Related Experiment Videos
Isolation of mitochondrial DNA-less mouse cell lines and their application for trapping mouse synaptosomal
1Institute of Biological Sciences, University of Tsukuba, Ibaraki 305, Japan.
Abstract:
For isolation of mouse mtDNA-less (rho0) cell lines, we searched for various antimitochondrial drugs that were expected to decrease the mtDNA content and found that treatment with ditercalinium, an antitumor bis-intercalating agent, was extremely effective for completely excluding mtDNA in all the mouse cell lines we tested. The resulting rho0 mouse cells were successfully used for trapping the mtDNA of living nerve cells into dividing cultured cells by fusion of the rho0 cells with mouse brain synaptosomes, which represent synaptic endings isolated from nerve cells. With neuronal mtDNA obtained, all of the cybrid clones restored mitochondrial translation activity similarly regardless of whether the mtDNA was derived from young or aged mice, thus at least suggesting that defects in mitochondrial genomes are not involved in the age-associated mitochondrial dysfunction observed in the brain of aged mice. Furthermore, we could trap a very small amount of a common 5823-base pair deletion mutant mtDNA (DeltamtDNA5823) that was detectable by polymerase chain reaction in the cybrid clones. As the amount of mutant mtDNA with large scale deletions was expected to increase during prolonged cultivation of the cybrids, these cells should be available for establishment of mice containing the deletion mutant mtDNA.
Insights
Ditercalinium effectively creates mouse mtDNA-less (rho0) cells for studying neuronal mitochondrial DNA. This research suggests age-associated mitochondrial dysfunction in mice may not stem from mitochondrial genome defects.
Area of Science:
- Cell Biology
- Neuroscience
- Genetics
Background:
- Mitochondrial dysfunction is implicated in age-associated neurological decline.
- Understanding the role of mitochondrial DNA (mtDNA) in this process is crucial.
- Developing methods to study neuronal mtDNA in controlled environments is needed.
Purpose of the Study:
- To establish a method for isolating mtDNA-less (rho0) mouse cells.
- To investigate the role of neuronal mtDNA in mitochondrial function and aging.
- To create a model for studying mtDNA mutations in vivo.
Main Methods:
- Treatment of mouse cell lines with ditercalinium to generate rho0 cells.
- Fusion of rho0 cells with mouse brain synaptosomes to introduce neuronal mtDNA.
- Analysis of cybrid clones for restored mitochondrial translation activity.
- Detection of deletion mutant mtDNA (DeltamtDNA5823) using polymerase chain reaction.
Main Results:
- Ditercalinium treatment efficiently produced mtDNA-less (rho0) mouse cells.
- Neuronal mtDNA successfully restored mitochondrial translation in cybrid clones, irrespective of donor age.
- A common 5823-base pair deletion mutant mtDNA was detectable in cybrid clones.
- The study suggests age-associated mitochondrial dysfunction in mouse brains is not primarily due to mtDNA defects.
Conclusions:
- Established rho0 mouse cells are effective for trapping and studying neuronal mtDNA.
- Neuronal mtDNA function appears preserved in aged mice, challenging the role of mtDNA defects in age-related dysfunction.
- The developed cybrid system provides a platform for studying mtDNA mutations and their potential in vivo effects.