A method for mutagenesis of mouse mtDNA and a resource of mouse mtDNA mutations for modeling human pathological conditions

Abstract

Mutations in human mitochondrial DNA (mtDNA) can cause mitochondrial disease and have been associated with neurodegenerative disorders, cancer, diabetes and aging. Yet our progress toward delineating the precise contributions of mtDNA mutations to these conditions is impeded by the limited availability of faithful transmitochondrial animal models. Here, we report a method for the isolation of mutations in mouse mtDNA and its implementation for the generation of a collection of over 150 cell lines suitable for the production of transmitochondrial mice. This method is based on the limited mutagenesis of mtDNA by proofreading-deficient DNA-polymerase γ followed by segregation of the resulting highly heteroplasmic mtDNA population by means of intracellular cloning. Among generated cell lines, we identify nine which carry mutations affecting the same amino acid or nucleotide positions as in human disease, including a mutation in the ND4 gene responsible for 70% of Leber Hereditary Optic Neuropathies (LHON). Similar to their human counterparts, cybrids carrying the homoplasmic mouse LHON mutation demonstrated reduced respiration, reduced ATP content and elevated production of mitochondrial reactive oxygen species (ROS). The generated resource of mouse mtDNA mutants will be useful both in modeling human mitochondrial disease and in understanding the mechanisms of ROS production mediated by mutations in mtDNA.

Presequence-Independent Mitochondrial Import of DNA Ligase Facilitates Establishment of Cell Lines with Reduced mtDNA Copy Number

“…We [ 18 ] and others [ 36 ] have shown that intracellular mtDNA cloning using partial depletion with EtBr is an effective means of inducing shifts in heteroplasmy (relative abundance of mtDNA haplotypes). However, prolonged treatment with EtBr represents a rate-limiting step of this procedure.…”

“…When indicated, uridine and pyruvate were omitted from this medium for selection of cells containing mtDNA (-UP medium). 3T3#52 is a Tet-On derivative of the NIH 3T3 cell line [ 18 ]. 4B6 mouse embryonic fibroblasts were derived from Lig3 flox/flox embryos [ 19 ].…”

Methods for Efficient Elimination of Mitochondrial DNA from Cultured Cells

“…A lentivirus encoding inducible secreted Gaussia luciferase was also described previously [ 24 ]. 3T3#52 is a Tet-On derivative of the NIH 3T3 cell line [ 25 ]. Plasmids and viral constructs were generated by standard techniques [ 26 ] and their diagrams are presented in the S1 Fig .…”

Landscape of Mitochondria Genome and Clinical Outcomes in Stage 1 Lung Adenocarcinoma

“…Characterization of mutations in mitochondrially encoded OXPHOS complex genes provided evidence for their role in tumor development and progression. Previous studies have demonstrated that mitochondrial mutations are associated with elevated ROS production [27,39], increased invasion ability [40,41], decimated mitochondria copy number [39,42], and higher risk of breast cancer [21], oral squamous cell carcinoma [39,43], colorectal cancer [12,44], and thyroid oncocytoma [39,45]. The existence of empirical evidence for mitochondrial genomic mutations and subsequent mitochondrial dysfunction in several cancers suggests that mitochondrial mutations may have potential in evaluating the risk of cancer progression [19,20,28,39,41,43].…”