Science Buddies: "Ask an Expert"

I was wondering if it's possible to use keratinocytes exposed to UV radiation in an experiment involving solar injury SNPs associated with skin cancer.

Thanks,
blueswim

Hi Blueswim,

Yes, this would be an excellent project if you had access to a laboratory with the necessary equipment to do a project like this. The cell culture reagents and materials for SNP analysis would represent a significant expense, as well. This is a very ambitious project and would require a significant amount of time.

Here is a similar project that studied SNP in keratinocytes in individuals in an investigation on wound healing. This article includes a description of the experimental technique and equipment required for this type of study.

http://www.plosone.org/article/info%3Ad ... ne.0000697

If you collected keratinocytes from humans, you would need preapproval for working with humans:

https://www.sciencebuddies.org/science- ... _src.shtml

Here are two more science buddies website articles that will give you important information before you start on this ambitious project:

https://www.sciencebuddies.org/science- ... dmap.shtml

https://www.sciencebuddies.org/science- ... tors.shtml

Do you have access to a lab with tissue culture facilities and PCR instruments? Have you ever done an experiment using tissue culture or PCR?

You need to narrow your topic down to a single experiment. What are you thinking about doing?

Donna Hardy

Thanks, Donna

I was thinking of doing one of the variations of the Bioinformatics - The Perfect Marriage of Computer Science & Medicine (https://www.sciencebuddies.org/science- ... p008.shtml) project.

Variation 1: Environmental Factor - Gene Interaction:
Identify how certain environmental factors may affect genes and their association to diseases by using Genetic Association Database (http://geneticassociationdb.nih.gov/cgi-bin/index.cgi). NOTE: This database is open-access and allows any user to input data. Use caution while using the data and only select data that has been endorsed by ‘Gene Expert’ or ‘Disease Expert’.
Click on ‘Environmental Factor Gene Interaction’ link on the left menu of the website. On the top of the page, click on the link to see a complete list of environmental factors.
Choose an environmental factor of interest (for e.g., tobacco smoke) by clicking on it.
You can see entries that describe gene association with specific diseases.
Are you able to identify any SNPs in this category? Follow links to research more for each category

However, I also wanted to do lab work. I don't know if I can work with skin cells yet, but I do know that I have access to equipment used to analyze SNPs and access to PCR equipment as well. I have worked with PCR but not tissue cultures (I've worked with bacteria before, but that's not a tissue...) So I was thinking, based on the variation above, that I would see if time skin was exposed to UV radiation would affect the SNPs (I've done the bioinformatics part of the SNP research).

Thanks,
blueswim

Hi Blueswim,

You have already done a lot of work on this project, if you have done the bioinformatics part of the search. Did you identify a SNP for a specific gene?

If you are able to do any lab work, how will the SNP’s be detected? For this analysis, you can use PCR, sequencing or direct hybridization.

I just noticed that your project is due at the end of November. If you will do lab work, you would need to have access to samples that have already been collected, and preferably with the DNA already extracted. The purity and integrity of the DNA is very important in SNP analysis. And you would need to have primers available if you are doing PCR.

With the imminent due date, perhaps you should concentrate on writing up your results to date. If you are not graduating this year, perhaps you could continue the project next year with the lab work.

Please let me know if you need any more information.

Donna Hardy

Thanks again.
Yes, I think I'll be using PCR and sequencing.
Oh, and I messed up on the project due date. That is just when my science fair procedures/hypothesis needs to be done.

Thanks,
blueswim

How long should I expose the keratinocytes for?
Also, at first I was going to use people for more genetic diversity, but I decided it was too much of a hassle.
How would I still get genetic diversity?

Thanks,
blueswim

HI - This looks like a really cool project but there are lots of logistics that I don't know if you've considered. If you're working with human cell lines or tissues, even if they're cell lines that have been in culture for 60 years and from a reliable source (like HeLa cells), you have to have Blood Borne Pathogen training at just about any institution you're planning on doing the work (at least in the US). These cell lines and tissues are possible sources of infectious viruses.

As for the actual experiment - are you looking for genetic diversity or similarity? You could probably get away with 3 individuals but I think it depends on the question you're trying to answer. If you're looking for the same marker to have a SNP in the same population, then try family members. If you want to find SNPs genome-wide that may contribute to a skin cancer risk, try genetically diverse individuals.

As for the exposure time, too much UV will kill a cell in culture. You might need to first do a "kill curve" - expose cells to UV for 5, 10, 20, 30 minutes and look for induction of apoptosis, survival and SNPs (you will probably have to wait 12-24 hours after UV exposure to examine the effects on the cells). The density of the cells in culture, or confluency, may also affect the rate at which damage and apoptosis occurs. There are going to have to be a lot of initial experiments and controls needed before you can start the "real" experiment.

Good luck!
Stephanie

Hi,
thanks for the reply.
I'm looking for SNPs in a certain population.
So it wouldn't be possible to order cell lines and take the experiment from there?

Thanks,
blueswim

Hi-
There are very few "normal" immortalized cell lines. This is because for cell lines to become immortalized, they often have to be tranformed, usually as a result of some kind of tumor-like activity. Then when injected into mice, they usually form carcinomas, or tumors. This might complicate things. There is on "normal" human keratinocyte cell line, called HaCaT. I can't figure out how to buy it in the US, but you might have luck contacting the original researcher who first described it:
Norbert E. Fusenig, Division of Differentiation and Carcinogenesis (A080), German Cancer Research Center (DKFZ) INF 280, 69120 Heidelberg, Germany. E-mail: [email protected].

There are also a couple of cell lines available from a company called ATCC (atcc.org) that sells cell lines. For example:
http://www.atcc.org/ATCCAdvancedCatalog ... ellBiology

or Invitrogen:
http://www.invitrogen.com/site/us/en/ho ... 5722990398

But, these are all Biosafety Level (BSL) 2 - will you have access to BSL2 hoods and equipment? And the BBP training? Are you working with somebody at the university level?

Otherwise, there are mouse keratinocyte cell lines that are BSL1:
https://www.atcc.org/ATCCAdvancedCatalo ... ellBiology

Good luck!
Stephanie

Hi,
Thanks agaiN!
I'm not working with someone at a university, and from descriptions of BSL1, I think the place I'm working at is at the BSL1 level.

Ok, so my project is about if theres a link between time exposed to UV light and the presence of SNPs. So is it enough to grow (a) keratinocyte sample(s) that isn't exposed to UV light, and another set of samples with different amount of exposure to UV light?

On the comments of the Mouse keratinocytes, it says

XB-2 cells can undergo a process of terminal differention similar to keratinocytes. When cultured at low cell density as adherent cells, few cells differentiate. When placed in suspension culture, however, cultures demonstrate colony forming ability decrease, nuclear pyknosis and other features of keratinocyte differentiation.

What does differentiation mean? I looked it up, and didn't really understand the definition.

Thanks,
blueswim

Hi there,

If you are interested in a project looking at the genetics of UV response, you might want to consider working with melanocytes. (These are the cells that produce melanin in the skin in response to UV damage) Human and mouse melanocytes cell lines are available from the Wellcome Trust functional genomic cell bank at
http://www.sgul.ac.uk/depts/anatomy/pages/WTFGCB.htm

Melanocytes also are responsible for pigmenting hair and fur, and the genetics of coat coloration in mice is one of the most powerful mammalian model systems. The cell bank noted above has scores of different mutant melanocyte lines isolated from coat color mutants -- this might be a useful source of relevant genetic variation for a study of UV response in melanocytes.