[Cancer Biology & Therapy 2:2,e74-e75:EPUB Ahead of Print, http://www.landesbioscience.com/journals/cbt/abstract.php?id=358. March/April 2003]; ©2003 Landes Bioscience
Commentary
Stable RNA interference (RNAi) in Mammalian Cells
Cancer Biol Ther 2003; 2: http://www.landesbioscience.com/journals/cbt/abstract.php?id=358.
Once the issue is complete and page numbers have been assigned, the citation will change accordingly.
KEY WORDS siRNA,
Lentivirus,
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RNA interference, Gene-silencing
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ABBREVIATIONS
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RNA interference small interfering RNA
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RNAi SiRNA
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This manuscript has been published online, prior to printing, for Cancer Biology & Therapy Volume 2, Issue 2. Definitive page numbers have not been assigned. The current citation for this manuscript is:
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Received 04/22/03; Accepted 04/22/03
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Correspondence to: Kumaravel Somasundaram; Department of Microbiology and Cell Biology; Indian Institute of Science; Bangalore 560 012 India; Tel.: 91.80.3942973; Fax: 91.80.3602697; Email:
[email protected]
RNA interference (RNAi), an evolutionarily conserved process, was first described in C. elegans1 and has subsequently been demonstrated in diverse eukaryotes such as insects, plants, fungi and vertebrates. RNAi is the mechanism of sequence-specific, post-transcriptional gene silencing initiated by double-stranded RNAs (dsRNA) homologous to the gene being suppressed. dsRNAs are processed by Dicer, a cellular ribonuclease III, to generate duplexes of about 21 nt with 3'-overhangs (small interfering RNA, siRNA) which mediate sequence-specific mRNA degradation. Although RNAi serves the purpose of protecting host cells from viruses and modulation of transposan activity, it enables a researcher to knock down a targeted gene without side effects, thereby revealing the function of the silenced gene. Initial attempts to demonstrate this phenomenon in mammalian cells failed due to non-specific suppression of gene expression (the interferon pathway) mediated by antiviral response to long double stranded RNA (dsRNA) molecules. This problem was circumvented by transfection of synthetic 21 nucleotide short interfering RNA (siRNA) duplexes into mammalian cells, which effectively inhibits endogenous genes in a sequence-specific manner without eliciting the generic antiviral response.2 Initial methods were based on expressing siRNAs in mammalian cells through transfection of either oligonucleotides or plasmids encoding siRNAs. Because these systems rely on transfection for delivery, the cell types available for study are restricted generally to transformed cell lines. Subsequently, retroviral vector based introduction of siRNA allowed stable inactivation of genes possible in primary cells and other cell types with low efficiency of transfection.3-5 However, retroviral vectors require active cell division for gene transfer and also suffer from the problem of gene-silencing. In this issue of Cancer Biology & Therapy, Matta et al.6, have developed a lentivirus based vector for delivering siRNA in to mammalian cells, which confirms the findings from others reported recently.7-9 Unlike other studies,7,9 this study is limited to tissue cultures cells. However, this study clearly proves the fact that efficient RNA interference for down regulating the expression of a specific gene can be achieved by using lentivirus based vector. The main advantages of using lentivirus-based vectors are of course that lentivirus can infect both dividing and non-dividing cells and achieve long-term multilineage gene expression. Matta and colleagues show that specific down regulation of p53 tumor suppressor protein in Hela cells infected with lentivirus expressing p53 specific siRNA can be achieved. It is worthwhile checking the p53 messenger levels to confirm that silencing occurs at the level of mRNA, considering the fact that the expression of p53 is largely regulated at posttranslational level when it gets activated. Similarly, the p53-silenced clones should be checked for loss of function. Perhaps, the inducibility of p53 protein levels in response to treatment of cells with DNA damaging agents like adriamycin could be considered. In addition, the vector developed by Matta et al. has additional advantages: the vector carry resistance marker, which makes it easy to select virus infected cells; the use of balsticidin resistance gene offers scientists ability to silence multiple genes using other resistance markers; it is possible, as the authors pointed out, to select for resistance to higher concentrations of blasticidin so as to get clones with multiple copies of integrated provirus. This strategy may particularly useful for silencing genes, which are expressed at higher levels. Analysis of individual p53 silenced clones in this study suggests that the presence of varying degree of silencing of target gene in different clones. This is particularly important for achieving complete silencing and hints at the importance of screening to identify the correct clone with complete silence. In summary, Matta and colleagues have demonstrated that lentivirus based vectors could be used to transfer siRNA to mammalian tissue culture cells and down regulate the expression of p53 gene. This study confirms similar reports from others published recently.7-9 Moreover, it raises an important question that what molar ratio of siRNA to target gene transcript is required for complete silencing. Future studies should perhaps address this problem also for a successful silencing of any particular gene.
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Kumaravel Somasundaram
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Cancer Biology & Therapy
2003; Vol. 2 Issue 2
STABLE RNA INTERFERENCE (RNAI) IN MAMMALIAN CELLS
References 1. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, et al. Potent and specific genetic interference by double stranded RNA in Caenorhabditis elegans. Nature 1998; 391:806–11. 2. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21 nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001; 411:494-8. 3. Brummelkamp T, Bernards R, Agami R. Stable suppression of tumorigenicity by virus mediated RNA interference. Cancer Cell 2002; 2:243. 4. Devroe E, Silver PA. Retrovirus-delivered siRNA. BMC Biotechnol 2002; 2:15. 5. Barton GM, Medzhitov R. Retroviral delivery of small interfering RNA into primary cells. Proc Natl Acad Sci USA 2002; 99:14943-5. 6. Matta H, Hozayev B, Tomar R, Chugh P, Chaudhary PM (2003). Use of lentiviral vectors for delivery of small interfering RNA. Cancer Biol Ther 2003; 2: http://www.landesbioscience.com/journals/cbt/abstract.php?id=348. 7. Rubinson DA, Dillon CP, Kwiatkowski AV, Sievers C, Yang L, Kopinja J, et al. A lentivirusbased system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet 2003; 33:401-6. 8. Abbas-Terki T, Blanco-Bose W, Deglon N, Pralong W, Aebischer P. Lentiviral-mediated RNA interference. Hum Gene Ther 2002; 13:2197-201 9. Tiscornia G, Singer O, Ikawa M, Verma IM. A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA. Proc Natl Acad Sci USA 2002; 100:1844-48.
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