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Analysis of Mitochondrial DNA in induced pluripotent and embryonic stem cells
chapter
posted on 2015-01-01, 00:00 authored by William Lee, Richard D W Kelly, Ka Yu Yeung, Gael Cagnone, Matthew McKenzieMatthew McKenzie, Justin C St JohnThe mitochondrial genome has a major role to play in establishing and maintaining pluripotency. Furthermore, mitochondrial DNA (mtDNA) copy is strictly regulated during differentiation. Undifferentiated, pluripotent cells possess fewer than 300 copies of mtDNA, which establishes the mtDNA set point and promotes cell proliferation and, as a result, these cells rely on glycolysis with some support from oxidative phosphorylation (OXPHOS) for the generation of ATP. The mtDNA set point provides the starting point from which cells increase their mtDNA copy number as they differentiate into mature functional cells. Dependent on cell types, mtDNA copy number ranges from ~10 copies in sperm to several thousand in cardiomyocytes. Consequently, differentiating cell types can acquire the appropriate numbers of mtDNA copy to meet their specific requirements for ATP generated through OXPHOS. However, as reprogrammed somatic cells do not always achieve this, it is essential to analyze them for their OXPHOS potential and ability to regulate mtDNA copy number. Here, we describe how to assess mtDNA copy number in pluripotent and differentiating cells using real-time PCR protocols; assess expression of the mtDNA specific replication factors through real-time RT-PCR; identify mtDNA variants in embryonic and induced pluripotent stem cells; determine DNA methylation patterns of the mtDNA-specific replication factors; and assess mitochondrial OXPHOS capacity.
History
Title of book
Cell reprogramming : methods and protocolsVolume
1330Series
Methods in molecular biology, 1064-3745 ; 1330Chapter number
18Pagination
219 - 252Publisher
SpringerPlace of publication
Berlin, GermanyPublisher DOI
eISSN
1940-6029Language
engPublication classification
B Book chapter; B1.1 Book chapterCopyright notice
2015, SpringerExtent
19Editor/Contributor(s)
Paul Verma, Huseyin SumerUsage metrics
Categories
Keywords
DifferentiationMitochondrial DNA,OXPHOS,POLGA,Pluripotency,mtDNA replication,AnimalsCell Culture TechniquesCell DifferentiationChromatin ImmunoprecipitationDNA MethylationDNA, MitochondrialEmbryonic Stem CellsGene DosageGene ExpressionGene LibraryGenes, MitochondrialHigh-Throughput Nucleotide SequencingInduced Pluripotent Stem CellsMiceMitochondriaOxidative PhosphorylationOxygen ConsumptionPolymerase Chain ReactionSequence Analysis, DNA