title: Efficient and specific gene knockdown by small interfering RNAs produced in bacteria.
abstract: Synthetic small interfering RNAs (siRNAs) are an indispensable tool to investigate gene function in eukaryotic cells and may be used for therapeutic purposes to knock down genes implicated in disease. Thus far, most synthetic siRNAs have been produced by chemical synthesis. Here we present a method to produce highly potent siRNAs in Escherichia coli. This method relies on ectopic expression of p19, an siRNA-binding protein found in a plant RNA virus. When expressed in E. coli, p19 stabilizes an ∼21-nt siRNA-like species produced by bacterial RNase III. When mammalian cells are transfected by them, siRNAs that were generated in bacteria expressing p19 and a hairpin RNA encoding 200 or more nucleotides of a target gene reproducibly knock down target gene expression by ∼90% without immunogenicity or off-target effects. Because bacterially produced siRNAs contain multiple sequences against a target gene, they may be especially useful for suppressing polymorphic cellular or viral genes.
authors.affiliation: Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, USA.
authors.forename: Linfeng
authors.initials: L
authors.lastname: Huang
authors.forename: Jingmin
authors.initials: J
authors.lastname: Jin
authors.forename: Padraig
authors.initials: P
authors.lastname: Deighan
authors.forename: Evgeny
authors.initials: E
authors.lastname: Kiner
authors.forename: Larry
authors.initials: L
authors.lastname: McReynolds
authors.forename: Judy
authors.initials: J
authors.lastname: Lieberman
mesh_terms: D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D055785:Gene Knockdown Techniques / Q000379:methods*; D005798:Genes, Bacterial / Q000235:genetics; D045325:HCT116 Cells; D006367:HeLa Cells; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008969:Molecular Sequence Data; D012329:RNA, Bacterial / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism*; D014758:Viral Core Proteins / Q000378:metabolism
chemical_list: D012329:RNA, Bacterial; D034741:RNA, Small Interfering; D014758:Viral Core Proteins