FIG.1.

RMCE in Lrp1. (A) Principle of RMCE. Due to recombination (indicated by X) between identical FRT sites by a transiently expressed FLP recombinase, an HygTK exchangeable cassette, present in a particular locus and flanked by heterospecific FRT sites (filled and open triangles), is exchanged by a Neo replacement cassette, present in a circular plasmid and also flanked by the same two heterospecific FRT sites. By application of positive (G418) and/or negative (ganciclovir) selection, clones resulting from exchange between the HygTK cassette and the Neo cassette can be selected for. (B) Homologous recombination (HR) replacing the 3′ end part of the Lrp1 gene (3′ part of intron 75 to sequences downstream of exon 89) by an exchangeable cassette (1), which can subsequently be replaced via RMCE (2) with initially removed Lrp1 coding sequences (genomic and cDNA sequences) restoring the Lrp1 gene, either wild type or mutant (3). For selection purposes, a Neo expression cassette flanked by LoxP sites (black triangles) was included in the replacement cassette, downstream of the Lrp1 sequences. The relative positions of the probes A and B and the PCR primer pairs (AB→GH) for monitoring the recombination events are indicated. Restriction enzyme sites are as follows: E, EcoRI; H, HindIII; K, KpnI; N, NotI; S, SacI. (C) Southern blot analyses of ES cell DNA proving correct homologous recombination in the Lrp1 gene in E14 ES cells (HindIII digest and probe A). (D) Southern blot analyses of ES cell DNA showing successful application of RMCE restoring the Lrp1 gene (KpnI digest and probe B). (E) PCR analyses of tail DNA with primer pair EF, proving the presence of the 5′ end FRT site in the Lrp1 gene restored by RMCE.