Premium
Development of strategies for conditional RNA interference
Author(s) -
Allen Danny,
Kenna Paul F.,
Palfi Arpad,
McMahon Helena P.,
MillingtonWard Sophia,
O'Reilly Mary,
Humphries Pete,
Farrar G. Jane
Publication year - 2007
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.1018
Subject(s) - rna silencing , rna interference , ribozyme , small hairpin rna , biology , rna , promoter , microbiology and biotechnology , polymerase , rna polymerase , computational biology , gene expression , gene , genetics
Background RNA interference (RNAi) represents a powerful tool with which to undertake sequence‐dependent suppression of gene expression. Synthesized double‐stranded RNA (dsRNA) or dsRNA generated endogenously from plasmid or viral vectors can be used for RNAi. For the latter, polymerase III promoters which drive ubiquitous expression in all tissues have typically been adopted. Given that dsRNA molecules must contain few 5′ and 3′ over‐hanging bases to maintain potency, employing polymerase II promoters to drive tissue‐specific expression of RNAi may be problematic due to potential inclusion of nucleotides 5′ and 3′ of siRNA sequences. Methods To circumvent this, polymerase II promoters in combination with cis‐acting hammerhead ribozymes and short‐hairpin RNA sequences have been explored as a means to generate potent dsRNA molecules in tissues defined by the promoter in use. Results The novel constructs evaluated in this study produced functional siRNA which suppressed the enhanced green fluorescent protein (eGFP) both in vitro and in vivo (in mice). Additionally, the constructs did not appear to elicit a significant type‐1 interferon response compared to traditional H1‐transcribed shRNA. Conclusions Given the potential ‘off‐target’ effects of dsRNAs, it would be preferable in many cases to limit expression of dsRNA to the tissue of interest and moreover would significantly augment the resolution of RNAi technologies. Notably, the system under evaluation in this study could readily be adapted to achieve this objective. Copyright © 2007 John Wiley & Sons, Ltd.