Increasing Telomere Growth

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jackkwan
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Increasing Telomere Growth

Post by jackkwan »

I am trying to come up with a natural substance to increasing the availability/activity of the telomerase enzyme. I have found antioxidants that work like vitamin b 12 and folate, I was wondering how I would go about mixing these antioxidants and together in a way where I could try it in worms and see if their telomeres grew?
SciB
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Re: Increasing Telomere Growth

Post by SciB »

Hi Jack,

Sounds like you have an ambitious project in mind. Telomere shortening is associated with aging and is a hot area of research right now.

I have some questions, though. What antioxidants are you using and why do you think they will stimulate the activity of telomerase? Did you read this in a published research paper? I know that many people take antioxidant supplements like vitamin C, green tea, turmeric, grape seed extract, etc., but I haven't read that this prevents telomere shortening. Do you have references for this? You need to have an idea of what concentration to use to see an effect.

You said you were going to use worms to test the hypothesis. I assume you mean Caenorhabditis elegans, right? Have you worked with these before? Do you have access to a research lab to work in? How will you determine the length of the telomeres?

Let us know more about the details of your project and we will be able to help you better. Also, how long do you have to work on this project? This is going to take a couple of months probably.

Sybee
jackkwan
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Re: Increasing Telomere Growth

Post by jackkwan »

This is a class at my high school that I am taking, we have 6 months to work on a research project. I have references to my finding is telomerase research and have thoroughly read enough articles to understand the relationship between telomerase and telomere length. I have done research and found that telomere shortening/attrition is caused a lot from oxidative stress in the cells. That's why antioxidants and stuff would help preserve the telomere length and overall decrease excess telomere attrition. What I found out is that there is a new substance out there called cycloastragenol and is the only know molecule that actually activates telomerase is humans. There are already many "telomere growth and telomerase increasing" products out there. Most of them are very similar in which their ingredients consist of a bunch of antioxidants and vitamins that relieve oxidative stress and preserve telomeres. The number one supplement on the market is TA-65. Which is a patented product that is marketed as the only product that has worked and been tested in humans to increase telomerase activity and overall growth. Ta-65 is largely composed of one ingredient, cycloastragenol. See what I am trying to do is create a mixture of top companies products and their active ingredients, to create the best of both worlds in terms of telomere growth and telomere preservation. Because when people compare these top products they also talk about what the other is missing. But no one ever thinks about combining them or what would happen if you took both of the supplements. So that is what I am trying to do. Create a product that has a good amount of antioxidants and the natural telomerase activator cycloastragenol and give it to C. elegans to see if their telomeres grow or are preserved. I will have a control group and and independent, in which I will be measuring their telomeric DNA as my dependent variable. My issue is, I had contacted a DNA sequencing firm to see if they could sequence my DNA of worms if I gave them it, to observe telomere length and added base pairs. But they told me It would be around 800$ for one sample sequence. So I have to look for another way to measure the worms telomeres. That's one issue- Another issue is actually creating a uniform substance of all my ingredients and actually "giving" it to worms.
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Re: Increasing Telomere Growth

Post by SciB »

I'm not sure I understand why you chose C elegans as the test organism. After it reaches adulthood, there is no more cell division so no telomere shortening can occur. There are different genetic variants of the worms--some have long telomeres, some have short--but this does not affect their lifespan or reproduction:

http://journals.plos.org/plosgenetics/a ... en.0010030

In adult mammals, on the other hand, there are numerous types of cells like stem cells that are continually dividing and thus subject to telomere shortening. I would suggest that you use a culture of normal (not a cancer or immortalized cell like HeLa) human cells like skin fibroblasts that divide and do show telomere shortening. Do you have access to a lab with a biosafety level 2 hood and the necessary tools and reagents to culture human cells?

As to how to measure the length of telomeres without sequencing them, the only way I can think of that you might be able to do it would be to find a restriction endonuclease (https://en.wikipedia.org/wiki/Restriction_enzyme) that cuts in or near the telomeres. You would have to isolate the DNA from the cells, which is easy to do with a kit, cut it with the RE and run it on an agarose gel. The problem then is how to identify which of the bands corresponds to telomeric DNA. Here is one method I found but it requires that you do a Southern blot, which your lab may not have the resources for: https://books.google.com/books?id=h53jl ... el&f=false

If you have access to a thermocycler and the resources to purchase the necessary reagents and primers, there is a method of using polymerase chain reaction, PCR, to measure telomere length: http://nar.oxfordjournals.org/content/30/10/e47.long

I hope this information is helpful. Post again with more questions and we will try to guide you in doing this ambitious and interesting project.

Sybee
jackkwan
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Re: Increasing Telomere Growth

Post by jackkwan »

Thank you so much for your feedback and suggestions! Unfortunately my school doesn't have the ability to perform cell culture. We can though perform the Southern Blot and PCR process and electrophoresis. Do you think it would be possibly to use fruit flies instead of C. elegans as my test subject for telomere growth? If not, could I just use C. elegans when they are young before they mature to adults? Thanks, Jack.
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Re: Increasing Telomere Growth

Post by SciB »

Hi Jack,

Sorry to hear that you can't do tissue culture. Doing your experiment with human cells would be the simplest and best method.

As to which would be better, immature C elegans or fruit flies, I really don't know. I don't really know how many cell divisions it takes to cause a noticeable shortening of telomeres in those organisms. The worms would be easier to expose to the antioxidants and telomerase activator than the flies, but the flies live longer and may show more telomere shortening. Look up some journal articles on fruit flies and see if any telomere research has been done using them. Also check telomere formation in developing C elegans to see if you might be able to use them.

Also, in order to identify the telomeres, you will need a specific DNA probe for the Southern blot or a set of primers for the PCR. You'll have to do some more research to come up with specific probes or primers for telomeres in the worms or the flies and I can help you do that. They can be fairly expensive to synthesize, but there are some companies that offer smaller amounts for researchers to test. If you are going to do a Southern blot you will also need to have a tag added to the probe so it will show up on the agarose gel. Here's a company that supplies one type: http://www.biosyn.com/hrp-oligonucleotide-probes.aspx

Here's another reference for nonradioactive DNA probe labeling and Southern blotting: http://www.dartmouth.edu/~staphy/protoc ... probe.html

Good luck!

Sybee
jackkwan
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Re: Increasing Telomere Growth

Post by jackkwan »

Thanks for the response! I am actually thinking about going down the path and using yeast instead of fruit flies and C. elegans to test for telomere growth. They seem easier to work with and are already sequenced. They also divide really quickly and are easy to replicate. Would you be able to come up with a way to extract the DNA, method for running PCR as well as performing a blotting technique? Thanks, Jack.
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Re: Increasing Telomere Growth

Post by SciB »

Hi Jack,

Yeah, I agree that yeast is easier to work with than fruit flies or worms. Just be sure when you write up your report that you research the differences between telomerase in fungus and telomerase in mammals. You want your conclusions to apply to higher organisms so you need to find out if the telomere systems of a yeast differ in any substantial ways from those of mammals.

Isolating DNA from yeast is pretty simple. Does the lab you plan to work in have a standard procedure for isolating DNA from cells? If they do then you should use that. Here's a simple procedure for isolating genomic DNA from yeast: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3182553/

Your lab should have the necessary chemicals and equipment to do this procedure and it will give you enough DNA to do the PCR and the Southern blot.

You'll have to decide what concentrations of test substances to use and I would do this by adding a range of concentrations to the yeast cultures and measuring the growth rate. You don't want your treatments to kill the yeast! Hopefully your lab has a hemocytometer which is a special microscope slide that is used to count cells. If they don't have a hemocytometer you could determine growth by using a spectrophotometer or nephelometer that can measure the turbidity of the cultures. If you don't have any of these instruments then you will just have to judge growth by eye. Put the cultures side by side and photograph them so you have a record of what they looked like after the treatment.

The next question is what set of primers to use for PCR so that you can determine the length of the telomeres. I don't know what they would be and you'll have to do some serious reading and talking to scientists who can help you. When I do PCR I am looking to see if a specific sequence is present, not to measure the length of a region of DNA. I don't know how familiar you are with how the polymerase chain reaction works but it requires two primers, one forward and one reverse. In order to choose these you need to know the sequence of the DNA that you are trying to amplify. The problem I see with measuring the length of a telomere by PCR is that the ending sequence is going to depend on how much has been cleaved off--how short the telomere has become. Naturally this will be different for different telomeres. So what reverse primer would you use? Maybe one of the other experts can answer this one or the scientists from your lab.

Doing a Southern blot may be the best way to measure telomere length. All you would need is a tagged DNA probe that is complementary to a specific region in the yeast telomeres and a way to cleave the telomeres away from the rest of the DNA at a point close to where the telomeres join the chromosomes. DNA cleavage is done using enzymes called restriction endonucleases and New England Biolabs has a program where you can plug in a DNA sequence and it will tell you what enzymes will cut that sequence: https://www.neb.com/tools-and-resources ... yme-finder

I hope I am not hitting you with too much information all at once. Molecular biology is college level science and I know a lot of this stuff is unfamiliar to you, but it really is not difficult to understand. Youtube has a lot of good videos on isolating DNA, doing Southern blots, selecting probes, doing restriction digests, etc., and you can learn a lot by watching those. Make a list of the questions you have and we will help you.

Good luck!

Sybee
jackkwan
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Re: Increasing Telomere Growth

Post by jackkwan »

Thank you for the feedback once again!I will continue to research the connection between the telomere systems in yeast and mammals. I will also look for the set of primers that I will to perform PCR with. I will be ordering the ingredients for my substance soon and will reply back to you with any questions that I have. Thanks, Jack.
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Re: Increasing Telomere Growth

Post by SciB »

You're welcome! Good luck finding all the information. Don't hesitate to call professors or grad students at universities. They are happy to help young students with questions about methods and the science behind them.

If you get stuck on anything, be sure to send us a post. We want you to understand why you do things in certain ways and not just follow a recipe or formula. You learn a lot more that way and can answer the judges' questions much better.

Sybee
jackkwan
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Re: Increasing Telomere Growth

Post by jackkwan »

Hey! What do you think about instead of measuring the telomere length via PCR and Southern Blot, I instead just measure the amount of times the yeast replicate as my dependent variable. I figured that would be justifiable for the efficacy of my product, because that would show which group the exp. or control can replicate longer, thus who has longer telomeres. This would be a ton simpler to do in the lab, and would totally be manageable. Thanks, Jack.
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Re: Increasing Telomere Growth

Post by SciB »

You could just measure yeast cell numbers as your dependent variable, but this would not prove that your treatment prevented telomere shortening. The treatment could stimulate cell division in some other way. I'm afraid the only way you can say that a substance keeps telomeres intact is by measuring their length.

If you do decide to use cell growth as your outcome you would want to measure the rate of growth as well as the number of cells at the endpoint. That would mean that you have to count the number of cells at specific time intervals and plot the number vs time. If the slope of the line for the treated cells is the same as for the non-treated controls then you can say that the treatment does not increase the rate of growth. Keep counting the cell numbers until they stop increasing and start to decline. If your treated cultures grow at the same rate as the controls but reach a higher final number then you could say that this might be due to telomere preservation.

If you have additional questions, please let us know.

Sybee
jackkwan
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Re: Increasing Telomere Growth

Post by jackkwan »

Thank you, I understand what you are saying about my end conclusions and will get back to you with any questions regarding my project soon. Jack
jackkwan
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Joined: Sun Sep 20, 2015 7:58 pm
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Re: Increasing Telomere Growth

Post by jackkwan »

Hey! I was wondering if I could get your insight on what the simplest procedure would be to go about my research? Thanks, Jack
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Re: Increasing Telomere Growth

Post by SciB »

Well, if you want simple just grow some yeast, treat it with antioxidants and see if those cultures produce more cells than a control without antioxidants. This experiment won't tell you whether telomeres in yeast treated with antioxidants are longer than those without, but you could test several different antioxidants and predict which one works best as a hypothesis.

If you want to measure telomeres you will have to do the experiments that I already mentioned.

Sybee
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