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Questions about proper technical approach


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6 replies to this topic

#1 Mindologist

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Posted 03 January 2013 - 04:28 PM

Helow,

I think I found an active promoter region on the human genome, and I guess a certain gene is expressed by the promoter.
Problem is I don't know how to design a series of experiment in order to the promoter that I found is leading to a gene expression.
Could you give me a clue?
Thanks a lot of your helps in advance.

#2 Pangea

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Posted 04 January 2013 - 05:50 AM

Maybe you have to introduce this region upstream to a mammalian expression vector. Cut and Paste the new Promotor. Little bit tricky would be to use recombination and deleting the region.

#3 Mindologist

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Posted 04 January 2013 - 12:15 PM

Thank you, Pangea, for the reply.
But, now that I see my question, it's kind of wrong, and even my english is terrible. I'm really sorry about that.

My situation is like this.
I found an active promoter on the genome, and I confirm its activity by reporter assay.
Now I'm thinking that there may be a gene which is located down stream of the promoter that I found, and the gene is under the control of the promoter.
To prove my idea about a gene that may be located down stream of the promoter, what should I do? What is the best approach to identify the gene that might be there?
I have no idea what to do regarding this.
Could you give me a clue?
Thanks a lot of your helps in advance.

#4 Pangea

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Posted 05 January 2013 - 01:32 AM

Maybe you can check first whether it is a gene or not by using biotool like finding ORF, start and stop codons etc. Than you should clone this region if possible with this promotor.

#5 Julio-Claudian

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Posted 05 January 2013 - 02:52 AM

Hi Mindologist,

I'll go straight to your first question. The first thing that came to my mind is to do what's called the genome walking. Bear in mind that I have not done this (or anything similar) before so someone here might have better alternatives.

Here are some stuffs that could be useful:

www.clontech.com/xxclt_ibcGetAttachment.jsp?cItemId=17326

http://www.protocol-...osts/37995.html

www.ucl.ac.uk/~ucbhjow/b200/Cloning_genomic_DNA.doc

http://www.biotechni...ques-44287.html

https://www.ncbi.nlm...cles/PMC306810/

Edited by Julio-Claudian, 05 January 2013 - 02:53 AM.


#6 Pangea

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Posted 05 January 2013 - 05:32 AM

Julio sounds good. Something new again. Thanks for sharing you knowledge.

#7 Mindologist

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Posted 05 January 2013 - 06:45 PM

Thanks a lot, Pangea and Julio, for the good information and knowledge.
One question for Pangea. Since I don't know anything about bioinformatic tools, do you have your favorite one? or could you recommend one of the tools?
Thanks again.




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