Establishment of a new method for precisely determining the functions of individual mitochondrial genes, using <it>Dictyostelium </it>cells

• Main Authors: Tanaka Masashi, Amagai Aiko, Chida Junji, Maeda Yasuo
• Format: Article
• Language: English
• Published: BMC 2008-03-01
• Series: BMC Genetics
• Online Access: http://www.biomedcentral.com/1471-2156/9/25

<p>Abstract</p> <p>Background</p> <p>Disruption of mitochondrial genes may become a powerful tool for elucidating precisely the functions of individual mitochondrial genes. However, it is generally difficult to manipulate genetically mitochondrial genes, because 1) a mitochondrion is surrounded by inner and outer membranes, and 2) there are a large number of mtDNA copies in a single cell. This is the reason why we tried to establish a novel method for disrupting a certain mitochondrial gene (<it>rps4</it>), using <it>Dictyostelium </it>cells.</p> <p>Results</p> <p>Here, we have developed a new method for specifically disrupting a mitochondrial gene (<it>rps4 </it>; ribosomal protein subunit S4), by a combination of homologous recombination and delivery of an appropriate restriction endonuclease (<it>Sfo</it>I) into mitochondria. First, mitochondrially targeted <it>Sfo</it>I whose expression is under control of the tetracycline (Tet)-regulated gene expression system was introduced into cells heteroplasmic with respect to the <it>rps4 </it>gene. Then, the heteroplasmic cells were produced by homologous recombination by use of the construct in which the unique <it>Sfo</it>I site and the 5'-half of the <it>rps4 </it>coding region were deleted not to be digested by <it>Sfo</it>I, and therefore their mitochondria have both the wild-type mtDNA and the mutant mtDNA with the disrupted <it>rps4 </it>gene. In response to removal of Tet from growth medium, <it>Sfo</it>I was selectively delivered into mitochondria and digested only the wild-type mtDNA but not the mutated <it>rps4</it>. Thus one can gain <it>rps4</it>-null cells with only the mutated mtDNA, under the Tet-minus condition.</p> <p>Conclusion</p> <p>The mitochondrial gene-disruption method presented here must be widely useful for precisely determining the functions of individual mitochondrial genes. This is the first report to demonstrate complete and specific mitochondrial gene disruption.</p>