Chapter title |
Analysis of the Mitochondrial DNA and Its Replicative Capacity in Induced Pluripotent Stem Cells.
|
---|---|
Chapter number | 156 |
Book title |
Induced Pluripotent Stem (iPS) Cells
|
Published in |
Methods in molecular biology, February 2015
|
DOI | 10.1007/7651_2014_156 |
Pubmed ID | |
Book ISBNs |
978-1-4939-3054-8, 978-1-4939-3055-5
|
Authors |
Gael Cagnone, Vijesh Vaghjiani, William Lee, Claire Sun, Jacqueline Johnson, Ka-Yu Yeung, Justin C St John, Justin C. St.John, Cagnone, Gael, Vaghjiani, Vijesh, Lee, William, Sun, Claire, Johnson, Jacqueline, Yeung, Ka-Yu, St.John, Justin C. |
Editors |
Kursad Turksen, Andras Nagy |
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. |
X Demographics
Geographical breakdown
Country | Count | As % |
---|---|---|
Unknown | 1 | 100% |
Demographic breakdown
Type | Count | As % |
---|---|---|
Members of the public | 1 | 100% |
Mendeley readers
Geographical breakdown
Country | Count | As % |
---|---|---|
India | 1 | 6% |
Unknown | 15 | 94% |
Demographic breakdown
Readers by professional status | Count | As % |
---|---|---|
Researcher | 7 | 44% |
Student > Ph. D. Student | 2 | 13% |
Other | 1 | 6% |
Student > Bachelor | 1 | 6% |
Professor | 1 | 6% |
Other | 2 | 13% |
Unknown | 2 | 13% |
Readers by discipline | Count | As % |
---|---|---|
Agricultural and Biological Sciences | 5 | 31% |
Biochemistry, Genetics and Molecular Biology | 4 | 25% |
Medicine and Dentistry | 2 | 13% |
Pharmacology, Toxicology and Pharmaceutical Science | 1 | 6% |
Computer Science | 1 | 6% |
Other | 1 | 6% |
Unknown | 2 | 13% |