Fig. 1. Erbb2^flox and HSA-CRE mice. (A) The Erbb2^flox locus and the gene targeting strategy. A loxP-neo/tk-loxP cassette was inserted into the intron 3' of the first coding exon of the Erbb2 gene, and additional loxP-site was inserted into the intron 5' to the exon (loxP-sites are shown as red triangles; the start site of transcription by an arrow). ES cells were transfected with a DNA fragment extending from the EcoRI site to the BamHI site. Targeted ES clones were identified by Southern blot, using a probe located immediately 3' of the targeting vector. One positive clone was retransfected with a Cre-expressing plasmid, and clones that had lost the neo-tk cassette, but retained two loxP sites flanking the first coding exon of the Erbb2 gene were identified by Southern blot using the same probe as described above. One positive clone was used to generate germ line transmitting chimeric mice. (B) Southern blot analysis of targeted ES cells containing two loxP sites; wt, no targeting; clones 173, 243, properly targeted ES cell clones. (C) The HSA-Cre construct used to generate the transgenic mouse lines. HAS, human skeletal -actin; NLS, nuclear localization signal; pA, polyadenylation signal. (D) Whole-mount lacZ staining of E12.5 (left panel) and E14.5 (right panel) embryos obtained from intercrosses between a Rosa26-lacZ^flox tester mouse with a HSA-Cre mouse. Arrows in the left panel indicate intercostal muscles, arrowheads highlight muscle groups in the forelimb; the arrow in the right panel marks recombined cells in the skin, the arrowheads mark muscle groups in forelimb. (E) Cross-section through forelimb of a E12.5 mouse embryo stained for lacZ. All muscle groups within the forelimb were lacZ positive (arrows), as well as some cells in the skin (arrowhead). (F) Extra- and intrafusal muscle fibers show strong nuclear lacZ staining (arrows), whereas perineural cells forming the spindle capsule are lacZ negative (arrowhead). Scale bars: 200 µm in D (left panel) and E; 500 µm in D (right panel); 50 µm in F.