Fig. 1.
Generation of Hcn4lox/lox,Cre transgenic mice and cardiac specificity of the knockout process. (A) Structure of the wild type (wt) and recombinant (rec) Hcn4 alleles. The long and short arms of homology used for the recombination process and the loxP sites and the neomycin resistance cassette (neo) used for cell selection are shown. (B) Southern blot analysis of XbaI restriction fragments of DNA extracted from five clones; the two bands in lanes 3 and 5 confirm the presence of one recombinant allele. (C) The Cre-induced removal of exon 2 (Upper) eliminates a large part of the protein (Lower, shaded area) and generates a frameshift leading to an early stop codon three residues downstream of R262; this results in a DNA sequence coding for a truncated protein including only the HCN4 N terminus. (D) Tissue slices obtained from sinoatrial node (SAN), ventricle, brain, and skeletal muscle extracted from an MCM, ROSA26-EYFP transgenic mouse at day 5 of Tam treatment. The expression of enhanced yellow fluorescent protein (EYFP) is restricted to the SAN and the ventricle as expected for a cardiac conditional activation of Cre-recombinase. Each fluorescent image (Upper panels) is paired with the corresponding bright-field image (Lower panels). Further details are in SI Materials and Methods.