AMPD activity is not detectable in erythrocytes (rbc) and is reduced in the heart of <i>Ampd3^−/−</i> mice.

<p>A. AMP deminase assays were carried out with cell lysates from the indicated organs. For all samples, except serum (WT n = 10; <i>Ampd3^−/−</i> n = 5), heart (n = 8) and erythrocytes (WT n = 15; <i>Ampd3^−/−</i> n = 7), the data represent the average of tissue samples (n = 3). B. The level of AMPD3 activities in erythrocytes from wild type, <i>Ampd3^+/−</i> and <i>Ampd3^−/−</i> mice. Error bars, mean ± SEM. T-test: *p<0.05.</p

Erythrocytes of <i>Ampd3^−/−</i> mice have elevated levels of ATP and ADP but normal levels of AMP.

<p>A. Nucleotides from methanol extracts of erythrocyte lysates from wild type (n = 4) and <i>Ampd3^−/−</i> (n = 4) were separated by HPLC and quantified by calibration with standards. B. Representative HPLC chromatograms from wild type and <i>Ampd3^−/−</i> erythrocyte nucleotide extracts. C. Fasting glucose levels in <i>Ampd3^−/−</i> (n = 16) and <i>Ampd3^+/−</i> mice (n = 9) and WT (n = 14) mice. Error bars, mean ± SEM. T-test: *p<0.05.</p

The “knockout-first” conditional construct for use at the <i>Ampd3</i> genomic loci.

<p>A. Recombination at the 5′ and 3′ homologous arms of the vector to the target sequences of the <i>Ampd3</i> gene allows the insertion of an en2-LacZ-neo cassette into a region spanning the intron between exon 5 and exon 6 of the <i>Ampd3</i> 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 <i>Ampd3</i> transcript. B. RT-PCR showing that <i>Ampd3</i> mRNA from the heart of wild type (WT) mice is absent in <i>Ampd3^−/−</i> heart tissue. “-RT” indicates the negative control where reverse transcriptase was lacking in parallel reactions.</p