MtDNA Transmission

MtDNA does not follow the normal Mendelian mechanism of inheritance and is thus transmitted through the female germ line (37,38). An oocyte contains far more cytoplasm than the fertilizing sperm cell. This results in the mother contributing approximately 100,000 mitochondria to the embryonic cytoplasm and greatly diluting the 100 mtDNA copies (39) or so provided by the father. Sperm mitochondria are also thought to be degraded and actively destroyed by ubiqui-tination in the oocyte cytoplasm and subsequent proteolysis (40). However, recent reports suggest that paternal mtDNA can avoid destruction during human embryonic development to the blastocyst stage (41). Furthermore, sperm mtDNA has been identified in a male patient presenting with a mitochondrial disease arising from a mutation present in his father's mtDNA (42). Additionally, nonhuman primates generated through blastomere nuclear transfer possess sperm mtDNA (9). This raises concerns for the generation of ESC lines using such technology and such cell lines should be screened for heteroplasmy.

Here, we define a series of protocols that can be used to describe the process of cellular mitochondrial and mtDNA differentiation during hESC differentiation. Furthermore, we describe polymerase chain reaction (PCR) protocols that will define the initiation of mtDNA transcription and replication as hESCs progress through migration to the fully committed cell. Last, we describe realtime PCR protocols that allow both the identification of mtDNA homoplasmy or heteroplasmy and mtDNA copy number.

Was this article helpful?

0 0

Post a comment