1 reply to this topic

[seashell83]

Hi All,

I am new to the world of alternative transcripts. I am working with a protein which has 2 transcripts and I really want to know if it is possible to test to the functional significance as well as functional differences between both the transcripts.
What kind of tests would i perform. Would it work if i knocked down just one of the transcripts utilising some area which is not common to them both and also if the transcript arises from a same longer mrna transcript would it essentially inhibit the main transcript if i try to knock down only one of them.

Can anyone help me out with this and let me know some assays which could help me differentiate  between them functionally and also if it could be that one could inhibit the other....!!

THANKS

[Jon Moulton]

Hi Seashell,

One potential approach is to modify splicing using a splice-modifying oligo.  These oligos usually cause exon skipping, splicing a targeted exon from the pre-mRNA to make a shorter-than-usual mRNA.  You could potentially change the entire population of mRNA produced from your gene of interest into one of the two isoforms.

There are several antisense types that can modify splicing, including Morpholino oligos, locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and 2'-O-methyl phosphorothioates.  All of these are RNase-H independent structural types (instead of modifying splicing, an RNase-H competent oligo will trigger cleavage of the target mRNA by RNase-H).

Here are some sources:
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.
http://www.gene-tool...os2007howto.pdf

Moulton JD, Yan YL.  Using morpholinos to control gene expression.  Curr Protoc Mol Biol. 2008 Jul;Chapter 26:Unit26.8.
http://currentprotoc...protocol/mb2608

Edited by Jon Moulton, 24 April 2009 - 07:00 AM.