siRNA-mediated gene silencing in insect and mammalian cells. The effect of esiRNA on R-luc (a and c) or F-luc (b and d) expression was determined in Drosophila S2 cells (a and b) and mammalian cell lines, HeLa and C33A (c and d). Plasmid DNAs with or without various sized dsRNAs were cotransfected into cells. (e) Reporter plasmids, siRNAs, dsRNAs, and short hairpin RNAs. The diagram illustrates DNA constructs used to produce in vivo mRNA and in vitro dsRNA for F-luc and R-luc genes. The positions of dsRNAs, chemically synthesized siRNAs (CS1 and CS2), in vitro-transcribed siRNAs (T1 to T6), and short hairpin RNAs (H1 to H6) are indicated. (f) The variable effect of synthetic siRNAs or short hairpin RNAs on different sequences within a gene. Plasmid DNAs with or without the various indicated short RNA species were cotransfected into HeLa cells. R is the F-luc esiRNA shorter than 30 bp. transfected with pGL-3-control (Promega) and pRL-CMV with or without 21- to 23-bp esiRNA corresponding to either the cytomegalovirus (CMV) promoter or R-luc. Luciferase activity was measured 24–96 h after cotransfection. (e and f) Relationship between RNAi and the IFN signaling pathway. DsF-592 and F-luc esiRNA (21–23 bp) were transfected individually or together into HeLa cells to silence F-luc expression from plasmid templates. Luciferase activity was measured 24–48 h after cotransfection. Data are expressed as either F-luc units (e) or relative F-luc activity (f), normalized to that of DNA-only transfections.

esiRNA induces degradation of its cognate mRNA. ³²P-labeled F-luc or R-luc mRNA was incubated in extracts of HeLa cells with or without the cognate esiRNAs for the indicated time periods and then analyzed in a 4% sequence gel.

Preparation of siRNA from dsRNA by hydrolysis with E. coli RNase III. (a) Overexpression and purification of GST-RNase III fusion. Lanes 1 and 2, E. coli extracts before and after isopropyl β-d-thiogalactoside induction; lane 3, purified GST-RNase III fusion; lane M, protein molecular weight marker. (b) Time course of RNase III digestion of dsRNA. DsR-457 or dsF-592 RNA was incubated with RNase III for the indicated time, and then separated by electrophoresis in a 4% agarose gel. Lanes 1, 4, and 7 are dsR-457; lanes 2, 5, and 8 are dsF-592; lanes 3, 6, and 9 are 21-bp siRNA marked by an arrow. (c) Agarose gel analysis of purified short RNA species processed from dsF-592. Lane M, 10-bp DNA marker; lanes 1 and 2 are chemically synthesized siRNAs; lane 3, 21–23 bp; lane 4, 24–26 bp; lane 5, 27–30 bp.

Characteristics of RNAi. (a) Dose–response for antisense RNA (asRNA) and esiRNA against F-luc. Indicated amounts of F-luc esiRNA (21–23 bp) or antisense strand of CS1 were transfected into RPE cells to target F-luc expression from chromatin templates, and luciferase activities were measured at 24 h after transfection. Data represent the average of 3–6 experiments and are expressed as relative luciferase activity after normalization to the luciferase units/μg of protein observed in control cells transfected with R-luc esiRNA. (b) Duration of esiRNA-mediated inhibition. F-luc esiRNA (21–23 bp) was transfected into RPE cells, and luciferase activities were measured at the indicated time period. When HeLa cells were used, F-luc target was expressed from cotransfected plasmid DNA. (c) esiRNA silences gene expression from mRNA templates. HeLa cells were transfected with 0.5 μg F-luc mRNA with or without 0.1 μg of esiRNA (21–23 bp) for either F-luc or R-luc for 3 h and then collected for luciferase assays. Data are expressed as arbitrary luciferase units and normalized to that produced in cells transfected with mRNA only. (d) esiRNA corresponding to the promoter region has no RNAi activity. HeLa cells were

Silencing of endogenous mammalian genes with esiRNA. (a) Specific inhibition of expression of LCa. Western blotting was used to analyze HeLa cells that had been mock-transfected or transfected with LCa esiRNA. Three antibodies were used: rabbit serum that recognizes both LCa and LCb, mAb against actin, and monoclonal TD.1 against the clathrin heavy chain (CHC). (b) Selective reduction of c-Myc expression; 293 cells were transfected with buffer, c-myc esiRNA, or R-luc esiRNA. Western blotting was performed with antibodies against c-myc and actin 72 h after transfection. (c) Dose-dependent inhibition of Cdk2 expression by esiRNA; 293 cells were transfected with indicated doses of Cdk2 esiRNA. Western blotting was performed with antibodies against Cdk2 and cyclin A 5 days after transfection.