This Article Free Full Text Free Full Text Full Text Full Text (PDF) Free Full Text All Versions of this Article: jhc.2009.954701v1 jhc.2009.954701v2 58/2/207    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Lentz, S. I. Articles by Feldman, E. L. Search for Related Content PubMed PubMed Citation Articles by Lentz, S. I. Articles by Feldman, E. L. Social Bookmarking                         What's this?

Mitochondrial DNA (mtDNA) Biogenesis: Visualization and Duel Incorporation of BrdU and EdU Into Newly Synthesized mtDNA In Vitro

Stephen I. Lentz, James L. Edwards, Carey Backus, Lisa L. McLean, Kristine M. Haines and Eva L. Feldman

Department of Internal Medicine, Division of Metabolism, Endocrinology, and Diabetes (SIL), Department of Neurology (JLE,CB,LLM,ELF), Michigan Research Community, Undergraduate Research Opportunity Program (KMH), and A. Alfred Taubman Medical Research Institute (ELF), University of Michigan, Ann Arbor, Michigan

Correspondence to: Stephen I. Lentz, PhD, University of Michigan Department of Internal Medicine, 3062 BSRB, 109 Zina Pitcher Place, Ann Arbor, MI 48109. E-mail: lentzs{at}umich.edu

Mitochondria are key regulators of cellular energy and are the focus of a large number of studies examining the regulation of mitochondrial dynamics and biogenesis in healthy and diseased conditions. One approach to monitoring mitochondrial biogenesis is to measure the rate of mitochondrial DNA (mtDNA) replication. We developed a sensitive technique to visualize newly synthesized mtDNA in individual cells to study mtDNA replication within subcellular compartments of neurons. The technique combines the incorporation of 5-bromo-2-deoxyuridine (BrdU) and/or 5-ethynyl-2'-deoxyuridine (EdU) into mtDNA, together with a tyramide signal amplification protocol. Employing this technique, we visualized and measured mtDNA biogenesis in individual cells. The labeling procedure for EdU allows for more comprehensive results by allowing the comparison of its incorporation with other intracellular markers, because it does not require the harsh acid or enzyme digests necessary to recover the BrdU epitope. In addition, the utilization of both BrdU and EdU permits sequential pulse–chase experiments to follow the intracellular localization of mtDNA replication. The ability to quantify mitochondrial biogenesis provides an essential tool for investigating the alterations in mitochondrial dynamics involved in the pathogenesis of multiple cellular disorders, including neuropathies and neurodegenerative diseases. (J Histochem Cytochem 58:207–218, 2010)

Key Words: mitochondrial DNA • mitochondrial biogenesis • dorsal root ganglion neurons • 5-bromo-2-deoxyuridine • 5-ethynyl-2'-deoxyuridine

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    Twitter    What's this?

Guidelines | Subscriptions | About | exPRESS - Current - Archive | Business Information | Contact

The Journal of Histochemistry & Cytochemistry is owned, published, and licensed by The Histochemical Society © 2010