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Specific targeting of alternative splice forms?


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#1 Coomb Raider

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Posted 11 March 2010 - 09:50 AM

Hi experts,
given that alternative splice forms all start out on the same piece of pre-mRNA, is it possible to target a particular one? Having trawled through the archives it seems that some people are saying yes, but at the same time it seems that the degradation happens intra-nuclearly, so surely at least some of the prespliced mRNA must be degraded? Can't seem to get the answer from the literature so if anyone does have a reference explaining why it does or doesn't that would be great. I need to know that I'm having zero effect on the splice form I wish to preserve.

It may or may not be important, but I'm planning to transfect in shRNAs to specifically target an exon about 2500bp into a final mRNA of 3kb

Many thanks

#2 Jon Moulton

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Posted 12 March 2010 - 07:44 AM

One option is to use Morpholinos targeted for splice modification to convert the undesired splice forms into the one you want (or trigger their degradation by nonsense-mediated decay(NMD)). A Morpholino can be targeted to a splice junction bordering an exon, causing the exon it borders to be spliced out of the pre-mRNA (1). If a Morpholino causes clean excision of an exon with a number of bases not evenly divisible by three, it causes downstream sequence to be frameshifted and often sets up the modified mature mRNA for NMD. Unexpected outcomes occasionally result from blocking splicing, such as activation of a cryptic splice site leading to partial exon excision or partial intron inclusion, so the product of splice-modification should be characterized by RT-PCR (2). Splice-modification is a commonly-used technique in developmental biology, especially for zebrafish and Xenopus embryos. For a more complete description of the techniqe, see the Current Protocols chapter (3).


(1) Morcos PA. Achieving targeted and quantifiable alteration of mRNA splicing with Morpholino oligos. Biochem Biophys Res Commun. 2007 Jun 29;358(2):521-7. Epub 2007 May 7.

(2) Draper BW, Morcos PA, Kimmel CB. Inhibition of zebrafish fgf8 pre-mRNA splicing with morpholino oligos: A quantifiable method for gene knockdown. Genesis. 2001 Jul;30(3):154-6.
http://www3.intersci...006231/PDFSTART

(3) Moulton JD, Yan YL. Using Morpholinos to control gene expression. Curr Protoc Mol Biol. 2008 Jul;Chapter 26:Unit26.8.

Edited by Jon Moulton, 12 March 2010 - 07:47 AM.

Jon D. Moulton
Gene Tools, LLC
www.gene-tools.com

#3 Coomb Raider

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Posted 15 March 2010 - 04:42 AM

[quote name='Jon Moulton' date='Mar 12 2010, 03:44 PM' post='62411']

Thanks a lot, I'll check these ones out.

Also, I've been using siRNA scales to design my targets. How do these predictions work out in general? I like the fact it gives an estimated level of knock-down, but being that I have had a couple of suggestions of sequences that knock it down by 102%, I have a feeling that they might be taking the predicitive nature of the algorithm a bit too far!




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