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Increased postischemic brain injury in mice deficient in uracil-DNA glycosylase
Matthias Endres, … , Andreas Meisel, Rudolf Jaenisch
Matthias Endres, … , Andreas Meisel, Rudolf Jaenisch
Published June 15, 2004
Citation Information: J Clin Invest. 2004;113(12):1711-1721. https://doi.org/10.1172/JCI20926.
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Article Neuroscience

Increased postischemic brain injury in mice deficient in uracil-DNA glycosylase

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Abstract

Uracil-DNA glycosylase (UNG) is involved in base excision repair of aberrant uracil residues in nuclear and mitochondrial DNA. Ung knockout mice generated by gene targeting are viable, fertile, and phenotypically normal and have regular mutation rates. However, when exposed to a nitric oxide donor, Ung–/– fibroblasts show an increase in the uracil/cytosine ratio in the genome and augmented cell death. After combined oxygen-glucose deprivation, Ung–/– primary cortical neurons have increased vulnerability to cell death, which is associated with early mitochondrial dysfunction. In vivo, UNG expression and activity are low in brains of naive WT mice but increase significantly after reversible middle cerebral artery occlusion and reperfusion. Moreover, major increases in infarct size are observed in Ung–/– mice compared with littermate control mice. In conclusion, our results provide compelling evidence that UNG is of major importance for tissue repair after brain ischemia.

Authors

Matthias Endres, Detlev Biniszkiewicz, Robert W. Sobol, Christoph Harms, Michael Ahmadi, Andreas Lipski, Juri Katchanov, Philipp Mergenthaler, Ulrich Dirnagl, Samuel H. Wilson, Andreas Meisel, Rudolf Jaenisch

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Figure 1

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Generation of Ung–/– mice and the resulting DNA, RNA, and activity analy...
Generation of Ung–/– mice and the resulting DNA, RNA, and activity analysis. (A) Targeting strategy for inactivation of the ung gene. Lines (from top to bottom) show the map of the ung genomic locus, which consists of six exons (and exon 1a), the structure of the targeting construct (neo construct), and the resulting mutant (neo) allele after homologous recombination. Homologous recombination leads to the deletion of both mitochondrial (UNG1, exon 1a) and nuclear (UNG2) isoforms of uracil DNA glycosylase. Relevant Southern blot analysis was performed with the indicated 3′ outside probe. EcoRI digestion results in an 8.2-kb fragment in wild-type and a 5.2-kb fragment in homologus recombinations. (B) Exemplary Southern blot analysis of EcoRI-digested DNA from ES cells and mouse tissue (tails). neo, mutant. (C) Activity assays that measured the ability of Ung+/+ and Ung–/– ES cell lysates to excise uracil from an end-labeled oligonucleotide substrate demonstrated no detectable enzymatic activity in the knockout cells (independent experiments). (D) RT-PCR in which Ung+/+ and Ung–/– ES cells were used, demonstrated residual transcription of exons 1a and 6 in the recombined ung genomic locus. The promoter region for exon 1a was left intact by the knockout strategy and allowed residual transcription.

Copyright © 2022 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)

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