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Nucleic Acids Research, 2000, Vol. 28, No. 10 2164-2170
© 2000 Oxford University Press

Cloning of neuronal mtDNA variants in cultured cells by synaptosome fusion with mtDNA-less cells

Ian Trounce, Janet Schmiedel, Hsiu-Chuan Yen, Seyed Hosseini, Michael D. Brown, Jeffrey J. Olson1 and Douglas C. Wallace*

Center for Molecular Medicine and 1Department of Neurosurgery, Emory University School of Medicine, Atlanta, GA 30329, USA

Synaptosome cybrids were used to confirm the presence of heteroplasmic mtDNA sequence variants in the human brain. Synaptosomes contain one to several mitochondria, and when fused to mtDNA-deficient ({rho}°) mouse or human cell lines result in viable cybrid cell lines. The brain origin of mouse synaptosome cybrid mtDNAs was confirmed using sequence polymorphisms in the mtDNA COIII, ND3 and tRNAArg genes. The brain origin of the human synaptosome cybrids was confirmed using a rare mtDNA MboI polymorphism. Fusion of synaptosomes from the brain of a 35-year-old woman resulted in 71 synaptosome cybrids. Sequencing the mtDNA control region of these cybrid clones revealed differences in the number of Cs in a poly C track between nucleotide pairs (nps) 301 and 309. Three percent of the cybrid clones had mtDNAs with 10 Cs, 76% had nine, 18% had eight and 3% had seven Cs. Comparable results were obtained by PCR amplification, cloning and sequencing of mtDNA control regions directly from the patient’s brain tissue, but not when the control region was amplified and cloned from a synaptosome cybrid homoplasmic for a mtDNA with nine Cs. Thus, we have clonally recovered mtDNA control region length variants from an adult human brain without recourse to PCR, and established the variant mtDNAs within living cultured cells. This confirms that some mtDNA heteroplasmy can exist in human neurons, and provides the opportunity to study its functional significance.

* To whom correspondence should be addressed at: Center for Molecular Medicine, 1462 Clifton Road, NE, Room 420, Atlanta, GA 30322, USA. Tel: +1 404 727 8368; Fax: +1 404 727 8367; Email: dwallace@gen.emory.edu Present address: Ian Trounce, Mutation Research Centre, St Vincent’s Hospital, 41 Victoria Parade, Fitzroy 3065, Melbourne, Australia


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