XB-ART-49925Methods. September 15, 2013; 63 (2): 101-9.
Organic small hairpin RNAs (OshR): a do-it-yourself platform for transgene-based gene silencing.
The RNA interference (RNAi) pathway in animal cells can be harnessed to silence gene expression with artificial small interfering RNAs (siRNAs) or transgenes that express small hairpin RNAs (shRNAs). The transgene-expressing shRNA approach has been adapted into large-scale resources for genome-wide loss-of-function screens, whereas focused studies on a narrow set of genes can be achieved by using individual shRNA constructs from these resources. Although current shRNA repositories generally work, they might fail in certain situations and therefore necessitate other alternatives. We detail here a new highly-accessible and rational design of custom shRNAs that utilizes a refined backbone configuration termed the ''organic'' shRNA (OshR) platform. The OshR platform is ''organic'' because it conforms more naturally to the endogenous vertebrate miRNAs by maintaining specific bulges and incorporating strategic mismatches to insure the desired guide strand is produced while reducing the accumulation of passenger strands that might contribute to off-target effects. We also demonstrate that the reliability of the OshR platform for gene silencing is increased when sequences target the 3'' UnTranslated Region (3''UTR) of a gene. We further compare the OshR platform with the current and emerging shRNA designs, and propose that the OshR platform is a novel approach that can allow investigators to generate custom and effective shRNAs for individual gene functional studies.
PubMed ID: 23707624
PMC ID: PMC3966114
Article link: Methods.
Grant support: EY017400 NEI NIH HHS , EY018000 NEI NIH HHS , R00 HD057298 NICHD NIH HHS , R00HD057298 NICHD NIH HHS , R21 DA026977 NIDA NIH HHS , R21DA026977 NIDA NIH HHS , T32 MH019929 NIMH NIH HHS, R01 EY017400 NEI NIH HHS , R00 HD057298 NICHD NIH HHS , R21DA026977 NIDA NIH HHS , EY017400 NEI NIH HHS , R01 EY018000 NEI NIH HHS , EY018000 NEI NIH HHS , T32 MH019929 NIMH NIH HHS, R00HD057298 NICHD NIH HHS , R21 DA026977 NIDA NIH HHS