Comparison of the complete mtDNA genome sequences of human cell lines--HL-60 and GM10742A--from individuals with pro-myelocytic leukemia and leber hereditary optic neuropathy, respectively, and the inclusion of HL-60 in the NIST human mitochondrial DNA standard reference material--SRM 2392-I

Forensic and clinical laboratories benefit from DNA standard reference materials (SRMs) that provide the quality control and assurance that their results from sequencing unknown samples are correct. Therefore, the mitochondrial DNA (mtDNA) genome of HL-60, a promyelocytic leukemia cell line, has been completely sequenced by four laboratories and will be available to the forensic and medical communities in the spring of 2003; it will be called National Institute of Standards and Technology (NIST) SRM 2392-I. NIST human mtDNA SRM 2392 will continue to be available and includes the DNA from two apparently healthy individuals. Both SRM 2392 and 2392-I contain all the information (e.g. the sequences of 58 unique primer sets) needed to use these SRMs as positive controls for the amplification and sequencing any DNA. Compared to the templates in SRM 2392, the HL-60 mtDNA in SRM 2392-I has two tRNA differences and more polymorphisms resulting in amino acid changes. Four of these HL-60 mtDNA polymorphisms have been associated with Leber Hereditary Optic Neuropathy (LHON), one as an intermediate mutation and three as secondary mutations. The mtDNA from a cell line (GM10742A) from an individual with LHON was also completely sequenced for comparison and contained some of the same LHON mutations. The combination of these particular LHON associated mutations is also found in phylogenetic haplogroup J and its subset, J2, and may only be indicative that HL-60 belongs to haplogroup J, one of nine haplogroups that characterize Caucasian individuals of European descent or may mean that haplogroup J is more prone to LHON. Both these mtDNA SRMs will provide enhanced quality control in forensic identification, medical diagnosis, and single nucleotide polymorphism detection.     

A Human Mitochondrial DNA Standard Reference Material for Quality Control in Forensic Identification,Medical Diagnosis,and Mutation Detection

A human mitochondrial DNA (mtDNA) standard reference material (SRM 2392) will provide quality control when mtDNA is sequenced for forensic identifications, medical diagnosis, or mutation detection. SRM 2392 includes DNA from two lymphoblast cell cultures (CHR and 9947A) and cloned DNA from the CHR HV1 region, which contains a C stretch and is difficult to sequence. The mtDNA sequence (but not the DNA) of a third human template GM03798 is provided for comparison. Fifty-eight unique primer sets allow any area or the entire mtDNA (16,569 bp) to be amplified and sequenced. While none of the differences in these three templates correspond to published mutations associated with specific diseases, some of these differences did result in animo acid changes compared with that published by S. Andersonet al.(1981,Nature290: 457–465). An interlaboratory evaluation of the amplification, sequencing, and data analysis of the CHR template was conducted by four laboratories. Corroboration of the SRM results will provide quality assurance that any unknown mtDNA is also being amplified and sequenced correctly.     

Mutation analysis of the entire mitochondrial genome using denaturing high performance liquid chromatography

In patients with mitochondrial disease a continuously increasing number of mitochondrial DNA (mtDNA) mutations and polymorphisms have been identified. Most pathogenic mtDNA mutations are heteroplasmic, resulting in heteroduplexes after PCR amplification of mtDNA. To detect these heteroduplexes, we used the technique of denaturing high performance liquid chromatography (DHPLC). The complete mitochondrial genome was amplified in 13 fragments of 1–2 kb, digested in fragments of 90–600 bp and resolved at their optimal melting temperature. The sensitivity of the DHPLC system was high with a lowest detection of 0.5% for the A8344G mutation. The muscle mtDNA from six patients with mitochondrial disease was screened and three mutations were identified. The first patient with a limb-girdle-type myopathy carried an A3302G substitution in the tRNA^Leu(UUR) gene (70% heteroplasmy), the second patient with mitochondrial myopathy and cardiomyopathy carried a T3271C mutation in the tRNA^Leu(UUR) gene (80% heteroplasmy) and the third patient with Leigh syndrome carried a T9176C mutation in the ATPase6 gene (93% heteroplasmy). We conclude that DHPLC analysis is a sensitive and specific method to detect heteroplasmic mtDNA mutations. The entire automatic procedure can be completed within 2 days and can also be applied to exclude mtDNA involvement, providing a basis for subsequent investigation of nuclear genes.     

Mitochondrial DNA integrity is not dependent on DNA polymerase-beta activity

Mutations in mitochondrial DNA (mtDNA) are involved in a variety of pathologies, including cancer and neurodegenerative diseases, as well as in aging. mtDNA mutations result predominantly from damage by reactive oxygen species (ROS) that is not repaired prior to replication. Repair of ROS-damaged bases occurs mainly via base excision repair (BER) in mitochondria and nuclei. In nuclear BER, the two penultimate steps are carried out by DNA polymerase-beta (Polbeta), which exhibits both 5'-deoxyribose-5-phosphate (5'-dRP) lyase and DNA polymerase activities. In mitochondria, DNA polymerase-gamma (Polgamma) is believed to be the sole polymerase and is therefore assumed to function in mitochondrial BER. However, a recent report suggested the presence of Polbeta or a "Polbeta-like" enzyme in bovine mitochondria. Consequently, in the present work, we tested the hypothesis that Polbeta is present and functions in mammalian mitochondria. Initially we identified two DNA polymerase activities, one corresponding to Polgamma and the other to Polbeta, in mitochondrial preparations obtained by differential centrifugation and discontinuous sucrose density gradient centrifugation. However, upon further fractionation in linear Percoll gradients, we were able to separate Polbeta from mitochondria and to show that intact mitochondria, identified by electron microscopy, lacked Polbeta activity. In a functional test for the presence of Polbeta function in mitochondria, we used a new assay for detection of random (i.e., non-clonal) mutations in single mtDNA molecules. We did not detect enhanced mutation frequency in mtDNA from Polbeta null cells. In contrast, mtDNA from cells harboring mutations in the Polgamma exonuclease domain that abolish proofreading displayed a >or=17-fold increase in mutation frequency. We conclude that Polbeta is not an essential component of the machinery that maintains mtDNA integrity.     

Detection of mutations in mitochondrial DNA by droplet digital PCR

Mutations in mitochondrial DNA (mtDNA) may result in various pathological processes. Detection of mutant mtDNAs is a problem for diagnostic practice that is complicated by heteroplasmy – a phenomenon of the inferring presence of at least two allelic variants of the mitochondrial genome. Also, the level of heteroplasmy largely determines the profile and severity of clinical manifestations. Here we discuss detection of mutations in heteroplasmic mtDNA using up-todate methods that have not yet been introduced as routine clinical assays. These methods can be used for detecting mutations in mtDNA to verify diagnosis of “mitochondrial disease”, studying dynamics of mutant mtDNA in body tissues of patients, as well as investigating structural features of mtDNAs. Original data on allele-specific discrimination of m.11778G>A mutation by droplet digital PCR are presented, which demonstrate an opportunity for simultaneous detection and quantitative assessment of mutations in mtDNAs.     

Nanopore sequencing identifies a higher frequency and expanded spectrum of mitochondrial DNA deletion mutations in human aging

Mitochondrial DNA (mtDNA) deletion mutations cause many human diseases and are linked to age-induced mitochondrial dysfunction. Mapping the mutation spectrum and quantifying mtDNA deletion mutation frequency is challenging with next-generation sequencing methods. We hypothesized that long-read sequencing of human mtDNA across the lifespan would detect a broader spectrum of mtDNA rearrangements and provide a more accurate measurement of their frequency. We employed nanopore Cas9-targeted sequencing (nCATS) to map and quantitate mtDNA deletion mutations and develop analyses that are fit-for-purpose. We analyzed total DNA from vastus lateralis muscle in 15 males ranging from 20 to 81 years of age and substantia nigra from three 20-year-old and three 79-year-old men. We found that mtDNA deletion mutations detected by nCATS increased exponentially with age and mapped to a wider region of the mitochondrial genome than previously reported. Using simulated data, we observed that large deletions are often reported as chimeric alignments. To address this, we developed two algorithms for deletion identification which yield consistent deletion mapping and identify both previously reported and novel mtDNA deletion breakpoints. The identified mtDNA deletion frequency measured by nCATS correlates strongly with chronological age and predicts the deletion frequency as measured by digital PCR approaches. In substantia nigra, we observed a similar frequency of age-related mtDNA deletions to those observed in muscle samples, but noted a distinct spectrum of deletion breakpoints. NCATS-mtDNA sequencing allows the identification of mtDNA deletions on a single-molecule level, characterizing the strong relationship between mtDNA deletion frequency and chronological aging.     

Accurate detection and quantitation of heteroplasmic mitochondrial point mutations by pyrosequencing

Disease-causing mutations in mitochondrial DNA (mtDNA) are typically heteroplasmic and therefore interpretation of genetic tests for mitochondrial disorders can be problematic. Detection of low level heteroplasmy is technically demanding and it is often difficult to discriminate between the absence of a mutation or the failure of a technique to detect the mutation in a particular tissue. The reliable measurement of heteroplasmy in different tissues may help identify individuals who are at risk of developing specific complications and allow improved prognostic advice for patients and family members. We have evaluated Pyrosequencing technology for the detection and estimation of heteroplasmy for six mitochondrial point mutations associated with the following diseases: Leber's hereditary optical neuropathy (LHON), G3460A, G11778A, and T14484C; mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS), A3243G; myoclonus epilepsy with ragged red fibers (MERRF), A8344G, and neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP)/Leighs: T8993G/C. Results obtained from the Pyrosequencing assays for 50 patients with presumptive mitochondrial disease were compared to those obtained using the commonly used diagnostic technique of polymerase chain reaction (PCR) and restriction enzyme digestion. The Pyrosequencing assays provided accurate genotyping and quantitative determination of mutational load with a sensitivity and specificity of 100%. The MELAS A3243G mutation was detected reliably at a level of 1% heteroplasmy. We conclude that Pyrosequencing is a rapid and robust method for detecting heteroplasmic mitochondrial point mutations.     

Mitochondrial DNA mutation frequencies in experimentally irradiated compost worms, Eisenia fetida

The compost worm Eisenia fetida is routinely used in ecotoxicological studies. A standard assay to assess genetic damage in this species would be extremely valuable. Since mitochondrial DNA (mtDNA) is known to exhibit an increased mutation rate following exposure to ionising radiation we assessed the validity of a mtDNA-based assay for measuring increases in mutation rate in laboratory-irradiated compost worms. To this end the mutation frequency in the mtDNA of the compost worm E. fetida was quantified following in vivo gamma-irradiation of adult worms in three dose groups. Five adult worms exposed to 1.4 mGy/h for 55 days (total dose 1.85 Gy), five adult worms exposed to 8.5 mGy/h for 55 days (total dose 11.22 Gy) and five adult control worms were used to assess the effect of irradiation on mtDNA mutation induction. DNA samples extracted from irradiated adult worms were used in high-fidelity PCR of a 486 bp region of mtDNA spanning the ATPase 8 gene, chosen for its high spontaneous mutation rate. PCR products were cloned and sequenced to identify mutations, with 89-102 clones successfully sequenced per individual. A significant elevation in mtDNA mutation frequency (p=0.032) was seen in worms exposed at the higher dose rate (8.5 mGy/h, total dose 11.22 Gy; mutation frequency 27.98+/-4.85 x 10(-5)mutations/bp) in comparison to controls (mutation frequency 12.68+/-3.06 x 10(-5)mutations/bp), but no elevation in mutation frequency (p=0.764) was seen for the lower dose rate (1.4 mGy/h, total dose 1.85 Gy; mutation frequency 13.74+/-1.29 x 10(-5)mutations/bp) compared with controls. This indicates that although the technique has the potential to detect an elevation in mutation frequency, it does not have sufficient sensitivity at the doses likely to be encountered in environmental monitoring scenarios.     

Mitochondrial DNA exhibits resistance to induced point and deletion mutations

The accumulation of somatic mitochondrial DNA (mtDNA) mutations contributes to the pathogenesis of human disease. Currently, mitochondrial mutations are largely considered results of inaccurate processing of its heavily damaged genome. However, mainly from a lack of methods to monitor mtDNA mutations with sufficient sensitivity and accuracy, a link between mtDNA damage and mutation has not been established. To test the hypothesis that mtDNA-damaging agents induce mtDNA mutations, we exposed Muta^TMMouse mice to benzo[a]pyrene (B[a]P) or N-ethyl-N-nitrosourea (ENU), daily for 28 consecutive days, and quantified mtDNA point and deletion mutations in bone marrow and liver using our newly developed Digital Random Mutation Capture (dRMC) and Digital Deletion Detection (3D) assays. Surprisingly, our results demonstrate mutagen treatment did not increase mitochondrial point or deletion mutation frequencies, despite evidence both compounds increase nuclear DNA mutations and demonstrated B[a]P adduct formation in mtDNA. These findings contradict models of mtDNA mutagenesis that assert the elevated rate of mtDNA mutation stems from damage sensitivity and abridged repair capacity. Rather, our results demonstrate induced mtDNA damage does not readily convert into mutation. These findings suggest robust mitochondrial damage responses repress induced mutations after mutagen exposure.     

Complete sequence analysis of mitochondrial DNA of aplastic anemia patients

This study was primarily undertaken to test the hypothesis that mitochondrial DNA (mtDNA) mutations may be associated with aplastic anemia (AA). We analyzed mtDNA sequences from 15 patients with AA. The samples were obtained from bone marrow, and patients' oral epithelial cells were collected for normal tissue comparison. Total DNA was amplified by PCR after extraction, and these segments were then sent for sequencing. The results were compared with those of oral epithelial tissues as well as mtDNA sequences in the revised Cambridge Reference Sequence (rCRS) database. We detected 61 heteroplasmic mutations in 11 genes, including those encoding NADH dehydrogenase (ND)1-2 and 4-6, tRNA glutamic acid (TRNE), ribosomal RNA (RNR) 1 and 2, cytochrome c oxidase (COX1), cytochrome b (CYTB), and tRNA glycine (TRNG); mutation rates were particularly high in ND2 (34.4%) and ND4 (21.3%) in the patients' mtDNA genomes. The products of these genes are involved in oxidation in the respiratory chain, and a large number of homoplasmic mutations were found. Interestingly, these 162 polymorphisms were mostly in the D-loop DNA structure (54.3%), in which numerous mutations associated with leukemia and myelodysplastic syndromes are found. We conclude that functional impairment of the mitochondrial respiratory chain induced by mutation may be an important reason for hematopoietic failure in AA patients.     

Optimized PCR fragments for heteroduplex analysis of the whole human mitochondrial genome with denaturing HPLC

Denaturing high pressure liquid chromatography (dHPLC) is an efficient method for discovery of unknown mutations by heteroduplex analysis of PCR fragments. For comprehensive mutation scanning of the whole 16.569 bp human mitochondrial genome, we developed a set of 67 primer pairs defining overlapping PCR fragments that are well suited for heteroduplex analysis. The aim of our optimization efforts was to ensure that point mutations are detectable at every nucleotide position of each amplicon. Some GC-rich regions of mitochondrial DNA (mtDNA) were found to have unfavourable melting profiles in all possible amplicons, therefore requiring GC-clamps at the end of one or both oligonucleotide PCR primers. Following detection of a heteroduplex pattern by dHPLC, our primers can also be employed for DNA sequencing to identify the underlying mutation. In case of heteroplasmic mutations with a low proportion of mutant mtDNA, a fragment collector is useful to recover the heteroduplex peak, which contains mutant and wildtype DNA molecules in a 1:1 ratio.     

Mitochondrial DNA Mutations Induce Mitochondrial Dysfunction,Apoptosis and Sarcopenia in Skeletal Muscle of Mitochondrial DNA Mutator Mice

Background

Aging results in a progressive loss of skeletal muscle, a condition known as sarcopenia. Mitochondrial DNA (mtDNA) mutations accumulate with aging in skeletal muscle and correlate with muscle loss, although no causal relationship has been established.

Methodology/Principal Findings

We investigated the relationship between mtDNA mutations and sarcopenia at the gene expression and biochemical levels using a mouse model that expresses a proofreading-deficient version (D257A) of the mitochondrial DNA Polymerase γ, resulting in increased spontaneous mtDNA mutation rates. Gene expression profiling of D257A mice followed by Parametric Analysis of Gene Set Enrichment (PAGE) indicates that the D257A mutation is associated with a profound downregulation of gene sets associated with mitochondrial function. At the biochemical level, sarcopenia in D257A mice is associated with a marked reduction (35–50%) in the content of electron transport chain (ETC) complexes I, III and IV, all of which are partly encoded by mtDNA. D257A mice display impaired mitochondrial bioenergetics associated with compromised state-3 respiration, lower ATP content and a resulting decrease in mitochondrial membrane potential (Δψ_m). Surprisingly, mitochondrial dysfunction was not accompanied by an increase in mitochondrial reactive oxygen species (ROS) production or oxidative damage.

Conclusions/Significance

These findings demonstrate that mutations in mtDNA can be causal in sarcopenia by affecting the assembly of functional ETC complexes, the lack of which provokes a decrease in oxidative phosphorylation, without an increase in oxidative stress, and ultimately, skeletal muscle apoptosis and sarcopenia.     

Somatic mutations in the D-loop and decrease in the copy number of mitochondrial DNA in human hepatocellular carcinoma

Somatic mutations in mitochondrial DNA (mtDNA) have been detected in many human cancers, including hepatocellular carcinoma (HCC). The D-loop region was found to be a "hot spot" for mutation in mtDNA of the tumors. However, effects of the D-loop mutations on the copy number of mtDNA in tumor tissues are poorly understood. Using direct sequencing, we examined mutations in the D-loop region of mtDNA in 61 HCCs and the corresponding non-tumor liver tissues. The results revealed that 39.3% of the HCCs carried somatic mutation(s) in the D-loop of mtDNA, and most of these mutations were homoplasmic. Moreover, 37.0% (10/27) of these mutations were T-to-C and G-to-A transitions and 40.7% (11/27) of them were located in the polycytidine stretch between nucleotide position (np) 303 and 309 of mtDNA. In addition, we found that mtDNA copy number of HCC was significantly decreased in 60.5% of the patients with hepatoma, especially in those with somatic mutation(s) in the D-loop of mtDNA (17/24). This decrease in mtDNA copy number was highly associated with the occurrence of point mutations near the replication origin of the heavy-strand of mtDNA. Interestingly, we found that 42.9% (6/14) of the HCCs without mutation in the D-loop had a reduced copy number of mtDNA, indicating that other unidentified factors involved in mitochondrial biogenesis might be defective in the tumor. The results obtained in this study strongly suggest that somatic mutations in the D-loop together with the decrease in the copy number of mtDNA may be an important event during the early phase of liver carcinogenesis.     

Modeling pathogenic mutations of human twinkle in Drosophila suggests an apoptosis role in response to mitochondrial defects

The human gene C10orf2 encodes the mitochondrial replicative DNA helicase Twinkle, mutations of which are responsible for a significant fraction of cases of autosomal dominant progressive external ophthalmoplegia (adPEO), a human mitochondrial disease caused by defects in intergenomic communication. We report the analysis of orthologous mutations in the Drosophila melanogaster mitochondrial DNA (mtDNA) helicase gene, d-mtDNA helicase. Increased expression of wild type d-mtDNA helicase using the UAS-GAL4 system leads to an increase in mtDNA copy number throughout adult life without any noteworthy phenotype, whereas overexpression of d-mtDNA helicase containing the K388A mutation in the helicase active site results in a severe depletion of mtDNA and a lethal phenotype. Overexpression of two d-mtDNA helicase variants equivalent to two human adPEO mutations shows differential effects. The A442P mutation exhibits a dominant negative effect similar to that of the active site mutant. In contrast, overexpression of d-mtDNA helicase containing the W441C mutation results in a slight decrease in mtDNA copy number during the third instar larval stage, and a moderate decrease in life span in the adult population. Overexpression of d-mtDNA helicase containing either the K388A or A442P mutations causes a mitochondrial oxidative phosphorylation (OXPHOS) defect that significantly reduces cell proliferation. The mitochondrial impairment caused by these mutations promotes apoptosis, arguing that mitochondria regulate programmed cell death in Drosophila. Our study of d-mtDNA helicase overexpression provides a tractable Drosophila model for understanding the cellular and molecular effects of human adPEO mutations.     

Multifactorial nature of high frequency of mitochondrial DNA mutations in somatic mammalian cells

The high frequency of mitochondrial DNA (mtDNA) mutations in somatic mammalian cells, which is more than two orders of magnitude higher than the mutation frequency of nuclear DNA (nDNA), significantly correlates with development of a variety of mitochondrial diseases (neurodegenerative diseases, cardiomyopathies, type II diabetes mellitus, cancer, etc.). A direct cause—consequence relationship has been established between mtDNA mutations and aging phenotypes in mammals. However, the unclear nature of the high frequency of mtDNA mutations requires a comprehensive consideration of factors that contribute to this phenomenon: oxidative stress, features of structural organization and repair of the mitochondrial genome, ribonucleotide reductase activity, replication errors, mutations of nuclear genes encoding mitochondrial proteins.     

Comprehensive, rapid and sensitive detection of sequence variants of human mitochondrial tRNA genes.

In the present study, a comprehensive, rapid and sensitive method for screening sequence variation of the human mitochondrial tRNA genes has been developed. For this purpose, the denaturing gradient gel electrophoresis (DGGE) technique has been appropriately modified for simultaneous mutation analysis of a large number of samples and adapted so as to circumvent the problems caused by the anomalous electrophoretic behavior of DNA fragments encoding tRNA genes. Eighteen segments of mitochondrial DNA (mtDNA), each containing a single uniform melting domain, were selected to cover all tRNA-encoding regions using the computer program MELT94. All 18 segments were simultaneously analyzed by electrophoresis through a single broad range denaturing gradient gel under rigorously defined conditions, which prevent band broadening and other migration abnormalities from interfering with detection of sequence variants. All base substitutions tested, which include six natural mutations and 14 artificially introduced ones, have been detected successfully in the present study. Several types of evidence strongly suggest that the anomalous behavior in DGGE of tRNA gene-containing mtDNA fragments reflects their tendency to form temporary or stable alternative secondary structures under semi-denaturing conditions. The high sensitivity of the method, which can detect as low as 10% of mutant mtDNA visually, makes it valuable for the analysis of heteroplasmic mutations.     

Sequence and functional analyses of mtDNA in a maternally inherited family with bipolar disorder and depression

Recent studies suggest that mutations/polymorphisms of mitochondrial DNA (mtDNA) are associated with neuropsychiatric diseases. We identified a patient with major depression and epilepsy. Some family members in the pedigree of the proband had bipolar disorder, depression, suicide, or psychotic disorder not otherwise specified. The mode of inheritance was compatible with maternal inheritance with low penetration. We assumed that the mental disorder in this family might be associated with maternally inherited mitochondrial DNA (mtDNA) mutation. We sequenced the entire mtDNA of the proband. Among the 34 base substitutions detected in the proband, two homoplasmic, nonsynonymous single substitutions of mtDNA, T3394C in MT-ND1 and A9115G in MT-ATP6, were suspected to cause functional impairment, because the former was reported to be disease-related and the latter is vary rare. To study the functional outcome of these substitutions, we examined mitochondrial membrane potential and the activity of mitochondrial ATP synthesis in the transmitochondrial cybrids, but no significant impairment was detected. The data did not support our hypothesis that these disorders in this family are caused by mtDNA mutation(s).     

A novel heteroplasmic tRNA(Leu(CUN)) mtDNA point mutation associated with chronic progressive external ophthalmoplegia

We have sequenced all mitochondrial tRNA genes from a patient with chronic progressive external ophthalmoplegia (CPEO) and mitochondrial myopathy, who had no detectable large mtDNA deletions. Direct sequencing failed to detect previously reported mutations and showed a heteroplasmic mutation at nucleotide 12,276 in the tRNA(Leu(CUN)) gene, in the dihydrouridine stem, which is highly conserved through the species during evolution. RFLP analyses confirmed that 18% of muscle mtDNA harbored the mutation, while it was absent from DNA of fibroblasts and lymphocytes of the proband and in 110 patients with other encephalomyopathies. To date, besides large and single nucleotide deletions, several point mutations on mitochondrial tRNA genes have been reported in CPEO patients, but only three were in the gene coding for tRNA(Leu(CUN)).     

Cell and animal models of mtDNA biology: progress and prospects

The past two decades have witnessed an evolving understanding of the mitochondrial genome’s (mtDNA) role in basic biology and disease. From the recognition that mutations in mtDNA can be responsible for human disease to recent efforts showing that mtDNA mutations accumulate over time and may be responsible for some phenotypes of aging, the field of mitochondrial genetics has greatly benefited from the creation of cell and animal models of mtDNA mutation. In this review, we critically discuss the past two decades of efforts and insights gained from cell and animal models of mtDNA mutation. We attempt to reconcile the varied and at times contradictory findings by highlighting the various methodologies employed and using human mtDNA disease as a guide to better understanding of cell and animal mtDNA models. We end with a discussion of scientific and therapeutic challenges and prospects for the future of mtDNA transfection and gene therapy. mitochondria; mitochondrial DNA; cybrid