Enhanced gene silencing by the application of multiple speci c smallinterfering RNAs
Enhanced gene silencing by the application of multiple speci¢c small interfering RNAs Jingmin Ji, Marion Wernli, Thomas Klimkait, Peter Erb Institute for Medical Microbiology, University of Basel, Petersplatz 10, 4003 Basel, Switzerland Received 30 April 2003; revised 5 August 2003; accepted 7 August 2003 First published online 22 August 2003 Edited by Felix Wieland Abstract Small interfering RNA duplexes (siRNA) induce gene silencing in various eukaryotic cells, although usually in an incomplete manner. Using chemically synthesized siRNAs targeting the HIV-1 co-receptor CXCR4 or the apoptosis-inducing Fas-ligand (FasL), co-transfection of cells with two or more siRNA duplexes targeting di¡erent sites on the same mRNA resulted in an enhanced gene silencing compared with each single siRNA. ... Transfection e⁄ciency determined for the FasL-speci¢c siRNAs was dose-dependent and varied among the siRNAs tested, but was not the main reason for the enhanced gene silencing. ... Key words: RNA interference; Small interfering RNA; Gene silencing; CXCR4; Fas-ligand; HIV infection 1. Introduction In 1998, Fire and coworkers reported that double-stranded RNA (dsRNA) introduced into C. elegans blocked gene expression by a process of sequence-speci¢c, post-transcriptional gene silencing (PTGS), which they termed RNA interference (RNAi) [1]. Since then, gene silencing by dsRNA has been established in plants, invertebrates, vertebrates and in mammals [2]. The long dsRNA introduced into cells is cut by the RNase III-related nuclease Dicer into short (21^25 nucleotides (nt)) interfering RNAs (siRNA). These siRNAs are incorporated into a multiprotein RNA-induced silencing complex (RISC), where the siRNA duplex is unwound, leaving the antisense strand to guide RISC to its homologous mRNA targets for endonucleolytic cleavage [3^5]. The discovery of RNAi and siRNA, as well as its successful application, provides a powerful tool to target genes for de-activation. ... We used speci¢c siRNAs to down-regulate CXCR4 in HeLa cells and Fas-ligand (FasL) in HEK293 cells. ... We provide evidence that in both systems the use of two or more speci¢c siRNAs signi¢cantly improved the gene silencing e¡ect induced by a single siRNA. Investigating the mechanism we found that the transfection e⁄ciency varied among the di¡erent siRNAs, was siRNA dose-dependent but not the main reason for the enhanced gene silencing e¡ect observed. ... To evaluate the transfection e⁄ciency, 3P-rhodamine-labeled FasL-speci¢c siRNAs were used. ... Cells and transfection The HeLa CD4- and CXCR4-expressing reporter cell line SX22-1 (stably transfected CD4 expressing plasmid and carrying a lacZ gene under the control of HIV-1 LTR), Hut/4-3 (constitutively producing HIV-1), the FasL-expressing human embryonic kidney 293 cells, HEK293-005, and BALB/c B-cell lymphoma A20 GFP, were cultured as described elsewhere [9,10]. ... 5 days, which permits cellular HIV-1 infection and LacZ reporter gene expression. ... Apoptosis induction by FasL At 2 days post-transfection with FasL-speci¢c siRNA duplexes or their combination, FasL-expressing HEK293 cells were washed three times with PBS and then co-cultured with the Fas-apoptosis reporter cells A20 GFPat a ratio of 1:2 (e¡ector: reporter cells) in FACS tubes overnight. ... Down-regulation of CXCR4 and FasL with sequence-speci¢c siRNA duplexes CXCR4-expressing HeLa SX22-1 cells were separately transfected with either 50 nM of siRNA-A, siRNA-B, siRNA- C or the control (FasL) siRNA-1. ... 1a,b, all three speci¢c siRNA duplexes down-regulated CXCR4 gene expression (siRNA-A evoked the highest inhibition, 73% at the protein, 79% at the mRNA level), whereas the control siRNA-1 did not. ... 1a,c shows that all three FasL-speci ¢c siRNA duplexes inhibited FasL expression at the protein and mRNA level, whereas the control siRNA-A did not. These data show that the inhibition induced by these siRNAs is sequence-speci¢c. ... Dose-dependent down-regulation of CXCR4/FasL expression by siRNA duplexes Titration experiments with graded concentrations of CXCR4-speci¢c siRNA duplexes (siRNA-A, -B and -C, Fig.