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Title: The regulation of mitochondrial DNA transmission to generate offspring that are genetically identical
Author: Lloyd, Rhiannon Eleanor Iris.
Awarding Body: University of Birmingham
Current Institution: University of Birmingham
Date of Award: 2004
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Donor cell mitochondrial DNA (D-mtDNA) outcomes in somatic cell nuclear transfer (SCNT)-embryos, foetuses and offspring are variable. The factors that regulate D-mtDNA transmission and its influence ,post-SCNT are uncertain. Therefore, ovine primary foetal fibroblast (PDFF2 and SSFl) cells were depleted of their mtDNA to varying degrees using low concentrations of ethidium bromide. JC 1 staining, transmission electron microscopy, reverse transcription PCR, immunocytochemistry and Western blotting were used to determine the effects of mtDNA-depletion on the ovine cells prior to SCNT. PDFF2 cells containing full (PDFF2 mtDNA), partially-depleted (PDFF2 mtDNAPD ) and almost completely depleted (PDFF2 mtDNA 0) mtDNA complements were used successfully to produce SCNT -embryos up to the hatched blastocyst stage. Quantitative allele-specific-real time PCR analysis revealed that 74% of the PDFF2 SCNT-embryos contained D-mtDNA (range 0.01 to 8.72%). Furthermore, the persistence (P < 0.002) and amount of D-mtDNA (P < 0.007) was significantly reduced in PDFF2 mtDNA 0 embryos compared to PDFF2 mtDNA+ embryos, although their blastocyst formation rates and number of cell per blastocyst were similar. In order to verify the PDFF2 SCNT-embryo outcomes, another set of SCNTembryos was produced using SSFI cells ~ontaining full (SSFI mtDNA+) and almost completely depleted (SSFI mtDNAo) mtDNA complements. 86% of the SSFI SCNT-embryos contained D-mtDNA (range 0.01 to 1.19%) and the amount ofD-mtDNA was reduced, though not significantly, in the SSFI mtDNAo embryos compared to the SSFI mtDNA + embryos. Interestingly, the SSFI mtDNA 0 blastocysts contained significantly more cells than SSFI mtDNA+ blastocysts (P < 0.05). Taken together, these data show that: I) persistent D-mtDNA is a common feature following ovine SCNT; 2) D-mtDNA outcomes in ovine SCNT -embryos are influenced by the amount of mtDNA in the nuclear donor cells, 3) small amounts of D-mtDNA « 9%) in ovine SCNT-embryos do not influence their blastocyst formation rates, and finally 4) depending on the cell line, using nuclear donors almost completely depleted ofmtDNA increases ovine SCNT -blastocyst cell number and therefore could enhance their developmental competence.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available