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.
Highlights
Introduction of SynaptosomalmtDNA into 0 Mouse Cells—The brain of a B6mtJ or B6 strain mouse was washed in phosphate-buffered saline and homogenized in medium containing 0.25 M sucrose, 50 mM Hepes, pH 7.5, and 0.1 mM EDTA in a Teflon-glass Potter-Elvehjem homogenizer
EtBr is effective for isolating 0 lines from avian [9] and human [10, 11] cells, but we failed to isolate them from EtBr-treated mouse cells (the copy number of mtDNA in C2 cells did not change substantially (Fig. 1A), whereas its transcription was completely inhibited by EtBr treatment, consistent with our previous observations [26])
There are no previous reports on successful isolation of 0 lines from mouse cells, probably because exposure of mouse cells to EtBr, which has been effectively used for isolation of 0 lines from yeast, avian, and human cells [33], induced EtBrresistant mutant cell lines containing mtDNA instead of isolating 0 lines
Summary
Cells and Cell Culture—The mouse myoblast line C2C12 (C2 cells) and mouse fibroblast lines (5P cells derived from the B6mtJ strain and NIH3T3 cells) were grown in normal medium (RPMI 1640 (Nissui Seiyaku, Tokyo) containing 10% fetal calf serum, 50 g/ml uridine, and 0.1 mg/ml pyruvate). The cloned cells were cultivated in normal medium without the drug. On days 14 –20 after fusion, the cybrid clones growing in the selection medium were clonally isolated by the cylinder method. Southern Blot Analyses of mtDNA—Total cellular DNA (1–2 g) extracted from 2 ϫ 105 cells was digested with the restriction enzyme (XhoI or BamHI (Nippon Gene, Tokyo, Japan)), and restriction fragments were separated in 0.6% agarose gel, transferred to a nylon membrane, and hybridized with [␣-32P]dATP-labeled mouse mtDNA. PCR Analyses—For detection of a small amount of normal mtDNA in 0 C2 cells, total cellular DNA (0.5 g) extracted from 2 ϫ 105 0 C2 cells was used as a template. 1. Screening of antimitochondrial drugs by Southern blot analysis of XhoI fragments for isolation of 0 mouse cell lines. A, effect of antimitochondrial drugs on the mtDNA content in the mouse myoblast cell line C2. For quantitative estimation of [35S]methionine-labeled polypeptides, the dried gel was exposed to an imaging plate for 12 h, and the radioactivities of polypeptides were measured with a bioimaging analyzer
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