Fig. 1. Targeting strategy and molecular analysis of recombinant ES cells and conditional ß4 knockout mice. (A) Restriction map of the 5' end of the ß4 locus, the targeting construct and different ß4 mutant alleles. Exons are indicated as numbered black boxes and loxP sites as triangles. Position of the unique SacII and KpnI sites used for insertion of the single loxP and the floxed neo-tk cassette are indicated, as are the primers used for PCR analyses to detect the different mutant alleles of the ß4 gene. Restriction sites are: H, HindIII; RI, EcoRI; X, XhoI; RV, EcoRV. (B) Southern blot and PCR analyses of recombinant ES clones. DNA from two independently targeted ES clones (lanes 1, 3) and two wild-type ES clones (lanes 2, 4) were digested with HindIII (left panel) or EcoRV (right panel), subjected to agarose gel electrophoresis, and transferred to nitrocellulose. Wild-type and mutant alleles were detected by hybridization of the filters with radiolabeled mouse ß4 genomic probes corresponding to exons 2-7 (5' probe) or exons 7-9 (3' probe) (left and right panels, respectively). The presence of the first loxP site in intron 1a of the targeted ES clones was confirmed by PCR using primers P1 and P2 (bottom panel). (C) PCR analysis on genomic DNA of conditional ß4 knockout mice before and after their crossing with K14-Cre mice. The conditional allele of the ß4 gene was detected by PCR analysis on tail DNA using primers P3 and P4 (Before and After K14-Cre, left and top right panel), and the K14-Cre transgene using the primers K14-cre3 and K14-cre5 (After K14-Cre, middle panel). The removal of exons 1b-5 by Cre-mediated recombination, thereby generating a ß4 null allele, was detected by PCR analysis using primers P1 and P4 (After K14-Cre, bottom panel). PCR fragments were resolved by agarose gel electrophoresis and visualized by ethidium bromide. Bands corresponding to the wt, floxed and null alleles of the ß4 gene as well as to the K14-Cre transgene are indicated (wt, flox, null, K14). Sizes of molecular weight markers are indicated in bp.