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What are methods to promote or suppress gene expression?


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What are methods to promote or suppress gene expression?

Postby 1101 » Wed Jul 28, 2010 10:20 pm

Hi I was just wondering what some common methods of promoting or suppressing genes are. I'm just going to list off some of my own ideas and would appreciate it if anyone could tell me if I was wrong/right, provide other techniques, or just provide more information on the subjects. Keep in mind I'm talking about live human genes so techniques that would only work on a single cell (like micro injections) or gamete cells aren't what I'm interested in.

Basically the following examples assume that a retrovirus is going to be used to inject viral RNA into a cell. I'm not really too familiar with other methods that work on muticelluar eukaryotes (if you happen to know any I'd love to hear them). Though the problem with viral vectors is that sooner or later the immune system is going to gang up on them so they'd only be effective for a little bit (which might be fine depending on what it is your doing).

Ideas for promotion of genes
1) Introduce RNA that is translated to a gene promoter in the cell.
2) Introduce RNA that is translated to a protein that activates a gene promoter in a cell.
3) Introduce RNA that deactivates the RNA of a suppressor for that gene or is translated into a protein that deactivates a suppressor.

Ideas for suppression of genes
1) Introduce RNA that binds to the corresponding mRNA of the gene you wish to suppress.
2) Introduce RNA that is translated into a protein that either destroys to a gene promoter or changes it shape so that it can't bind to DNA.
3) Introduce RNA that is translated into a suppressor for desired gene.

You can see the pattern. So far the only thing I really know is sending in viral RNA to mess with proteins or mRNA.
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Postby JackBean » Thu Jul 29, 2010 7:22 am

1) what is that? How do want to translate gene into promoter?

What about simply expression of your gene of interest?

1) simply any RNAi technology should work (miRNA, siRNA etc.)

2) you can't destroy gene promoter, that would mean to break DNA and you sincerely do not want that.

3) or protein that deactivates activator etc. ;)

4) you could try knock-out of your gene, but I don't know, whether it works in human.

5) in plants you could use simply T-DNA insertion :) but that's not specific.
But some people belive, that Agrobacterium can infect even humans...

Cis or trans? That's what matters.
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Re: What are methods to promote or suppress gene expression?

Postby jonmoulton » Thu Jul 29, 2010 3:01 pm

There are some techniques you can apply at the RNA level. Steric blocking antisense oligos can be used to suppress gene expression, either by blocking the translation initiation complex by binding to the 5'-UTR or by modifying splicing of pre-mRNA by blocking elements of the splicing system. The expression of genes controlled by miRNA translation suppression can be enhanced by protecting the miRNA target on the mRNA, again using a steric-blocking antisense oligo.

Your interest in doing this in an intact animal makes it harder, as that requires systemic delivery of the oligo. There are some moieties which can be attached to a steric-blocking oligo to help the oligo enter cells (likely be enhancing endosomal escape). One example is to use an arginine-rich cell penetrating peptide covalently linked to the oligo. So far the delivery moieties developed have been somewhat toxic, though they have been found effective at doses where the toxicity is not detectable. However, extended clinical trials have not been conducted using peptide-oligo conjugates so toxicity questions remain. An advantage of these techniques over viral-mediated delivery is that, at least for Morpholino-peptide conjugates, no immune response has been detected; immune responses have been a complicating factor for systemic viral delivery of nucleic acids.
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