Mitochondrial DNA Transmission and Transcription after Somatic Cell Fusion to One or More Cytoplasts

¹ The Mitochondrial and Reproductive Genetics Group, The Medical School, The University of Birmingham, Birmingham, B15 2TT, UK
² Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, Victoria 3800, Australia
³ Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, Victoria 3800, Australia; Stemagen Corporation, 4150 Regents Park Row, La Jolla, California, 92037-1417, USA
⁴ Centre for Reproduction and Development, Monash Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, Vic 3168, Australia
⁵ The Mitochondrial and Reproductive Genetics Group, Clinical Sciences Research Institute, Warwick Medical School, The University of Warwick, CV2 2DX, UK

^* To whom correspondence should be addressed. E-mail: J.C.St-John{at}Warwick.ac.uk.

	Abstract

Following fertilisation, mitochondrial DNA is inherited from the oocyte and transmitted homoplasmically. However, following nuclear transfer, mitochondrial DNA can be transmitted from both the donor cell and recipient oocyte, resulting in the state of heteroplasmy. To determine whether the genetic diversity between donor cell and recipient cytoplast mitochondrial DNA influences development, we generated bovine embryos by fusing a donor cell to one or more enucleated cytoplasts. Analysis of mitochondrial DNA from embryos, foetal tissues and blood samples from offspring revealed that early preimplantation embryos from two or three cytoplasts had significantly more mitochondrial DNA variants than foetal tissues. Phylogenic analysis of embryos generated using single cytoplasts divided the mtDNA sequence variants into three separate groups of varying genetic divergence from the donor cell line. In heteroplasmic tissue and blood samples, the predominant mitochondrial DNA population was significantly more divergent from the donor cell than the less frequent allele. Furthermore, analysis of the mitochondrially-encoded cytochrome B gene showed that two heteroplasmic alleles encoded for different amino acids and the ratios of mitochondrial DNA:mRNA for each allele differed significantly between tissues. The degree of evolutionary distance between the donor cell and the cytoplast and the variability in heteroplasmy between tissues may impact on more divergent inter-generic nuclear transfer and the use of this approach for the generation of embryonic stem cells.