Achieving targeted and quantifiable alteration of mRNA splicing with Morpholino oligos
Section snippets
Background
Morpholino oligos allow experimental manipulation of pre-mRNA splicing. Such manipulation is powerful given that about 74% of all transcripts from multi-exon genes undergo alternative splicing [1].
A powerful technique for studying functions of individual transcripts is to use steric-blocking antisense oligos to block RNA processing events, splicing events in particular, and thereby force the expression of altered transcripts. Morpholino oligos have been shown to be the most effective
The test system and general guidelines
Our 3-exon test system is a human β-globin intron fusion with firefly luciferase stably integrated in HeLa cells [14]. Blocking a dominant splice mutant with a Morpholino oligo edits out a stop codon and brings luciferase in-frame, up-regulating luciferase expression. In this test system, splice-modification can be characterized by luciferase activity or RT-PCR analysis (Fig. 1A) and it provides all the components necessary to define optimal splice targeting.
The 3-exon test system looks no
Conclusion
We have focused on the use of Morpholino oligos as tools to alter splicing events and have shown that with appropriate sequence analysis and by targeting internal exons, the likely outcome is an exon deletion. The targeting methods described in this paper have been used to design oligos for hundreds of successful splice-blocking experiments in tissue culture and in developmental model systems including zebrafish and frog embryos. A large percentage of splice-blocking results have yielded
Experimental procedures
Materials. HeLa cells stably transfected with pLUC/705 [14], a plasmid comprising the firefly luciferase gene interrupted with human β-globin intron2 containing a favored splice site mutation (IVS2-705) and referred to as the positive test system, were from Dr. Ryszard Kole. Morpholino oligos were synthesized by GENE TOOLS, LLC (http://www.gene-tools.com). The sequences of the Morpholino oligos targeting the β-globin test system were derived from pLUC/705 sequences as follows: e1i1,
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