Author

Jon Moulton
http://www.gene-tools.com     Email

Published on SDB CoRe: Apr 17 2012

Tools & Techniques: Molecular Techniques
Organism: Zebrafish, Xenopus, Chick, Mouse, Axolotl, Human, Medaka, Lamprey, Salamander
Stage of Development: Embryo

Object Description

The figure compares normal gene expression (top) with gene expression where splicing is modified by a Morpholino oligo (bottom).  The Morpholino is shown in green.

Morpholinos for modifying splicing

Morpholino oligos are typically targeted to splice junctions to modify pre-mRNA splicing.  They can also block the binding sites of splice-regulatory proteins.

A snRNP binds at each intron-exon junction in a pre-mRNA; these direct the splicing system to the splice junctions.  Binding a Morpholino to a snRNP binding site prevents the snRNP from binding, so the splicosome splices between the remaining snRNPs.  Typically this results in exclusion of an exon (exon skipping) if an internal splice site is targeted, or inclusion of an intron if the very first or very last splice site is targeted.  Sometimes cryptic splice sites are activated, producing RT-PCR products of unexpected mass.  Sometimes intron inclusions occur even if internal splice sites are targeted. 

If an exon is skipped which has a number of bases not evenly divisible by three, it frameshifts downstream sequence.  This often brings a premature termination codon in-frame and activates the nonsense-mediated decay system causing rapid degradation of the transcript.

The activity of a splice-modifying Morpholino is typically assessed by RT-PCR with primers on the exons flanking the exon targeted by the Morpholino.  If excluding an exon produces an in-frame transcript, you can usually detect an RT-PCR product at the mass determined by the exon-skipped product.  If a transcript is rapidly decayed after frameshifting, the mass-shifted RT-PCR product might not be observed.

References

Draper, B.W., Morcos, P.A., Kimmel, C.B.  Inhibition of zebrafish fgf8 pre-mRNA splicing with morpholino oligos: A quantifiable method for gene knockdown. Genesis, 2001, 30:154-6.

Morcos, P.A.   Achieving targeted and quantifiable alteration of mRNA splicing with Morpholino oligos. Biochem Biophys Res Commun., 2007, 358:521-7.

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