Fig. 1

Generation of the Dmd ^EGFP reporter mouse. a Construction of the targeting vector through insertion of a FLAG-EGFP coding sequence into the 3′UTR of the Dmd gene. The termination codon of exon 79 was altered to a leucine coding triplet. The asterisk indicates the modified exon 79. The Dmd wild-type allele was targeted in ES cells after electroporation of the SalI linearized targeting vector and subsequent homologous recombination. Dashed lines represent homologous regions. BglII and BamHI restriction sites were artificially inserted into the construct to facilitate identification of targeted ES cells by Southern blot analysis using either 5′ and 3′ hybridization probes (blue squares) located outside of the targeting vector. Recombinant ES cells were then used for generation of transgenic mice. The neo cassette was removed by crossing the F1 generation mice with ubiquitous Cre-deleter mice (constructs are not drawn to scale). b PCR genotyping of the wild-type (202 bp) and recombinant (350 bp) allele. −/− homozygous wild-type female, y/+ hemizygous male for the Dmd ^EGFP allele, +/− heterozygous female. c Western blot analysis of whole TA muscle extract using dystrophin antibodies against the rod (Dys1) and the C-terminal domain (H4) with normal expression of the full-length Dp427 dystrophin in the Dmd ^EGFP mice. The anti-GFP antibody detected the tagged Dp427-EGFP protein only in the transgenic animal. d TA cross and longitudinal sections show natural EGFP fluorescence (green) in Dmd ^EGFP mice that can be detected at the sarcolemma without amplification of the signal. No fluorescent signal above background was observed in the wild-type cross section using the identical illumination parameters