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MitochondrialGeneTherapy: Questions

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#1 caliban

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Posted 20 September 2013 - 12:29 AM


Post questions to the researchers below.

Please give them some time to answer- want the scientists focussed on the science, not browsing forums all day.

#2 reason

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Posted 26 September 2013 - 01:16 AM

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?
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#3 Elus

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Posted 28 September 2013 - 08:45 PM

Heya guys... undergrad biology student here :). Few questions!

1. Which diseases of aging do you suspect are implicated in mtDNA damage?

2. Can your mRNA import approach be used for the other 11 mitochondrial proteins?

3. What are the chances that the mitochondrial DNA in the nucleus will itself undergo mutation? i.e. what's the chance of the backup copy failing, and what would happen if it does?

4. How do you ensure that reverse transcriptase does not cause the mtDNA to insert itself into a critically important part of the nuclear genome, accidentally disrupting cellular function?

5. How do you control how many copies of mtDNA are integrated into the nucleus?

5. How do you regulate the rate at which the nuclear mtDNA is transcribed into mRNA? Any particular promoter region in mind, or some transcription factors?

6. If you can't find mutant cells lacking the other 11 genes, could you synthesize a mitochondrial genome from scratch which contains the appropriate mutations? Craig Venter style...

7. Suppose the following hypothetical situation: Your experiment works flawlessly, you manage to rescue the mitochondria, and then you go on to do the same experiment for the rest of the mitochondrial genome and again it works flawlessly.

Would you then create a mtDNA retroviral vector that indiscriminately infects cells in the human body, and test that in clinical trials? Or would you create a vector that infects a specific cell type? How would you deliver these backup genes to the 100 trillion cells in the human body? What about immune response to viral infection - how do you cloak your vector from the immune system?

8. If you don't need to infect the majority of the cell in the human body, you can ignore this question. Instead of flooding the patient's bloodstream with vector, could you give the virus the ability to self-replicate and inject just a few copies, and essentially use the human body as a bioreactor to grow the virus? Any advantages or disadvantages to this?

Edited by Elus, 28 September 2013 - 09:11 PM.


#4 niner

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Posted 28 September 2013 - 10:24 PM

Assuming that the ultimate therapy would involve transfecting every cell, how feasible is this? I'm thinking that if you only get 90 or 95% transfection, then although there will be a lot of healthy mitochondria, there will still be a significant fraction of potentially sick mitochondria that might wreak all manner of havoc. Am I off base with this thinking?
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#5 Elus

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Posted 28 September 2013 - 11:25 PM

Assuming that the ultimate therapy would involve transfecting every cell, how feasible is this? I'm thinking that if you only get 90 or 95% transfection, then although there will be a lot of healthy mitochondria, there will still be a significant fraction of potentially sick mitochondria that might wreak all manner of havoc. Am I off base with this thinking?


I wonder this too. Aubrey speaks of the selective advantage that "broken" mitochondria have - they do not incur oxidative damage to their membrane because of impaired OXPHOS, impairing the ability of the cell to discard these broken mitochondria which have high membrane fidelity (allowing them to to evade proteases) but low ATP production.

Perhaps by introducing this therapy, the mitochondrial population within the majority of cells will be maintained above some critical threshold such that disease does not occur.

Also, once transcribed from the nucleus, it appears that this mRNA isn't capable of differentiating between a broken mitochondrion and a healthy one (unless I'm mistaken). Would extra copies of mitochondrial mRNA enhance the function of healthy mitochondria above their normal capacity?

Edited by Elus, 28 September 2013 - 11:31 PM.


#6 mitomutant

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Posted 30 September 2013 - 08:25 AM

Assuming that the ultimate therapy would involve transfecting every cell, how feasible is this? I'm thinking that if you only get 90 or 95% transfection, then although there will be a lot of healthy mitochondria, there will still be a significant fraction of potentially sick mitochondria that might wreak all manner of havoc. Am I off base with this thinking?


I wonder this too. Aubrey speaks of the selective advantage that "broken" mitochondria have - they do not incur oxidative damage to their membrane because of impaired OXPHOS, impairing the ability of the cell to discard these broken mitochondria which have high membrane fidelity (allowing them to to evade proteases) but low ATP production.

Perhaps by introducing this therapy, the mitochondrial population within the majority of cells will be maintained above some critical threshold such that disease does not occur.

Also, once transcribed from the nucleus, it appears that this mRNA isn't capable of differentiating between a broken mitochondrion and a healthy one (unless I'm mistaken). Would extra copies of mitochondrial mRNA enhance the function of healthy mitochondria above their normal capacity?


I asked Aubrey about this "selective advantage" after reading "Ending aging". In the same thread, there is an interesting alternative to his theory (mitochondria dynamics).

As much I wish this transfection therapy would work, I don´t think it will. This project is just too ambitious for our current knowledge about mitochondria in particular and genetics in general.

Can´t find the paper now but targeting defective mitochondria and inducing death on them would be a much more effective way to treat mitochondrial diseases. The "threshold effect" is key here: You "only" need to maintain a healthy ratio of wild-type/mutant mitochondria to keep the clinical symptons at bay. In fact, "genetic shifting" - in this case by increasing wild-type population via satellite cell activation - is a proposed therapy to treat mitochondrial diseases.

This is my opinion for treating mitochondrial diseases. I understand that SENS is not about treating diseases.

Edited by mitomutant, 30 September 2013 - 08:26 AM.

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#7 niner

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Posted 30 September 2013 - 01:23 PM

Can´t find the paper now but targeting defective mitochondria and inducing death on them would be a much more effective way to treat mitochondrial diseases. The "threshold effect" is key here: You "only" need to maintain a healthy ratio of wild-type/mutant mitochondria to keep the clinical symptons at bay. In fact, "genetic shifting" - in this case by increasing wild-type population via satellite cell activation - is a proposed therapy to treat mitochondrial diseases.

This is my opinion for treating mitochondrial diseases. I understand that SENS is not about treating diseases.


SENS isn't about treating diseases, as you point out, but targeting sick mitochondria, much like targeting senescent cells, strikes me as a viable rejuvenation therapy, which would seem to put it squarely in the SENS wheelhouse. I don't want to get too off topic in this thread, so if anyone wants to discuss ablation of trouble-making cells, please start a new thread on it.

#8 Oki

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Posted 03 October 2013 - 06:29 PM

Ah! Sorry, everyone, great questions. I didn't notice them building up. I'll start on them as much as I can today and definitely get to all of them soon.
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#9 Oki

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Posted 03 October 2013 - 09:28 PM

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?



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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#10 Oki

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Posted 09 October 2013 - 09:42 PM

Heya guys... undergrad biology student here file:///C:\DOCUME~1\ADMINI~1\LOCALS~1\Temp\msohtmlclip1\01\clip_image002.gif. Few questions!

Great questions Elus. I'll number my answers to correspond w/ your questions.

1. Which diseases of aging do you suspect are implicated in mtDNA damage?

I think the primary affect of mitochondrial mutation is age-related decline in muscle and neuronal function. I think that the muscular aspect was beautifully illustrated in this recent paper: PMID 23692570. In a way, you might say that our main target here would be age-induced "frailty"
Mutations in OxPhos Complex I have also been implicated in Parkinson's disease.

2. Can your mRNA import approach be used for the other 11 mitochondrial proteins?

Well we are using an mRNA targeted protein import approach for this project (rather than an mRNA import approach which is also potentially feasible), but yes, assuming that it works for these 2 then we fully expect it to work for all 13. See my answer to Reason's question.

3. What are the chances that the mitochondrial DNA in the nucleus will itself undergo mutation? i.e. what's the chance of the backup copy failing, and what would happen if it does?

I'm not overly worried about this. The whole reason that we are putting the genes in the nucleus is that they'll be safer there. Are they 100% safe there? Probably not. It is essentially impossible than any rejuvenation ever invented will ever be eternal. Will it be good enough to last 40 years? Probably. Then you do it over again. The nice thing about transgenic tech is that you can do it over and over again. I don't think there is any danger of mutation to the constructs mostly just loss of function which needs to be replaced when the rare events that cause it happen.


4. How do you ensure that reverse transcriptase does not cause the mtDNA to insert itself into a critically important part of the nuclear genome, accidentally disrupting cellular function?

That's more of a question for the gene therapy people who will implement my tech. What I do know is that there are dozens of tricks in the world of gene therapy and my answer to that could range from "well that would be fairly rare and therefore probably nothing to worry about…" to "we're going to use a site specific endonuclease coupled to an integrase." That's all downstream of where I am now. Fortunately there are literally hundreds of labs working on this problem so I don't really need to worry about it. There are many good options.


5. How do you control how many copies of mtDNA are integrated into the nucleus?

With site specific tech that is easy. With random integration that is essentially impossible. My research should help determine whether or copy number control is important to regulate.

5. How do you regulate the rate at which the nuclear mtDNA is transcribed into mRNA? Any particular promoter region in mind, or some transcription factors?

Mostly at the promoter level, but also the integration site. Right now I'm just going for overkill, but if we think there is any toxicity then we can dial back the promoter and/or control the copy number. Don't forget though, that the cell has an mtDNA copy number of ~5000(!) so overkill might be just what the doctor ordered.


6. If you can't find mutant cells lacking the other 11 genes, could you synthesize a mitochondrial genome from scratch which contains the appropriate mutations? Craig Venter style...

Well it's actually very easy to make cells that are null for all 13 genes (Rho 0 cells), but creating a specific single desired mutant is the difficult part. I think that once we optimize the system for these 2 genes we won't need to test all of the others individually and can jump straight to groups of them in Rho 0 cells.

Synthesizing a mitochondrial genome in vitro is actually pretty easy with today's technology. It's just a 16.5kb plasmid. The trick is delivering it into the mitochondria. If you think gene therapy is hard then try that! There are a lot of ideas on how to do it out there, but I've never seen any evidence of even proof of concept success.

7. Suppose the following hypothetical situation: Your experiment works flawlessly, you manage to rescue the mitochondria, and then you go on to do the same experiment for the rest of the mitochondrial genome and again it works flawlessly. Would you then create a mtDNA retroviral vector that indiscriminately infects cells in the human body, and test that in clinical trials? Or would you create a vector that infects a specific cell type? How would you deliver these backup genes to the 100 trillion cells in the human body? What about immune response to viral infection - how do you cloak your vector from the immune system?

Like I said in #4 above, I'm a mitochondria expert, not a gene therapy expert. We'd, of course, start with animals (mice) long before moving to humans. I don't know whether it would be easier to do whole body or prioritize certain tissues (eg muscle and brain), but what you really want to avoid is gene therapies that preferentially work in dividing cells because the ones we are most worried about mitochondria in are the rarely replicating cells.

8. If you don't need to infect the majority of the cell in the human body, you can ignore this question. Instead of flooding the patient's bloodstream with vector, could you give the virus the ability to self-replicate and inject just a few copies, and essentially use the human body as a bioreactor to grow the virus? Any advantages or disadvantages to this?


A safe live-replicating viral vector would be awesome, but I think we are a long way away from approval for one since it is so hard to safely control a replicating virus. Those suckers have a nasty tendency to evolve and spread!

Edited by Oki, 09 October 2013 - 09:50 PM.

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#11 Oki

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Posted 10 October 2013 - 08:23 AM

Assuming that the ultimate therapy would involve transfecting every cell, how feasible is this? I'm thinking that if you only get 90 or 95% transfection, then although there will be a lot of healthy mitochondria, there will still be a significant fraction of potentially sick mitochondria that might wreak all manner of havoc. Am I off base with this thinking?


Honestly I think even 50% efficiency would be a dramatic rejuvenation. We won't really know much until we can do some good animal experiments, but imagine that you're 90 and all of a sudden you have half as many "sick" cells as you did before. My guess is that you're feeling a lot better. Also, the nice thing about gene therapy is that you can keep doing more treatments. Not happy w/ 90%, then we go til you're 95%. Not happy w/ 95%? Then we go to 99%. Finally, SENS Research Foundation is sponsoring projects in how to kill toxic cells of various types so we can tackle this problem from 2 directions.

Assuming that the ultimate therapy would involve transfecting every cell, how feasible is this? I'm thinking that if you only get 90 or 95% transfection, then although there will be a lot of healthy mitochondria, there will still be a significant fraction of potentially sick mitochondria that might wreak all manner of havoc. Am I off base with this thinking?


I wonder this too. Aubrey speaks of the selective advantage that "broken" mitochondria have - they do not incur oxidative damage to their membrane because of impaired OXPHOS, impairing the ability of the cell to discard these broken mitochondria which have high membrane fidelity (allowing them to to evade proteases) but low ATP production.

Perhaps by introducing this therapy, the mitochondrial population within the majority of cells will be maintained above some critical threshold such that disease does not occur.

Also, once transcribed from the nucleus, it appears that this mRNA isn't capable of differentiating between a broken mitochondrion and a healthy one (unless I'm mistaken). Would extra copies of mitochondrial mRNA enhance the function of healthy mitochondria above their normal capacity?


Fantastic question! Will the first transgenic mice that we make with this be "super" mice or something? Who knows! I can't wait to find out. :)
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#12 Turnbuckle

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Posted 10 October 2013 - 01:45 PM

Are there any supplements that might promote natural mitochondrial transfer?

#13 Oki

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Posted 10 October 2013 - 04:42 PM

Can´t find the paper now but targeting defective mitochondria and inducing death on them would be a much more effective way to treat mitochondrial diseases. The "threshold effect" is key here: You "only" need to maintain a healthy ratio of wild-type/mutant mitochondria to keep the clinical symptons at bay. In fact, "genetic shifting" - in this case by increasing wild-type population via satellite cell activation - is a proposed therapy to treat mitochondrial diseases.

This is my opinion for treating mitochondrial diseases. I understand that SENS is not about treating diseases.


SENS Research Foundation is, in fact, 100% dedicated to curing all of the diseases of aging. We are also working on connecting the research that we do to ways in which it can help people with rare disease. For example, I have been in contact with the United Mitochondrial Disease Foundation and they have expressed interest in the potential of my approach for treating inherited and spontaneous mitochondrial disease.

Edited by Michael, 15 October 2013 - 11:49 PM.

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#14 Oki

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Posted 10 October 2013 - 04:48 PM

Are there any supplements that might promote natural mitochondrial transfer?


In short, no. What we are doing is completely artificial and highly engineered. There are many topics on the longecity forums that discuss supplements and drugs that might help improve mitochondrial function, but nothing other than an engineered solution can fix a mitochondria that has lost 100% of its ability to synthesize its own proteins.

#15 okok

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Posted 10 October 2013 - 05:14 PM

A safe live-replicating viral vector would be awesome, but I think we are a long way away from approval for one since it is so hard to safely control a replicating virus. Those suckers have a nasty tendency to evolve and spread!


Haha, wouldn't it be great - immortality goes viral.

(off track, but this made me think if there's a way to harness these suckers in a different way - keywords mutation, massive parallelism, QM, selection.)

#16 mitomutant

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Posted 10 October 2013 - 05:32 PM

proposed therapy[/url] to treat mitochondrial diseases.

This is my opinion for treating mitochondrial diseases. I understand that SENS is not about treating diseases.

SENS Research Foundation is, in fact, 100% dedicated to curing all of the diseases of aging. We are also working on connecting the research that we do to ways in which it can help people with rare disease. For example, I have been in contact with the United Mitochondrial Disease Foundation and they have expressed interest in the potential of my approach for treating inherited and spontaneous mitochondrial disease.


Thanks for the clarification. I had no idea that SENS tries to target specific diseases along its way to defeat aging.

UMDF is a great organization for patient care and family support, but consider contacting Foundation for Mitochondrial Medicine and JDM Fund for mitochondrial research. The are much more focused on funding projects like yours. For example, JDM helped to fund the latest Dr. Moraes research with TALE nucleases to target and destroy mutant mitochondria.

Edited by Michael, 15 October 2013 - 11:48 PM.

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#17 Turnbuckle

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Posted 10 October 2013 - 05:34 PM

Are there any supplements that might promote natural mitochondrial transfer?


In short, no. What we are doing is completely artificial and highly engineered. There are many topics on the longecity forums that discuss supplements and drugs that might help improve mitochondrial function, but nothing other than an engineered solution can fix a mitochondria that has lost 100% of its ability to synthesize its own proteins.


Are you familiar with the work of Prockop, et al?

Mitochondrial transfer between cells can rescue aerobic respiration

We report here that mitochondria are more dynamic than previously considered: mitochondria or mtDNA can move between cells. The active transfer from adult stem cells and somatic cells can rescue aerobic respiration in mammalian cells with nonfunctional mitochondria...In this article, we ask whether stem/progenitor cells or other somatic cells can repair cells with nonfunctional mitochondria by transfer of functional mitochondria or mtDNA.


If so, I'd expect there to be a supplement or drug that could accelerate this process.

#18 Oki

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Posted 11 October 2013 - 08:29 PM

UMDF is a great organization for patient care and family support, but consider contacting Foundation for Mitochondrial Medicine and JDM Fund for mitochondrial research. The are much more focused on funding projects like yours. For example, JDM helped to fund the latest Dr. Moraes research with TALE nucleases to target and destroy mutant mitochondria.


Thanks for the suggestion Mitomutant (and for the generous donation)! I'm always looking for more support. I'll contact them soon.
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#19 Carl Kenner

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Posted 15 October 2013 - 07:42 PM

Which of the "13" mitochondrial proteins have you already done? And which haven't you started on yet? How much more is there still to do before you can show that all "13" proteins can be individually imported successfully? Are there any that you expect to have difficulties with?

And what about the 14th mitochondrial DNA encoded protein? Are you going to do anything about Humanin? It appears to protect neurons. I haven't heard anything from SENS about it. Fortunately, even though it is encoded by mitochondrial DNA, it doesn't seem to need importing into the mitochondria, since it acts on both the inside and the outside of cells. And fortunately, other people have already made circular DNA of it that the nucleus can process, and that is effective in rejuvenating neurons.

(In case you are wondering where the genes for another protein are hiding... it's in the rRNA of MT-RNR2... the ribosome has to translate part of another ribosome)
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#20 Oki

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Posted 16 October 2013 - 05:54 AM

Hi Carl,

We have done various amounts of work with 5 of the 13: ND1, ND4, CyB, ATP6, and ATP8. As explained above, for several reasons we've decided to focus on CyB and ATP8 for now.

We haven't worked at all on Humanin yet. I've always been perplexed by the logic of evolving a protein to be secreted by the mitochondria. If it does need to be restored, however, it should (as you point out) be much easier than the other 13 since it doesn't seem to require any special targeting. Good question, much more needs to be learned about Humanin.


Which of the "13" mitochondrial proteins have you already done? And which haven't you started on yet? How much more is there still to do before you can show that all "13" proteins can be individually imported successfully? Are there any that you expect to have difficulties with?

And what about the 14th mitochondrial DNA encoded protein? Are you going to do anything about Humanin? It appears to protect neurons. I haven't heard anything from SENS about it. Fortunately, even though it is encoded by mitochondrial DNA, it doesn't seem to need importing into the mitochondria, since it acts on both the inside and the outside of cells. And fortunately, other people have already made circular DNA of it that the nucleus can process, and that is effective in rejuvenating neurons.

(In case you are wondering where the genes for another protein are hiding... it's in the rRNA of MT-RNR2... the ribosome has to translate part of another ribosome)



#21 caliban

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Posted 17 October 2013 - 04:43 PM

Hello Oki
It is wonderful to see that thanks to marvellous support from the LongeCity community and generous donations large and small, we have now almost reached the point where the only item still outstanding is the cell counter (application).(product link)

This was flagged up in the review and approval process as an item that would benefit from clarification.

Can you explain why the counter is strictly necessary - can't you count the cells by hand or, much better yet, using the flow cytometer that the lab already owns? If the counter was procured, could it be used for other projects as well? Could it be sold or loaned to other researchers?

#22 Oki

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Posted 18 October 2013 - 01:46 AM

Hi Caliban,

Yes, everyone here is a very excited about the rapid progress on this innovative crowd funded matched grant that you rewarded us with.

A mechanical cell counter is not essential for the project to succeed, but it will be faster and better with one. The old-fashioned way to count cells (and the way we are currently doing it) is manually using a hemocytometer (essentially a fancy glass slide), a light microscope, and a little hand-held clicker counting device. One disadvantage of the manual method is that it is slow and laborious. My team is wasting a lot of valuable time manually counting many sets of cells every day. The other disadvantage is that the quality of your experiments suffer while you are counting cells. This is due to the fact that you almost always have to count cells on the fly while you are in the middle of your experiment. For example, let's say that I have many different cell lines that I need to purify mitochondria from and that I want to start with 20 million cells per sample. We like to do it in sets of 6 cell lines at a time. That is our standard protocol. To collect the cells we have to treat them with an enzyme (trypsin) which is damaging to cells if left on them for longer than a few minutes. Counting 6 cell lines can take up to 30 minutes. This may not seem like such a horrific amount of time, but to the cells sitting there waiting for me to work with them it is deadly! This is particularly bad for mitochondrial experiments because as cells sit and suffer the mitochondria quickly start to degrade and brake-down (because they use up all the stored ATP and the energy balance gets disturbed). So we end up either compromising accuracy by guesstimating the number of cells to use or compromising quality by letting the mitochondria brake-down while we count. A FACS machine is not at all appropriate for this purpose. While a FACS machine is, strictly speaking, a dedicated cell counter, it is much too fancy to use for this purpose. When you use a FACS machine, you generally collect ALL of your cells (a whole experiment) and then measure whatever you are trying to measure (eg. expression of a gene(s), absorbance of a some dye, cell viability). You use FACS at the end of an experiment and it takes a significant amount of time to analyze the data. A cell counter (and hemocyctometer) use a tiny number of cells (usually < 1% of whatever you are using) and are measured on the fly.

We'd be happy to let other researchers in the area use it, but I doubt anyone would want to travel any distance to use one, for the same reason that we wouldn't.

A cell counter would be a good purchase because it will be used every day for the duration of the project and indefinitely into the future. The cell counter will absolutely be valuable for almost every other project being performed now and in the future at SRF RC. The only cell type in the lab that might be difficult to count is the Retinal Pigmented Epithelial cells that are used for most experiments being performed by the LysoSENS group that we share lab space with. Those cells are fairly small, but not as small as red blood cells, so it would probably still work and just have a little more variability error involved. When I told the guys from the oncoSENS project that I might be getting a cell counter, one of them almost hugged me. He wastes a lot of time counting cells...
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#23 Elus

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Posted 28 October 2013 - 04:09 AM

Counting cells with a mechanical clicker while manipulating a slide and changing the microscope's focus is pretty annoying and time consuming - I can definitely see where Oki is coming from - I had to use a hemocytometer for cell counting during an internship and it was a pain. When I went to a lab with an optical counter, where you just plop a little test tube full of cell suspension into the machine and get a cell count, life was much improved. All you would need to do is multiply the cell density by the total volume of your cell suspension, and that's your total cell count and you're done!

From what I remember, in either case it's pretty important that the cell suspension is homogenous before you take your measurement sample, so that you can accurately measure the cell density.

Edited by Elus, 28 October 2013 - 04:16 AM.


#24 jiminbrizzy

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Posted 02 December 2013 - 07:40 AM

Apologies if I have got this wrong, I am no scientist, but what is to stop the dysfunctional proteins produced by the damaged genes in the mitchondria completing with the corrected imported proteins?

And how do you know that this isn't a prion Huntington style disease where a misfolded protein is wrecking havock (but only within the mtichondria)?

#25 olaf.larsson

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Posted 06 December 2013 - 01:27 AM

A question; Why do you go mitochondrial gene therapy? I thought that the mito theory of aging has been largely refuted. As I remember the number/percentage of mutated mitos in old people is not significant enough to cause an aging phenotype. Sorry if I am not updated about some things I have been absent from the field for several years.

Edited by olaf.larsson, 06 December 2013 - 01:30 AM.


#26 Oki

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Posted 06 December 2013 - 01:58 AM

Apologies if I have got this wrong, I am no scientist, but what is to stop the dysfunctional proteins produced by the damaged genes in the mitchondria completing with the corrected imported proteins?

And how do you know that this isn't a prion Huntington style disease where a misfolded protein is wrecking havock (but only within the mtichondria)?



Hi Jiminbrizzy,

I can give the same answer to both questions: by far the most common DNA mutations in aging are ones that completely delete protein production within the mitochondria, rather than creating mutant proteins. Thus the main problem we are dealing with is one of insufficiency, rather than rogue mutant proteins.

Matthew O'Connor

#27 jiminbrizzy

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Posted 21 December 2013 - 12:17 AM

Thanks for the reply Matthew.

I'm sure you've seen this recent research on Mtichondrial dysfunction which seems to suggested aging is caused by a breakdown in communication between the nucleus' genome and the mtiochondrial genomes (decreased NAD decreases the ability of SIRT1 to keep HIF-1 in check, leading to a breakdown in communication).

What implications (if any) does this have for your group's research?

http://hms.harvard.edu/news/genetics/new-reversible-cause-aging-12-19-13

Edited by jiminbrizzy, 21 December 2013 - 12:18 AM.


#28 Multivitz

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Posted 19 December 2015 - 01:03 AM

D3 can effect DNA, are the team very familiar with natural substances and there mehtods of action/purpose around the DNA?





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