Mitochondrial Genetic Manipulation

In the context of current genomics, mitochondrial genetics might seem accessory, given the small size and limited gene content of the organelle genome. This is not the case, as functional efficiency of mitochondria and cross-talk with the other compartments are essential. Mitochondrial genetic manipulation is appealing for a range of fundamental investigations, but also to treat neurodegenerative diseases caused by organelle DNA mutations. In plants, mitochondrial genetics underlies key breeding tools, like cytoplasmic male sterility. Given the importance of these issues, manipulating mitochondrial genetics has been a long standing goal, but regular transformation was reached only in a couple of unicellular organisms through conventional biolistics. Thus, the challenge gave rise to a range of alternative strategies progressing towards mitochondrial transformation or circumventing the problem. We address both approaches. Earlier in vitro assays with isolated organelles established that mitochondria can functionally import DNA. On that basis, we develop nanocarrier-mediated cell uptake and mitochondrial targeting of functional gene constructs. DNA incorporation into mammalian cells and expression of a marker gene placed under the control of the mitochondrial heavy-strand promoter were obtained. A second approach exploits the natural tRNA import pathway of plant mitochondria. Nuclearly-expressed RNAs driven by a tRNA mimic are transported into the organelles along this pathway in plant cells and whole plants. Using trans-cleaving ribozymes or a CMS-associated orf as cargo sequences, we manipulated the level of specific mitochondrial RNAs and brought evidence for coordinated regulation of organelle and nuclear transcripts. Prospects and further alternative strategies will be discussed.