title: Analysis of the Mitochondrial DNA and Its Replicative Capacity in Induced Pluripotent Stem Cells.
abstract: The mitochondrial genome resides in the mitochondrion of nearly all mammalian cells. It is important for energy production as it encodes 13 of the key subunits of the electron transfer chain, which generates the vast majority of cellular ATP through the process of oxidative phosphorylation. As cells establish pluripotency, they regulate their mtDNA copy number so that they possess few copies but sufficient that they can be replicated to match the differentiated cell-specific requirements for ATP derived through oxidative phosphorylation. However, the failure to strictly regulate this process prevents pluripotent cells from differentiating. We describe a series of protocols that analyze mtDNA copy number, DNA methylation within the nuclear-encoded mtDNA-specific polymerase, and gene expression of the other factors that drive replication of the mitochondrial genome. We demonstrate how to measure ATP-generating capacity through oxygen respiratory capacity and total cellular ATP and lactate levels. Finally, we also describe how to detect mtDNA variants in pluripotent and differentiating cells using next-generation sequencing protocols and how the variants can be confirmed by high-resolution melt analysis.
authors: The Mitochondrial Genetics Group, Centre for Genetic Diseases, MIMR-PHI Institute of Medical Research, 27-31 Wright Street, Clayton, VIC, 3168, Australia.|The Department of Molecular and Translational Science, Monash University, 27-31 Wright Street, Clayton, VIC, 3168, Australia.
authors: Gael
authors: G
authors: Cagnone
authors: The Mitochondrial Genetics Group, Centre for Genetic Diseases, MIMR-PHI Institute of Medical Research, 27-31 Wright Street, Clayton, VIC, 3168, Australia.|The Department of Molecular and Translational Science, Monash University, 27-31 Wright Street, Clayton, VIC, 3168, Australia.
authors: Vijesh
authors: V
authors: Vaghjiani
authors: The Mitochondrial Genetics Group, Centre for Genetic Diseases, MIMR-PHI Institute of Medical Research, 27-31 Wright Street, Clayton, VIC, 3168, Australia.|The Department of Molecular and Translational Science, Monash University, 27-31 Wright Street, Clayton, VIC, 3168, Australia.
authors: William
authors: W
authors: Lee
authors: The Mitochondrial Genetics Group, Centre for Genetic Diseases, MIMR-PHI Institute of Medical Research, 27-31 Wright Street, Clayton, VIC, 3168, Australia.|The Department of Molecular and Translational Science, Monash University, 27-31 Wright Street, Clayton, VIC, 3168, Australia.
authors: Claire
authors: C
authors: Sun
authors: The Mitochondrial Genetics Group, Centre for Genetic Diseases, MIMR-PHI Institute of Medical Research, 27-31 Wright Street, Clayton, VIC, 3168, Australia.|The Department of Molecular and Translational Science, Monash University, 27-31 Wright Street, Clayton, VIC, 3168, Australia.
authors: Jacqueline
authors: J
authors: Johnson
authors: The Mitochondrial Genetics Group, Centre for Genetic Diseases, MIMR-PHI Institute of Medical Research, 27-31 Wright Street, Clayton, VIC, 3168, Australia.|The Department of Molecular and Translational Science, Monash University, 27-31 Wright Street, Clayton, VIC, 3168, Australia.
authors: Ka-Yu
authors: KY
authors: Yeung
authors: The Mitochondrial Genetics Group, Centre for Genetic Diseases, MIMR-PHI Institute of Medical Research, 27-31 Wright Street, Clayton, VIC, 3168, Australia. justin.stjohn@monash.edu.|The Department of Molecular and Translational Science, Monash University, 27-31 Wright Street, Clayton, VIC, 3168, Australia. justin.stjohn@monash.edu.
authors: Justin C
authors: JC
authors: St John
mesh_terms: D000255:Adenosine Triphosphate / Q000096:biosynthesis; D002478:Cells, Cultured; D065150:Cellular Reprogramming / Q000235:genetics; D056915:DNA Copy Number Variations; D019175:DNA Methylation; D004261:DNA Replication; D004272:DNA, Mitochondrial / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000502:physiology; D015870:Gene Expression; D015723:Gene Library; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D008863:Microspheres; D009691:Nucleic Acid Denaturation; D020411:Oligonucleotide Array Sequence Analysis; D010085:Oxidative Phosphorylation; D010101:Oxygen Consumption; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D004272:DNA, Mitochondrial; D000255:Adenosine Triphosphate; D004259:DNA-Directed DNA Polymerase
keywords: ATP; Differentiation; Mitochondrial DNA; Next-generation sequencing; Oxygen consumption; Pluripotent cells; Transcription and replication; Variants