Need help and opinions ASAP.

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moe20
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Need help and opinions ASAP.

Post by moe20 » Wed Dec 26, 2007 10:09 am

hi, im a third year biology student and i need ur opinion on a question that my professor gave me for my presentation...the question is as follows: """Design a set of experiments to verify the following statement: Rap2, not Rap1, is critically needed for the adehsion of smooth muscle cells to fibronectin."""

now this statement may not be scientifically valid and the experiments may not be true too, also the compounds that are used may not be real either....the purose of this question is to test the ability to design experiments...

so any suggestions and opinions to prove the validity of this statement???

I appreciate ur help!

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Re: Need help and opinions ASAP.

Post by moe20 » Wed Dec 26, 2007 10:12 am

OH ONE MORE THING: THE EXPERIMENTS ARE JUST [u]HYPOTHETICAL[u], NO EXPERIMENTS MUST BE DONE IN LABRATORY.
Last edited by moe20 on Thu Dec 27, 2007 7:49 am, edited 1 time in total.

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mith
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Re: Need help and opinions ASAP.

Post by mith » Wed Dec 26, 2007 9:24 pm

Well obviously you can silence rap1/rap2 either thru homozygous knockout mutants or RNAi. Then it's just a problem of cause and effect isn't it?
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Accepting hardships as the pathway to peace;
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moe20
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Re: Need help and opinions ASAP.

Post by moe20 » Thu Dec 27, 2007 7:59 am

mith wrote:Well obviously you can silence rap1/rap2 either thru homozygous knockout mutants or RNAi. Then it's just a problem of cause and effect isn't it?


well you seem much more experienced than i am....just follow with me plz, honestly this is the 1st time i hear abt RNAi, so i went and read about it...this dsRNA -if specifically targeted on Rap1- will inhibit Rap1??...cld you plz just give me a brief explanation on its mechanism to inhibit Rap1??? and how can it be specified to work only on Rap1 or Rap2???

"i really appreciate your hlep"!

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mith
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Re: Need help and opinions ASAP.

Post by mith » Thu Dec 27, 2007 4:44 pm

Assuming rap is a gene, and you know the DNA sequence, you can make a RNAi sequence that is complementary to it. I'm not intricately familiar with the specifics but the sequence also has some other identifying features. Anyways the RNA would be taken up by the cell, binds to specific mRNA that is produced by the gene(for protein coding) then a slicer protein cuts them up making them useless. Therefore there is no rap protein production. You can achieve the same result if you have a homozygous mutant for that gene.
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Re: Need help and opinions ASAP.

Post by BioLad » Fri Dec 28, 2007 9:15 pm

RNAi basically makes a second strand of RNA in the opposite orientation that will bind to the strand generated by the cell. This triggers cell mechanisms to chop up the strand into smaller pieces and uses those pieces to bind to other RNA molecules, prime elongation from those bound RNA pieces (primers) and continue the cycle. Since, as Mith pointed out, there is no RNA to translate, no protein is made. This is thought to be part of an antiviral defense mechanism.

I have done a bit of work with RNAi. If I were performing this I would create two plasmids; one with the antisense Rap1 and one with antisense Rap2 either a constitutive (always on) promoter or an inducible (turns on under a certain trigger, chemical physical ect...) promoter. Inducible would be good in case Rap1 or Rap2 are needed for cell growth. Then I would insert the plasmids into two different sets of cells and check whether they adhere with and without the chemical trigger for the inducible promoter. I would also run controls with cells with the original plasmid (with no Rap1 or Rap2 sequence) with and without the chemical trigger to show that the trigger and plasmid backbone do not interfere through some other mechanism.
-BL

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