High-throughput screening of effective siRNAs from RNAi libraries delivered via bacterial invasion.
Use of RNA interference (RNAi) as a reverse genetics tool for silencing genes in mammalian cells is achieved by in vitro transfection of small interfering RNAs (siRNAs). For a target gene, several siRNAs must be designed according to the empirical rules. We demonstrated that functional short hairpin RNAs (shRNAs) could be synthesized in Escherichia coli and delivered directly via bacterial invasion to the near entirety of a mammalian cell population to trigger RNAi. Furthermore, using a luciferase-target gene transcript, we identified effective shRNAs and siRNAs from RNAi libraries delivered conveniently through bacterial invasion in 96-well plates without need for preparation, purification and transfection of shRNAs. Notably, several of the most highly effective shRNAs and siRNAs identified do not fit the empirical rules commonly used for siRNA design, suggesting that this approach is a powerful tool for RNAi research, and could be used complementarily to the empirical rules for RNAi applications.
Zhao
Hui-Fen
HF
Health Sector, Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montréal, Québec, Canada, H4P 2R2. hui-fen.zhao@cnrc-nrc.gc.ca
L'Abbé
Denis
D
Jolicoeur
Normand
N
Wu
Meiqun
M
Li
Zhen
Z
Yu
Zhenbao
Z
Shen
Shi-Hsiang
SH
D020650:Combinatorial Chemistry Techniques / Q000379:methods; D004926:Escherichia coli / Q000472:pathogenicity*; D015723:Gene Library; D006367:HeLa Cells / Q000382:microbiology; D006801:Humans; D016376:Oligonucleotides, Antisense / Q000235:genetics; D034622:RNA Interference; D012329:RNA, Bacterial / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D012342:RNA, Small Nuclear / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics*; D014162:Transfection
D016376:Oligonucleotides, Antisense; D012329:RNA, Bacterial; D034741:RNA, Small Interfering; D012342:RNA, Small Nuclear