The principle of dRMCE to re-engineer mouse conditional alleles. (a) Schematic of the target locus shows the configuration of a conditional mouse allele with a genomic region flanked by two loxP sites and an outside selection cassette flanked by two FRT sites. Upon transfection, the combination of iCre- and Flpo-mediated recombination in cis results in a deleted allele flanked by single loxP and FRT sites, which serves as a ‘docking site’ for insertion of the replacement vector. ex, exon. (b) Schematic representation of replacement in the Smad4 locus by dRMCE. The target locus is a Smad4 conditional allele (Smad4^f) with a promoterless selection cassette. Co-transfection of the pDIRE and pDREV-1 plasmids induces replacement, probably through production of the Smad4⁻ deleted allele as intermediate. Correct trans insertion of the replacement vector results in the Smad4^YFP allele. F1–F4 and R1–R3 denote primers used for PCR screening of colonies (see supplementary table 2 for sequences). H2B-Venus, YFP fusion protein with histone 2B; lacZ, β-galactosidase coding region; neo, neomycin resistance coding region; puro, puromycin resistance cassette; rox, Dre recombinase target sites¹¹; SA, splice acceptor; T, autocleavable T2A peptide coding region¹⁴. (c) PCR screening reveals a large number of clones with correct 3′ and 5′ replacement (69%). Col, colony; 3′ recombination, 5′ recombination, correct replacement at the 3′ and 5′ ends, respectively. (d) The parental Smad4^f cells are β-galactosidase positive. (e,f) Clones with correct replacement (Smad4^YFP) lack β-galactosidase activity but show YFP fluorescence. (g) Micrograph shows single cells expressing the H2B-Venus fusion protein engineered by dRMCE. Scale bars: 100 μm (d–f), 5 μm (g).

dRMCE for efficient modification of difficult-to-target loci. (a) Schematic shows the conditional Hand2 allele (Hand2^f) used as a target locus for insertion of a Flag epitope tag into the Hand2 coding region. After co-transfection of the replacement vector (pRVH2) and pDIRE plasmid into heterozygous Hand2^f mouse embryonic stem cells, dRMCE-mediated replacement results in the Hand2^Flag allele. The PGK-hygro (hygromycin resistance gene) selection cassette is flanked by the attB and attP target sites for excision by the ɸC31 recombinase. F5–F7 and R5–R7 denote primers used for PCR screening and genotyping. E, EcoRV site required to detect correct 5′ replacement by combining PCR amplification with an EcoRV restriction digestion. (b) PCR screening at both ends of the locus identified Hand2 colonies with correct replacement (13%). Scheme at right shows PCR fragment patterns indicative of particular genomic configurations. 3′ recombination, 5′ recombination, correct replacement at the 3′ and 5′ ends, respectively. A, B, primers to amplify a region serving as positive control (see supplementary table 2 for sequences). (c) Gels show germline transmission of the Hand2^Flag allele (lanes 1, 5, 6). Above, PCR analysis to detect Hand2^Flag allele. Below, PCR detection of wild-type allele. (d) In situ detection of Hand2 transcripts in a wild-type (Hand2^+/+; left) and Hand2^Flag/Flag (right) mouse embryo at embryonic day 10.5. Note expression in the posterior limb bud mesenchyme (arrow), branchial arches (black arrowhead) and heart (white arrowhead). Scale bar, 500 μm.