Transmitochondrial mice as models for primary prevention of diseases caused by mutation in the tRNA(Lys) gene - PubMed (original) (raw)

Transmitochondrial mice as models for primary prevention of diseases caused by mutation in the tRNA(Lys) gene

Akinori Shimizu et al. Proc Natl Acad Sci U S A. 2014.

Abstract

We generated transmitochondrial mice (mito-mice) that carry a mutation in the tRNA(Lys) gene encoded by mtDNA for use in studies of its pathogenesis and transmission profiles. Because patients with mitochondrial diseases frequently carry mutations in the mitochondrial tRNA(Lys) and tRNA(Leu(UUR)) genes, we focused our efforts on identifying somatic mutations of these genes in mouse lung carcinoma P29 cells. Of the 43 clones of PCR products including the tRNA(Lys) or tRNA(Leu(UUR)) genes in mtDNA of P29 cells, one had a potentially pathogenic mutation (G7731A) in the tRNA(Lys) gene. P29 subclones with predominant amounts of G7731A mtDNA expressed respiration defects, thus suggesting the pathogenicity of this mutation. We then transferred G7731A mtDNA into mouse ES cells and obtained F0 chimeric mice. Mating these F0 mice with C57BL/6J (B6) male mice resulted in the generation of F1 mice with G7731A mtDNA, named "mito-mice-tRNA(Lys7731)." Maternal inheritance and random segregation of G7731A mtDNA occurred in subsequent generations. Mito-mice-tRNA(Lys7731) with high proportions of G7731A mtDNA exclusively expressed respiration defects and disease-related phenotypes and therefore are potential models for mitochondrial diseases due to mutations in the mitochondrial tRNA(Lys) gene. Moreover, the proportion of mutated mtDNA varied markedly among the pups born to each dam, suggesting that selecting oocytes with high proportions of normal mtDNA from affected mothers with tRNA(Lys)-based mitochondrial diseases may be effective as a primary prevention for obtaining unaffected children.

Keywords: mtDNA heteroplasmic mutation; mutated mtDNA segregation; preimplantation genetic diagnosis; selection of oocytes.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Characterization of P29-69-183 cells to determine the pathogenicity of a G7731A mutation in the mitochondrial tRNA Lys gene. (A) Estimation of the proportion of G7731A mtDNA in P29 and P29-69-183 cells by DraI digestion of the PCR products. The G7731A mtDNA produced 96-bp and 34-bp fragments owing to the gain of a DraI site through G7731A substitution in the tRNA Lys gene, whereas mtDNA without the mutation produced a 130-bp fragment. Quantitative estimation of G7731A mtDNA showed that P29-69-183 cells contained 92% G7731A mtDNA. (B) Estimation of O2 consumption rates of P29 and P29-69-183 cells. *P < 0.05. (C) Estimation of mitochondrial superoxide levels in P29 and P29-69-183 cells after their treatment with MitoSOX Red. **P < 0.01.

Fig. 2.

Fig. 2.

Characterization of transmitochondrial B82mt7731 cybrids for their use as G7731A mtDNA donors to ES cells. (A) Estimation of the proportion of G7731A mtDNA in cybrids clones B82mt7731-1 and B82mt7731-2 by DraI digestion of the PCR products. Quantitative estimation of G7731A mtDNA showed that B82mt7731-1 and B82mt7731-2 had 70% and 95% G7731A mtDNA, respectively. (B) Estimation of O2 consumption rates. **P < 0.01. (C) Estimation of mitochondrial superoxide levels after treatment with MitoSOX Red. **P < 0.01.

Fig. 3.

Fig. 3.

Variation of G7731A mtDNA proportions among pups or oocytes from individual female mice. (A) Variation of G7731A mtDNA proportions among F5 pups born to three F4 dams. G7731A mtDNA proportions were estimated by using tails from F5 pups that were obtained from three F4 dams with high proportions of G7731A mtDNA. (B) Variation of G7731A mtDNA proportions among oocytes obtained from three F5 female mice. G7731A mtDNA proportions were estimated by using oocytes obtained by ovarian hyperstimulation of F5 female mito-mice-tRNALys7731 with high proportions of G7731A mtDNA.

Fig. 4.

Fig. 4.

Characterization of F5 male mito-mice-tRNALys7731 according to phenotypes associated with mitochondrial diseases. Comparison of (A) body length and (B) muscle (grip) strength between B6 mice (n = 3) and F5 mito-mice-tRNALys7731 carrying low (13–15%; n = 3), intermediate (37–56%; n = 3), and high (76–84%; n = 3) proportions of G7731A mtDNA. Body length and grip strength were measured at 4 mo after birth. Data are presented as means ± SD. *P < 0.05; **P < 0.01. (C) Comparison of activities of mitochondrial respiratory complexes (I + III, II + III, and IV) between B6 mice and F5 mito-mice-tRNALys7731 carrying high proportions (76–84%) of G7731A mtDNA in the skeletal muscle and the kidney at 4 mo after birth. Respiratory complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (cytochrome c reductase), and complex IV (cytochrome c oxidase) are components of the electron-transport chain. Enhanced activity of complex II + III in mito-mice-tRNALys7731 would be due to compensatory activation of complex II, which is controlled exclusively by nuclear DNA. Data are presented as means ± SD (n = 3). *P < 0.05; **P < 0.01.

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