High-throughput screening of effective siRNAs from RNAi libraries delivered via bacterial invasion
- PMID: 16299483
- DOI: 10.1038/nmeth812
High-throughput screening of effective siRNAs from RNAi libraries delivered via bacterial invasion
Abstract
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.
Similar articles
-
Expressing functional siRNAs in mammalian cells using convergent transcription.BMC Biotechnol. 2003 Nov 6;3:21. doi: 10.1186/1472-6750-3-21. BMC Biotechnol. 2003. PMID: 14604435 Free PMC article.
-
[Detection of RNA interference in nasopharyngeal carcinoma cell lines using reporter genes].Ai Zheng. 2005 Mar;24(3):371-5. Ai Zheng. 2005. PMID: 15757546 Chinese.
-
A plasmid-based system for expressing small interfering RNA libraries in mammalian cells.BMC Cell Biol. 2004 Apr 30;5:16. doi: 10.1186/1471-2121-5-16. BMC Cell Biol. 2004. PMID: 15119963 Free PMC article.
-
Gene silencing through RNA interference (RNAi) in vivo: strategies based on the direct application of siRNAs.J Biotechnol. 2006 Jun 25;124(1):12-25. doi: 10.1016/j.jbiotec.2005.12.003. Epub 2006 Jan 18. J Biotechnol. 2006. PMID: 16413079 Review.
-
RNA interference for the identification of disease-associated genes.Curr Opin Mol Ther. 2004 Apr;6(2):136-40. Curr Opin Mol Ther. 2004. PMID: 15195924 Review.
Cited by
-
Systematic gene silencing identified Cryptosporidium nucleoside diphosphate kinase and other molecules as targets for suppression of parasite proliferation in human intestinal cells.Sci Rep. 2019 Aug 21;9(1):12153. doi: 10.1038/s41598-019-48544-z. Sci Rep. 2019. PMID: 31434931 Free PMC article.
-
Live-Attenuated Bacterial Vectors: Tools for Vaccine and Therapeutic Agent Delivery.Vaccines (Basel). 2015 Nov 10;3(4):940-72. doi: 10.3390/vaccines3040940. Vaccines (Basel). 2015. PMID: 26569321 Free PMC article. Review.
-
Efficient and specific gene knockdown by small interfering RNAs produced in bacteria.Nat Biotechnol. 2013 Apr;31(4):350-6. doi: 10.1038/nbt.2537. Epub 2013 Mar 10. Nat Biotechnol. 2013. PMID: 23475073 Free PMC article.
-
Forward and robust selection of the most potent and noncellular toxic siRNAs from RNAi libraries.Nucleic Acids Res. 2009 Jan;37(1):e8. doi: 10.1093/nar/gkn953. Epub 2008 Nov 29. Nucleic Acids Res. 2009. PMID: 19043072 Free PMC article.
-
Involvement of p114-RhoGEF and Lfc in Wnt-3a- and dishevelled-induced RhoA activation and neurite retraction in N1E-115 mouse neuroblastoma cells.Mol Biol Cell. 2010 Oct 15;21(20):3590-600. doi: 10.1091/mbc.E10-02-0095. Epub 2010 Sep 1. Mol Biol Cell. 2010. PMID: 20810787 Free PMC article.
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
