RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase - PubMed (original) (raw)
RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase
Olivier Donzé et al. Nucleic Acids Res. 2002.
Abstract
Methods that allow the specific silencing of a desired gene are invaluable tools for research. One of these is based on RNA interference (RNAi), a process by which double-stranded RNA (dsRNA) specifically suppresses the expression of a target mRNA. Recently, it has been reported that RNAi also works in mammalian cells if small interfering RNAs (siRNAs) are used to avoid activation of the interferon system by long dsRNA. Thus, RNAi could become a major tool for reverse genetics in mammalian systems. However, the high cost and the limited availability of the short synthetic RNAs and the lack of certainty that a designed siRNA will work present major drawbacks of the siRNA technology. Here we present an alternative method to obtain cheap and large amounts of siRNAs using T7 RNA polymerase. With multiple transfection procedures, including calcium phosphate co-precipitation, we demonstrate silencing of both exogenous and endogenous genes.
Figures
Figure 1
Strategy to generate T7 siRNAs (see Results and Discussion for details). The sequence of the gene of interest is shown in red (sense) or blue (antisense), while the two unrelated nucleotides are in black. RISC stands for the RNA-induced silencing complex that targets the mRNA for cleavage.
Figure 2
T7 siRNAs silence transfected genes in mammalian cells. (A) Plasmid pEGFP-C1 and decreasing amounts of siRNAs were co-transfected using a lipofectamine-based protocol. GFP was revealed with an anti-GFP antibody. T7 siRNA against PKR was transfected as a specificity control. (B) T7 siRNAs directed against GFP (1–3 represent siRNAs from three independent transcription reactions) or PKR were transfected using the calcium phosphate co-precipitation technique into HeLa cells with the plasmids pEGFP-C1 and pcDNA3-Luc. GFP and luciferase were detected with anti-GFP and anti-luciferase polyclonal antibodies, respectively.
Figure 3
T7 siRNA silences an endogenous gene in mammalian cells. Aliquots of 0.2 or 2 µl of T7 siRNA directed against PKR were transfected using the calcium phosphate co-precipitation technique into 293T cells. Forty hours after transfection, cells were harvested and equal amounts of proteins were loaded on a 10% polyacrylamide gel. PKR was detected using a polyclonal antibody against PKR. Hsp90 was revealed as a specificity and loading control.
Similar articles
- Functional siRNA expression from transfected PCR products.
Castanotto D, Li H, Rossi JJ. Castanotto D, et al. RNA. 2002 Nov;8(11):1454-60. doi: 10.1017/s1355838202021362. RNA. 2002. PMID: 12458798 Free PMC article. - RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs.
Lipardi C, Wei Q, Paterson BM. Lipardi C, et al. Cell. 2001 Nov 2;107(3):297-307. doi: 10.1016/s0092-8674(01)00537-2. Cell. 2001. PMID: 11701121 - Subtype- and species-specific knockdown of PKC using short interfering RNA.
Irie N, Sakai N, Ueyama T, Kajimoto T, Shirai Y, Saito N. Irie N, et al. Biochem Biophys Res Commun. 2002 Nov 15;298(5):738-43. doi: 10.1016/s0006-291x(02)02531-7. Biochem Biophys Res Commun. 2002. PMID: 12419315 - Small RNA: can RNA interference be exploited for therapy?
Wall NR, Shi Y. Wall NR, et al. Lancet. 2003 Oct 25;362(9393):1401-3. doi: 10.1016/S0140-6736(03)14637-5. Lancet. 2003. PMID: 14585643 Review. - RNA interference: the molecular immune system.
Bagasra O, Prilliman KR. Bagasra O, et al. J Mol Histol. 2004 Aug;35(6):545-53. doi: 10.1007/s10735-004-2192-8. J Mol Histol. 2004. PMID: 15614608 Review.
Cited by
- Recurrent UBE3C-LRP5 translocations in head and neck cancer with therapeutic implications.
Dharavath B, Butle A, Chaudhary A, Pal A, Desai S, Chowdhury A, Thorat R, Upadhyay P, Nair S, Dutt A. Dharavath B, et al. NPJ Precis Oncol. 2024 Mar 4;8(1):63. doi: 10.1038/s41698-024-00555-4. NPJ Precis Oncol. 2024. PMID: 38438481 Free PMC article. - Direct dsRNA preparation by promoter-free RCT and RNase H cleavage using one circular dsDNA template with a mismatched bubble.
Chen H, Gu Z, Yang L, Liu F, An R, Ge Y, Liang X. Chen H, et al. RNA. 2023 Nov;29(11):1691-1702. doi: 10.1261/rna.079670.123. Epub 2023 Aug 3. RNA. 2023. PMID: 37536954 - RNAi-Based Therapeutics and Novel RNA Bioengineering Technologies.
Traber GM, Yu AM. Traber GM, et al. J Pharmacol Exp Ther. 2023 Jan;384(1):133-154. doi: 10.1124/jpet.122.001234. Epub 2022 Jun 9. J Pharmacol Exp Ther. 2023. PMID: 35680378 Free PMC article. Review. - Effect of down-regulating VEGF on proliferation of colon carcinoma cell HT-29.
Zhang X, Ge Y, Wang H, Cao M, Zhang J, Zhang Z, Wang X. Zhang X, et al. Front Biol China. 2007;2(1):35-38. doi: 10.1007/s11515-007-0006-6. Front Biol China. 2007. PMID: 32288755 Free PMC article. - Transcriptional profiling and physiological roles of Aedes aegypti spermathecal-related genes.
Pascini TV, Ramalho-Ortigão M, Ribeiro JM, Jacobs-Lorena M, Martins GF. Pascini TV, et al. BMC Genomics. 2020 Feb 10;21(1):143. doi: 10.1186/s12864-020-6543-y. BMC Genomics. 2020. PMID: 32041546 Free PMC article.
References
- Carthew R.W. (2001) Gene silencing by double-stranded RNA. Curr. Opin. Cell Biol., 13, 244–248. - PubMed
- Hammond S.M., Bernstein,E., Beach,D. and Hannon,G.J. (2000) An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature, 404, 293–296. - PubMed
- Zamore P.D., Tuschl,T., Sharp,P.A. and Bartel,D.P. (2000) RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell, 101, 25–33. - PubMed
- Bernstein E., Caudy,A.A., Hammond,S.M. and Hannon,G.J. (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature, 409, 363–366. - PubMed
- Williams B.R. (1999) PKR; a sentinel kinase for cellular stress. Oncogene, 18, 6112–6120. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources