Medium-chain acyl-CoA dehydrogenase deficiency in gene-targeted mice

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common inherited disorder of mitochondrial fatty acid β-oxidation in humans. To better understand the pathogenesis of this disease, we developed a mouse model for MCAD deficiency (MCAD-/-) by gene targeting in embryonic stem (ES) cells. The MCAD-/- mice developed an organic aciduria and fatty liver, and showed profound cold intolerance at 4 °C with prior fasting. The sporadic cardiac lesions seen in MCAD-/- mice have not been reported in human MCAD patients. There was significant neonatal mortality of MCAD -/- pups demonstrating similarities to patterns of clinical episodes and mortality in MCAD-deficient patients. The MCAD-deficient mouse reproduced important aspects of human MCAD deficiency and is a valuable model for further analysis of the roles of fatty acid oxidation and pathogenesis of human diseases involving fatty acid oxidation. © 2005 Tolwani et al

Immunoblots of Liver Homogenates from MCAD^+/+ and MCAD^−/− Mice

<p>These were probed with anti-MCAD antibody or anti-SCAD antibody. Homozygous MCAD^−/− mice had no detectable MCAD protein. MCAD protein is only detectable under the MCAD protein–spiked (positive control) lane. As a control analysis, liver homogenates probed with anti-SCAD antibody revealed that SCAD protein was present in both MCAD^+/+ and MCAD^−/− mice. No MCAD positive-control protein is detected by anti-SCAD antibodies (MCAD lane). mw, molecular weight standards.</p

Northern Blot Analysis from MCAD^−/− (<i>n</i> = 2) and MCAD^+/+ (<i>n</i> = 2) Mice

<p><i>Acadm</i> message was detected from the heart, liver, brown fat, brain, kidney, and muscle (and white fat and testes, data not shown) of only MCAD^+/+ mice. Most robust expression occurred in brown fat, kidney, heart, and skeletal muscle. MCAD^−/− mice had no detectable message in all tissues examined.</p

Histopathology of MCAD^+/+ and MCAD^−/− Mice

<div><p>(A) MCAD^+/+ mice had no evidence of hepatic steatosis following a 24-h fast. Liver section with Oil-Red O stain.</p><p>(B) Hepatosteatosis in MCAD^−/− mouse following a 24-h fast. Oil-Red O stained liver sections revealed severe and diffuse microvesicular and macrovesicular hepatic steatosis in MCAD^−/− mice.</p><p>(C and D) Diffuse cardiomyopathy with chronic active multifocal myocyte degeneration and necrosis in MCAD^−/− mice.</p></div

Strategy for Disruption of the Mouse <i>Acadm</i> Gene

<div><p>(A) The MCAD IV2 insertion targeting vector with a deleted 1.3-kb region encompassing exon 10 and flanking sequences. MCAD IV2 undergoes gap repair upon homologous recombination at the endogenous <i>Acadm</i> locus resulting in a duplication of exons 8, 9, and 10 at the disrupted allele.</p><p>(B) Southern blot analysis of EcoRI-digested genomic DNA from ES cells screened by PCR. Probe A, a DNA fragment consisting of a portion of exon 10 that is not present in the targeting vector, hybridizes to an endogenous 3.1-kb fragment and, upon homologous recombination, to a 13.2-kb fragment. Lane 1 represents a wild-type ES cell line, and Lane 2 and 3 represent targeted ES cell lines.</p></div

Acylcarnitine Analyses

<div><p>(A) Serum acylcarnitine analysis of MCAD^+/+ (<i>n</i> = 4) and MCAD^−/− mice (<i>n</i> = 4)</p><p>There are significant elevations in acylcarnitine species as indicated in MCAD^−/− mice. Values shown are mean values ± standard deviation (SD). Asterisk indicates <i>p</i> < 0.002 and ‡ indicates <i>p</i> < 0.01.</p><p>(B) There are significant elevations in bile acylcarnitines of the same mice shown in (A) as indicated. Values shown are mean values ± SD. Asterisk indicates <i>p</i> < 0.001.</p><p>(C) Bile acylcarnitine profile of an MCAD^−/− mouse compared to a human patient with MCAD deficiency. Internal standards are indicated by an asterisk.</p></div