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Suppression of a gene by Antisense strategy

Molecular biology Cloning Genetics

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3 replies to this topic

#1 awan

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Posted 01 November 2013 - 08:19 PM

Hi Experts

 

I want to clone a plant gene of 1.5 kb in antisense orientation. I am not interested in protein expression, but only antisense RNA. Do I need to include start codon and stop codon in my insert?

I would be thankful for your suggestions.



#2 jerryshelly1

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Posted 04 November 2013 - 08:35 AM

Are you saying that you want to clone your gene of interest into a vector that will allow for suppression of your gene?

 

Edit - That is confusing. Are you saying that you want to clone your gene into a vector, in the REVERSE direction with the hopes that it will hybridize and suppress your mRNA?



#3 awan

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Posted 04 November 2013 - 11:32 PM

Yes, Exactly... this is what I want to do?



#4 jerryshelly1

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Posted 05 November 2013 - 08:16 AM

This isn't as simple as you would imagine. Researchers pay a decent amount of money for companies to manufacture antisense oligos for gene suppression. Usually when you request antisense oligo's, the manufacturer will send you multiple different plasmids containing different oligo sequences that could potentially disrupt gene expression. As you would expect, it is difficult to determine the correct anti-sense sequence that will disrupt gene function and multiple need to be used to determine which sequence can convey the greatest degree of supression.

 

If you are serious about doing this yourself, you should scan all the relevant literature and try to find a group who has successfully "knocked down" your gene of interest and request the oligo sequence that accomplished this. You may even be able to request a small sample of their plasmid if the lab is feeling generous. I honestly wouldn't attempt this myself, unless I had a significant amount of free time and even then I do not think it is worth the time to develop these myself.







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