Medium-chain acyl-CoA dehydrogenase deficiency in gene-targeted mice - PubMed (original) (raw)

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

Ravi J Tolwani et al. PLoS Genet. 2005 Aug.

Abstract

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common inherited disorder of mitochondrial fatty acid beta-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 degrees 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.

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Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1. Strategy for Disruption of the Mouse Acadm Gene

(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 Acadm locus resulting in a duplication of exons 8, 9, and 10 at the disrupted allele. (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.

Figure 2. Northern Blot Analysis from MCAD−/− (n = 2) and MCAD+/+ (n = 2) Mice

Acadm 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.

Figure 3. Immunoblots of Liver Homogenates from MCAD+/+ and MCAD−/− Mice

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.

Figure 4. Acylcarnitine Analyses

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

Figure 5. Histopathology of MCAD+/+ and MCAD−/− Mice

(A) MCAD+/+ mice had no evidence of hepatic steatosis following a 24-h fast. Liver section with Oil-Red O stain. (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. (C and D) Diffuse cardiomyopathy with chronic active multifocal myocyte degeneration and necrosis in MCAD−/− mice.

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Medium-chain acyl-CoA dehydrogenase deficiency in gene-targeted mice - PubMed (original) (raw)