Development of strategies for conditional RNA interference.
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.
Allen
Danny
D
Department of Genetics, Trinity College Dublin, Dublin 2, Ireland. danny.allen@tcd.ie
Kenna
Paul F
PF
Palfi
Arpad
A
McMahon
Helena P
HP
Millington-Ward
Sophia
S
O'Reilly
Mary
M
Humphries
Pete
P
Farrar
G Jane
GJ
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D008815:Mice, Inbred Strains; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism
D016337:RNA, Catalytic; C427204:hammerhead ribozyme; D049452:Green Fluorescent Proteins; D012313:RNA; D012319:RNA Polymerase II