title: Production of highly potent recombinant siRNAs in Escherichia coli.
abstract: We recently invented a method to produce highly potent siRNAs in Escherichia coli, based on the serendipitous discovery that ectopic expression of p19, a plant viral siRNA-binding protein, stabilizes otherwise unstable bacterial siRNAs, which we named pro-siRNAs for prokaryotic siRNAs. We present a detailed protocol describing how to produce pro-siRNAs for efficiently knocking down any gene, beginning with the design of a pro-siRNA expression plasmid and ending with siRNA purification. This protocol uses one plasmid to co-express a recombinant His-tagged p19 protein and a long hairpin RNA containing sense and antisense sequences of the target gene. pro-siRNAs are isolated and purified using nickel beads and HPLC, using methods used to produce recombinant proteins. Once a pro-siRNA plasmid is obtained, production of purified pro-siRNAs takes a few days. The pro-siRNA technique provides a reliable and renewable source of siRNAs, and it can be implemented in any laboratory whose members are skilled in routine molecular biology techniques.
authors.affiliation: 1] Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts, USA. [2] Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Linfeng
authors.initials: L
authors.lastname: Huang
authors.forename: Judy
authors.initials: J
authors.lastname: Lieberman
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D006367:HeLa Cells; D006801:Humans; D010957:Plasmids; D034622:RNA Interference; D012329:RNA, Bacterial / Q000737:chemistry; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics*
chemical_list: D012329:RNA, Bacterial; D034741:RNA, Small Interfering