Figure 1. The “knockout-first” conditional construct for use at the Ampd3 genomic loci.

A. Recombination at the 5′ and 3′ homologous arms of the vector to the target sequences of the Ampd3 gene allows the insertion of an en2-LacZ-neo cassette into a region spanning the intron between exon 5 and exon 6 of the Ampd3 locus. With the “knockout-first” design, the 3′ end of exon 5, the first coding exon, is spliced into the splice acceptor (SA) site of the en2-LacZ-neo cassette in order to disrupt and destabilize the Ampd3 transcript. B. RT-PCR showing that Ampd3 mRNA from the heart of wild type (WT) mice is absent in Ampd3^−/− heart tissue. “-RT” indicates the negative control where reverse transcriptase was lacking in parallel reactions.