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Mitochondrial gene replacement in primate offspring and embryonic stem cells

A Corrigendum to this article was published on 10 December 2014

Abstract

Mitochondria are found in all eukaryotic cells and contain their own genome (mitochondrial DNA or mtDNA). Unlike the nuclear genome, which is derived from both the egg and sperm at fertilization, the mtDNA in the embryo is derived almost exclusively from the egg; that is, it is of maternal origin. Mutations in mtDNA contribute to a diverse range of currently incurable human diseases and disorders. To establish preclinical models for new therapeutic approaches, we demonstrate here that the mitochondrial genome can be efficiently replaced in mature non-human primate oocytes (Macaca mulatta) by spindle–chromosomal complex transfer from one egg to an enucleated, mitochondrial-replete egg. The reconstructed oocytes with the mitochondrial replacement were capable of supporting normal fertilization, embryo development and produced healthy offspring. Genetic analysis confirmed that nuclear DNA in the three infants born so far originated from the spindle donors whereas mtDNA came from the cytoplast donors. No contribution of spindle donor mtDNA was detected in offspring. Spindle replacement is shown here as an efficient protocol replacing the full complement of mitochondria in newly generated embryonic stem cell lines. This approach may offer a reproductive option to prevent mtDNA disease transmission in affected families.

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Figure 1: Spindle–chromosomal complex transfer and meiotic analysis of reconstructed monkey oocytes.
Figure 2: Mito and Tracker, the first primates to be produced by spindle–chromosomal complex transfer (ST) into enucleated oocytes followed by fertilization and embryo transfer.
Figure 3: MtDNA analysis in ST offspring.

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Acknowledgements

The authors acknowledge the Division of Animal Resources, Surgical Team, Endocrine Technology Core, Imaging & Morphology Core and Molecular & Cellular Biology Core at the Oregon National Primate Research Center for providing expertise and services that contributed to this project. We are grateful to W. Sanger and M. Nelson for karyotyping services, C. Penedo for microsatellite analysis and S. Wong for providing Sendai virus. We thank J. Hennebold, R. Stouffer and D. Wolf for consulting, helpful discussions and critical reading of the manuscript. This study was supported by start-up funds from Oregon National Primate Research Center, Oregon Stem Cell Center and grants from the National Institutes of Health.

Author Contributions S.M. and M.T. conceived the study, designed experiments and conducted ST micromanipulations. M.S. performed ICSI, mitochondrial staining and analysis in oocytes. H.S., J.W. and Y.L. conducted ES cell derivation, characterization and differentiation. M.T., H.M., L.C., H.S. and Y.L. performed DNA/RNA isolations and mtDNA analyses. C.R. and O.K. conducted ovarian stimulations, oocyte recovery, ICSI and embryo transfers. S.M. and M.T. analysed the data and wrote the paper.

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Correspondence to Shoukhrat Mitalipov.

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This file contains Supplementary Figures 1-6 with Legends, Supplementary Tables 1-5, Supplementary Methods and Supplementary References. (PDF 1163 kb)

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Tachibana, M., Sparman, M., Sritanaudomchai, H. et al. Mitochondrial gene replacement in primate offspring and embryonic stem cells. Nature 461, 367–372 (2009). https://doi.org/10.1038/nature08368

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