ABSTRACT
Mutations in subunits of succinyl-CoA synthetase/ligase (SCS), a component of the citric acid cycle, are associated with mitochondrial encephalomyopathy, elevation of methylmalonic acid (MMA), and mitochondrial DNA (mtDNA) depletion. A FACS-based retroviral-mediated gene trap mutagenesis screen in mouse embryonic stem (ES) cells for abnormal mitochondrial phenotypes identified a gene trap allele of Sucla2 (Sucla2SAβgeo), which was used to generate transgenic mice. Sucla2 encodes the ADP-specific β-subunit isoform of SCS. Sucla2SAβgeo homozygotes exhibited recessive lethality, with most mutants dying late in gestation (e18.5). Mutant placenta and embryonic (e17.5) brain, heart and muscle showed varying degrees of mtDNA depletion (20–60%). However, there was no mtDNA depletion in mutant liver, where the gene is not normally expressed. Elevated levels of MMA were observed in embryonic brain. SCS-deficient mouse embryonic fibroblasts (MEFs) demonstrated a 50% reduction in mtDNA content compared with wild-type MEFs. The mtDNA depletion resulted in reduced steady state levels of mtDNA encoded proteins and multiple respiratory chain deficiencies. mtDNA content could be restored by reintroduction of Sucla2. This mouse model of SCS deficiency and mtDNA depletion promises to provide insights into the pathogenesis of mitochondrial diseases with mtDNA depletion and into the biology of mtDNA maintenance. In addition, this report demonstrates the power of a genetic screen that combines gene trap mutagenesis and FACS analysis in mouse ES cells to identify mitochondrial phenotypes and to develop animal models of mitochondrial dysfunction.
Footnotes
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Competing interests
The authors declare no competing financial interests.
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Author contributions
T.D., C.S., W.J.C. and B.H.G. designed research; T.D., C.S., M.G., K.W.E. and B.H.G. performed research; T.D., C.S., W.J.C. and B.H.G. analyzed data; and T.D. and B.H.G. wrote the paper.
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Funding
This work was supported in part by the National Institutes of Health [grant numbers R03 AR052112 to B.H.G., R21 HL084239 to W.J.C. and R01 GM098387 to B.H.G.]. This work was also supported in part by March of Dimes [grant numbers 5-FY05-96 to B.H.G. and 1-FY07-507 to B.H.G.]. The project described was also supported by the Eunice Kennedy Shriver National Institute of Child Health & Human Development [award number P30HD024064]. This project was also supported by the Cytometry and Cell Sorting Core at Baylor College of Medicine with funding from the National Institute of Health [grant numbers AI036211, CA125123 and RR024574].
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Supplementary material
Supplementary material available online at http://dmm.biologists.org/lookup/suppl/doi:10.1242/dmm.013466/-/DC1
- Received June 27, 2013.
- Accepted November 18, 2013.
- © 2014. Published by The Company of Biologists Ltd
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.
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