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

Fayzulin RZ, Perez M, Kozhukhar N, Spadafora D, Wilson GL, Alexeyev MF - Nucleic Acids Res. (2015)

Bottom Line: 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.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).

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Neuroscience, University of South Alabama, Mobile, AL 36688, USA.

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Validation of the resource. (A) respiratory profiling of the 14 missense mutants. Note the predominance of near-WT activities and a range of respiratory activities in the remaining mutants. (B) Respiratory profiling of nonsense mutants. (B) Respiration by tRNA mutants. (C) ROS production by tRNA mutants. (D) Membrane potential in tRNA mutants. (E) Respiration of homoplasmic nonsense mutants. (F) ROS production by nonsense mutants. Dashed horizontal line, WT (100%). (G and H) Increased ROS production in nonsense mutants depends on mtDNA. mtDNA depletion by mUNG1 (G) is accompanied by reduced ROS production (H). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. (A–D) One-way ANOVA with post-hoc Tukey test. (G and H) Two-tailed unpaired Student's t-test assuming unequal variances.
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Figure 4: Validation of the resource. (A) respiratory profiling of the 14 missense mutants. Note the predominance of near-WT activities and a range of respiratory activities in the remaining mutants. (B) Respiratory profiling of nonsense mutants. (B) Respiration by tRNA mutants. (C) ROS production by tRNA mutants. (D) Membrane potential in tRNA mutants. (E) Respiration of homoplasmic nonsense mutants. (F) ROS production by nonsense mutants. Dashed horizontal line, WT (100%). (G and H) Increased ROS production in nonsense mutants depends on mtDNA. mtDNA depletion by mUNG1 (G) is accompanied by reduced ROS production (H). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. (A–D) One-way ANOVA with post-hoc Tukey test. (G and H) Two-tailed unpaired Student's t-test assuming unequal variances.

Mentions: For initial validation of the generated collection of mtDNA mutants, baseline respiration rates were examined in 15 clones containing homoplasmic missense mutations with a Seahorse XF-24 extracellular flux analyzer. As expected of an unbiased collection generated without enrichment for functional mutants, clones exhibited a range of respiratory activities with 60% of the mutants retaining WT rates of oxygen consumption, and the remaining 40% demonstrating various degrees of impairment of respiratory chain activity in vivo (Figure 4A).


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

Fayzulin RZ, Perez M, Kozhukhar N, Spadafora D, Wilson GL, Alexeyev MF - Nucleic Acids Res. (2015)

Validation of the resource. (A) respiratory profiling of the 14 missense mutants. Note the predominance of near-WT activities and a range of respiratory activities in the remaining mutants. (B) Respiratory profiling of nonsense mutants. (B) Respiration by tRNA mutants. (C) ROS production by tRNA mutants. (D) Membrane potential in tRNA mutants. (E) Respiration of homoplasmic nonsense mutants. (F) ROS production by nonsense mutants. Dashed horizontal line, WT (100%). (G and H) Increased ROS production in nonsense mutants depends on mtDNA. mtDNA depletion by mUNG1 (G) is accompanied by reduced ROS production (H). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. (A–D) One-way ANOVA with post-hoc Tukey test. (G and H) Two-tailed unpaired Student's t-test assuming unequal variances.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482060&req=5

Figure 4: Validation of the resource. (A) respiratory profiling of the 14 missense mutants. Note the predominance of near-WT activities and a range of respiratory activities in the remaining mutants. (B) Respiratory profiling of nonsense mutants. (B) Respiration by tRNA mutants. (C) ROS production by tRNA mutants. (D) Membrane potential in tRNA mutants. (E) Respiration of homoplasmic nonsense mutants. (F) ROS production by nonsense mutants. Dashed horizontal line, WT (100%). (G and H) Increased ROS production in nonsense mutants depends on mtDNA. mtDNA depletion by mUNG1 (G) is accompanied by reduced ROS production (H). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. (A–D) One-way ANOVA with post-hoc Tukey test. (G and H) Two-tailed unpaired Student's t-test assuming unequal variances.
Mentions: For initial validation of the generated collection of mtDNA mutants, baseline respiration rates were examined in 15 clones containing homoplasmic missense mutations with a Seahorse XF-24 extracellular flux analyzer. As expected of an unbiased collection generated without enrichment for functional mutants, clones exhibited a range of respiratory activities with 60% of the mutants retaining WT rates of oxygen consumption, and the remaining 40% demonstrating various degrees of impairment of respiratory chain activity in vivo (Figure 4A).

Bottom Line: 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.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).

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Neuroscience, University of South Alabama, Mobile, AL 36688, USA.

Show MeSH
Related in: MedlinePlus