Re: [DIYbio] DIY longevity biology

Ah, and I see that SENS Research Foundation folk are answering questions about the modest-sized mitochondrial gene therapy project hat is presently being crowdfunded - it's interesting stuff, worth a look for the rest of it, not just the quote below. After all, there are many people on this list who, given the lab support and connections provided by an SRF-like group, could be doing work in the medical life sciences that is just as cutting edge as this:

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http://www.longecity.org/forum/topic/65892-mitochondrialgenetherapy-questions/

Q: So why did you pick CyB and ATP8 as the two genes to work on here? Was that a fortuitous happenstance in terms of a suitable source of mutant cells to work with?

A: This is one of my favorite questions because we've spent so much time and effort figuring out which genes to focus on.

One reason is that these may be both the easiest and hardest genes to achieve efficient import with. CyB has a reputation (whether or not it is deserved is a matter for some debate) in the field of being the most difficult and hydrophobic protein to import into the mitochondria. It is one of the bigger mitochondrially encoded genes, so at the very least it is a challenge. ATP8, on the other hand, is tiny and so may be considered the easiest to import. Thus we've set ourselves a task that spans the range of challenges that we think we'll encounter.

The second reason is that, strategically, OxPhos complexes III and V are the most interesting for proof of concept rescue of the entire mito genome. The reason is that they have the fewest genes that are encoded by the mitochondria. Complex III has only CyB (and thus ONLY CyB is needed to rescue the entire complex) and Complex V has only 2: ATP6 and ATP8. So if we want to study functional rescue of entire complexes then III and V are the easiest.

The last reason is a common one in biology: the availability of useful tools for study. Mitochondrial mutations are relatively rare, difficult to create artificially, and the vast majority of naturally occurring mutations (in the protein coding genes) are partially functional point mutants. These are messy to study because they still produce protein and the proteins they produce are simply less functional than the wt version. Thus the phenotypes are mild compared to null mutations. We have worked with many different cell lines derived from patients in the SRF Research Center. We have a difficult time telling the difference (functionally) b/w point mutant cells and wt cells. The only way we can do it is with the difficult and expensive oxygen consumption assay. All our other assays are essentially useless in these cells. We have found (fortuitously as you guessed correctly), truly null cell lines for ATP8 and CyB. Researchers have shared their heteroplasmic mutant cell lines of these two genes with us and we are working on making them perfectly homoplasmic. They are already very useful in our experiments. The other tool we have is that we have antibodies that work against these two proteins. For some reason this seems to be rare for mito encoded proteins. No idea why. We have an excellent antibody against ATP8 that we had custom made for us and we tested many commercially available CyB antibodies and finally found conditions under which we get one to work passingly well.

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Reason