suited for LoF vectors. By replacing the region between the dotted lines, named the creative region, these vectors can be adapted to become GoF vectors. Two examples are provided, an inducible cDNA and a tissue-specific CreERT2 gene for spatiotemporal controlled deletion of genes flanked by loxP sites.

Side-by-side comparison for investment in time and costs between conventional targeting in wild-type ESCs and the GEMM-ESC approach. A typical example is given for a tumor model with four modified alleles, i.e. Rb^F/F;Trp53^F/F, in which one additional allele is introduced, a luciferase reporter. A conditional luciferase expression construct is introduced in wild-type ESCs or Rb^F/F;Trp53^F/F ESCs via either classic targeting or RMCE, respectively. Both approaches require blastocyst injections, though the GEMM-ESCs require more injections to directly establish a cohort of F0 chimeras. For the conventional wild-type ESC approach the number of crosses is shown to establish a similar cohort of compound mutant mice of the desired genotype. The ratio of mice with the right genotype generated from each cross is indicated. Note that the GEMM-ESC approach will deliver a considerable gain in time to establish a cohort (approximately 14 months), and a reduction in costs as the initial higher costs for chimera production are gained back by the absence of any breeding.

Non-germline chimeric GEMMs. A: Current method to obtain non-germline chimeric GEMMs by step-wise modification of wild-type ESCs by introduction of targeted mutations in tumor suppressor genes (TSGs) and random integration of transgenes for doxycycline (DOX) regulatable expression of luciferase reporter and oncogenes [8, 12]. B: Outline for GEMM-ESC strategy. ESCs are derived from an existing conditional GEMM containing all the modifications needed to develop a particular type of tumor. The GEMM-derived ESC are validated and targeted with a RMCE acceptor allowing for easy reengineering of the GEMM-ESCs using exchange vectors containing GoF or LoF alleles. Tumors are induced in chimeric animals.

Generation of repositories of RMCE-compatible ESCs from various GEMMs and collections of GoF and LoF vectors enables rapid in vivo validation of candidate cancer genes and drug targets identified in functional genomics screens or cancer genome sequencing projects [5]. Collections of RMCE-compatible reporter vectors permit in vivo monitoring of tumor-related processes via non-invasive imaging techniques.