Gene Silencing through RNA Interference

Potential for Therapeutics and Functional Genomics
  • David O. Azorsa
  • Spyro Mousses
  • Natasha J. Caplen
Part of the Medical Intelligence Unit book series (MIUN)


A major focus of biology is determining the role that specific genes play within a cell. The ability to control gene expression is a powerful tool for biologists, and methods that facilitate the expression of genes in trans have been very useful in delineating gene function. However, methods that enable researchers to decrease or inhibit gene expression in a controlled and specific manner have been lacking. A significant advancement in the field of gene silencing is the recent discovery of RNA interference (RNAi), a post-transcriptional gene silencing mechanism first described in C. elegans and Drosophila and more recently shown to occur in mammals, including mouse and human cells. This chapter will examine several aspects of RNAi and its related gene silencing mechanisms and will discuss the impact that technologies based on these endogenous cellular processes have had on studies of gene function and on new approaches to the treatment of disease.


Gene Silence Caenorhabditis Elegans Peptide Nucleic Acid Curr Biol Inhibit Gene Expression 
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  1. 1.
    Napoli C, Lemieux C, Jorgensen R. Introduction of a chimeric chalcone synthase gene into petunia results in reversible cosuppression of homologous genes in trans. Plant Cell 1990; 2(4):279–289.PubMedCrossRefGoogle Scholar
  2. 2.
    van der Krol AR, Mur LA, Beld M et al. Flavonoid genes in petunia: Addition of a limited number of gene copies may lead to a suppression of gene expression. Plant Cell 1990; 2(4):291–9.PubMedCrossRefGoogle Scholar
  3. 3.
    Romano N, Macino G. Quelling: Transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences. Mol Microbiol 1992; 6(22):3343–53.PubMedCrossRefGoogle Scholar
  4. 4.
    Vaucheret H, Beclin C, Fagard M. Post-transcriptional gene silencing in plants. J Cell Sci 2001;H4 (Pt 17):3083–91.Google Scholar
  5. 5.
    Fire A, Xu S, Montgomery MK et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998; 391(6669):806–811.PubMedCrossRefGoogle Scholar
  6. 6.
    Kennerdell JR, Carthew RW. Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway. Cell 1998; 95(7):1017–1026.PubMedCrossRefGoogle Scholar
  7. 7.
    Misquitta L, Paterson BM. Targeted disruption of gene function in Drosophila by RNA interference (RNA-i): A role for nautilus in embryonic somatic muscle formation. Proc Natl Acad Sci USA 1999; 96(4):1451–1456.PubMedCrossRefGoogle Scholar
  8. 8.
    Hamilton AJ, Baulcombe DC. A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 1999; 286(5441):950–2.PubMedCrossRefGoogle Scholar
  9. 9.
    Hammond SM, Bernstein E, Beach D et al. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 2000; 404(6775):293–6.PubMedCrossRefGoogle Scholar
  10. 10.
    Zamore PD, Tuschl T, Sharp PA et al. RNAi: Double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 2000; 101(1):25–33.PubMedCrossRefGoogle Scholar
  11. 11.
    Yang D, Lu H, Erickson JW. Evidence that processed small dsRNAs may mediate sequence-specific mRNA degradation during RNAi in Drosophila embryos. Curr Biol 2000; 10(19):1191–1200.PubMedCrossRefGoogle Scholar
  12. 12.
    Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21-and 22-nucleotide RNAs. Genes Dev 2001; 15(2):188–200.PubMedCrossRefGoogle Scholar
  13. 13.
    Elbashir SM, Martinez J, Patkaniowska A et al. Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. Embo J 2001; 20(23):6877–88.PubMedCrossRefGoogle Scholar
  14. 14.
    Cogoni C. Homology-dependent gene silencing mechanisms in fungi. Annu Rev Microbiol 2001;55:381–406.PubMedCrossRefGoogle Scholar
  15. 15.
    Parrish S, Fleenor J, Xu S et al. Functional anatomy of a dsRNA trigger. Differential requirement for the two trigger strands in RNA interference. Mol Cell 2000; 6(5):1077–1087.PubMedCrossRefGoogle Scholar
  16. 16.
    Bernstein E, Caudy AA, Hammond SM et al. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 2001; 409(6818):363–6.PubMedCrossRefGoogle Scholar
  17. 17.
    Cerutti L, Mian N, Bateman A. Domains in gene silencing and cell differentiation proteins: The novel PAZ domain and redefinition of the Piwi domain. Trends in Biochem Sci 2000; 25:481–482.CrossRefGoogle Scholar
  18. 18.
    Nykanen A, Haley B, Zamore PD. ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 2001; 107(3):309–21.PubMedCrossRefGoogle Scholar
  19. 19.
    Martinez J, Patkaniowska A, Urlaub H et al. Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell 2002; 110(5):563–74.PubMedCrossRefGoogle Scholar
  20. 20.
    Schwarz DS, Hutvagner G, Haley B et al. Evidence that siRNAs function as guides, not primers, in the Drosophila and human RNAi pathways. Mol Cell 2002; 10(3):537–48.PubMedCrossRefGoogle Scholar
  21. 21.
    Hammond SM, Boettcher S, Caudy AA et al. Argonaute2, a link between genetic and biochemical analyses of RNAi. Science 2001; 293(5532):1146–50.PubMedCrossRefGoogle Scholar
  22. 22.
    Caudy AA, Myers M, Hannon GJ et al. Fragile X-related protein and VIG associate with the RNA interference machinery. Genes Dev 2002; 16(19):2491–6.PubMedCrossRefGoogle Scholar
  23. 23.
    Ishizuka A, Siomi MC, Siomi H. A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins. Genes Dev 2002; 16(19):2497–508.PubMedCrossRefGoogle Scholar
  24. 24.
    Cogoni C, Irelan JT, Schumacher M et al. Transgene silencing of the al-1 gene in vegetative cells of Neurospora is mediated by a cytoplasmic effector and does not depend on DNA-DNA interactions or DNA methylation. Embo J 1996; 15(12):3153–63.PubMedGoogle Scholar
  25. 25.
    Palauqui JC, Elmayan T, Pollien JM et al. Systemic acquired silencing: Transgene-specific post-transcriptional silencing is transmitted by grafting from silenced stocks to non silenced scions. Embo J 1997; 16(15):4738–45.PubMedCrossRefGoogle Scholar
  26. 26.
    Montgomery MK, Xu S, Fire A. RNA as a target of double-stranded RNA-mediated genetic interference in Caenorhabditis elegans. Proc Natl Acad Sci USA 1998; 95(26):15502–15507.PubMedCrossRefGoogle Scholar
  27. 27.
    Cogoni C, Macino G. Gene silencing in Neurospora crassa requires a protein homologous to RNA-dependent RNA polymerase. Nature 1999; 399(6732):166–9.PubMedCrossRefGoogle Scholar
  28. 28.
    Smardon A, Spoerke JM, Stacey SC et al. EGO-1 is related to RNA-directed RNA polymerase and functions in germ-line development and RNA interference in C. elegans [published erratum appears in Curr Biol 2000 May 18;10(10):R393-4]. Curr Biol 2000; 10(4):169–78.PubMedCrossRefGoogle Scholar
  29. 29.
    Sijen T, Fleenor J, Simmer F et al. On the role of RNA amplification in dsRNA-triggered gene silencing. Cell 2001; 107(4):465–76.PubMedCrossRefGoogle Scholar
  30. 30.
    Makeyev EV, Bamford DH. Cellular RNA-dependent RNA polymerase involved in posttranscriptional gene silencing has two distinct activity modes. Mol Cell 2002; 10(6):1417–27.PubMedCrossRefGoogle Scholar
  31. 31.
    Alder MN, Dames S, Gaudet J et al. Gene silencing in Caenorhabditis elegans by transitive RNA interference. RNA 2003; 9(1):25–32.PubMedCrossRefGoogle Scholar
  32. 32.
    Vance V, Vaucheret H. RNA silencing in plants-defense and counterdefense. Science 2001;292(5525):2277–80.PubMedCrossRefGoogle Scholar
  33. 33.
    Li WX, Ding SW. Viral suppressors of RNA silencing. Curr Opin Biotechnol 2001; 12(2):150–4.PubMedCrossRefGoogle Scholar
  34. 34.
    Ahlquist P. RNA-dependent RNA polymerases, viruses, and RNA silencing. Science 2002;296(5571):1270–3.PubMedCrossRefGoogle Scholar
  35. 35.
    Tabara H, Sarkissian M, Kelly WG et al. The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell 1999; 99(2):123–132.PubMedCrossRefGoogle Scholar
  36. 36.
    Ketting RF, Haverkamp TH, van Luenen HG et al. Mut-7 of C. elegans, required for transposon silencing and RNA interference, is a homolog of Werner syndrome helicase and RNaseD. Cell 1999; 99(2):133–141.PubMedCrossRefGoogle Scholar
  37. 37.
    Dennis C. The brave new world of RNA. Nature 2002; 418(6894):122–4.PubMedCrossRefGoogle Scholar
  38. 38.
    Hannon GJ. RNA interference. Nature 2002; 418(6894):244–51.PubMedCrossRefGoogle Scholar
  39. 39.
    McManus MT, Sharp PA. Gene silencing in mammals by small interfering RNAs. Nat Rev Genet 2002; 3(10):737–47.PubMedCrossRefGoogle Scholar
  40. 40.
    Volpe TA, Kidner C, Hall IM et al. Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science 2002; 297(5588):1833–7.PubMedCrossRefGoogle Scholar
  41. 41.
    Reinhart BJ, Bartel DP. Small RNAs correspond to centromere heterochromatic repeats. Science 2002; 297(5588):1831.PubMedCrossRefGoogle Scholar
  42. 42.
    Hall IM, Shankaranarayana GD, Noma K et al. Establishment and maintenance of a heterochromatin domain. Science 2002; 297(5590):2232–7.PubMedCrossRefGoogle Scholar
  43. 43.
    Hall IM, Noma K, Grewal SI. RNA interference machinery regulates chromosome dynamics during mitosis and meiosis in fission yeast. Proc Natl Acad Sci USA 2003; 100(1):193–8.PubMedCrossRefGoogle Scholar
  44. 44.
    Zilberman D, Cao X, Jacobsen SE. ARGONAUTE4 control of locus-specific siRNA accumulation and DNA and histone methylation. Science 2003; 299(5607):716–9.PubMedCrossRefGoogle Scholar
  45. 45.
    Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 1993; 75(5):843–54.PubMedCrossRefGoogle Scholar
  46. 46.
    Pasquinelli AE, Reinhart BJ, Slack F et al. Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature 2000; 408(6808):86–9.PubMedCrossRefGoogle Scholar
  47. 47.
    Lee Y, Jeon K, Lee JT et al. MicroRNA maturation: Stepwise processing and subcellular localization. Embo J 2002; 21(17):4663–70.PubMedCrossRefGoogle Scholar
  48. 48.
    Grishok A, Pasquinelli AE, Conte D et al. Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 2001; 106(1):23–34.PubMedCrossRefGoogle Scholar
  49. 49.
    Hutvagner G, McLachlan J, Pasquinelli AE et al. A cellular function for the RNA-interference enzyme dicer in the maturation of the let-7 small temporal RNA. Science 2001; 293(5531):834–8.PubMedCrossRefGoogle Scholar
  50. 50.
    Ketting RF, Fischer SE, Bernstein E et al. Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes Dev 2001; 15(20):2654–9.PubMedCrossRefGoogle Scholar
  51. 51.
    Knight SW, Bass BL. A role for the RNase III enzyme DCR-1 in RNA interference and germ line development in Caenorhabditis elegans. Science 2001; 293(5538):2269–71.PubMedCrossRefGoogle Scholar
  52. 52.
    Wightman B, Ha I, Ruvkun G. Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell 1993; 75(5):855–62.PubMedCrossRefGoogle Scholar
  53. 53.
    Ha I, Wightman B, Ruvkun G. A bulged lin-4/lin-l4 RNA duplex is sufficient for Caenorhabditis elegans lin-14 temporal gradient formation. Genes Dev 1996; 10(23):3041–50.PubMedGoogle Scholar
  54. 54.
    Reinhart BJ, Slack FJ, Basson M et al. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 2000; 403(6772):901–6.PubMedCrossRefGoogle Scholar
  55. 55.
    Lai EC. Micro RNAs are complementary to 3′ UTR sequence motifs that mediate negative post-transcriptional regulation. Nat Genet 2002; 30(4):363–4.PubMedCrossRefGoogle Scholar
  56. 56.
    Lagos-Quintana M, Rauhut R, Lendeckel W et al. Identification of novel genes coding for small expressed RNAs. Science 2001; 294(5543):853–8.PubMedCrossRefGoogle Scholar
  57. 57.
    Lau NC, Lim LP, Weinstein EG et al. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 2001; 294(5543):858–62.PubMedCrossRefGoogle Scholar
  58. 58.
    Lee RC, Ambros V. An extensive class of small RNAs in Caenorhabditis elegans. Science 2001; 294(5543):862–4.PubMedCrossRefGoogle Scholar
  59. 59.
    Reinhart BJ, Weinstein EG, Rhoades MW et al. MicroRNAs in plants. Genes Dev 2002; 16(13):1616–26.PubMedCrossRefGoogle Scholar
  60. 60.
    Lagos-Quintana M, Rauhut R, Yalcin A et al. Identification of tissue-specific microRNAs from mouse. Curr Biol 2002; 12(9):735–9.PubMedCrossRefGoogle Scholar
  61. 61.
    Mourelatos Z, Dostie J, Paushkin S et al. miRNPs: A novel class of ribonucleoproteins containing numerous microRNAs. Genes Dev 2002; 16(6):720–8.PubMedCrossRefGoogle Scholar
  62. 62.
    Llave C, Xie Z, Kasschau KD et al. Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA. Science 2002; 297(5589):2053–6.PubMedCrossRefGoogle Scholar
  63. 63.
    Lagos-Quintana M, Rauhut R, Meyer J et al. New microRNAs from mouse and human. Rna 2003; 9(2):175–9.PubMedCrossRefGoogle Scholar
  64. 64.
    Dostie J, Mourelatos Z, Yang M et al. Numerous microRNPs in neuronal cells containing novel microRNAs. Rna 2003; 9(2):180–6.PubMedCrossRefGoogle Scholar
  65. 65.
    Lim LP, Lau NC, Weinstein EG et al. The microRNAs of Caenorhabditis elegans. Genes Dev 2003; 17(8):991–1008.PubMedCrossRefGoogle Scholar
  66. 66.
    Lim LP, Glasner ME, Yekta S et al. Vertebrate microRNA genes. Science 2003; 299(5612):1540.PubMedCrossRefGoogle Scholar
  67. 67.
    Ambros V, Bartel B, Bartel DP et al. A uniform system for microRNA annotation. Rna 2003; 9(3):277–9.PubMedCrossRefGoogle Scholar
  68. 68.
    Rhoades MW, Reinhart BJ, Lim LP et al. Prediction of plant microRNA targets. Cell 2002; 110(4):513–20.PubMedCrossRefGoogle Scholar
  69. 69.
    Brennecke J, Hipfner DR, Stark A et al. Bantam encodes a developmentally regulated microRNA that controls cell proliferation and regulates the proapoptotic gene hid in Drosophila. Cell 2003; 113(1):25–36.PubMedCrossRefGoogle Scholar
  70. 70.
    Xu P, Vernooy SY, Guo M et al. The drosophila microrna MIR-14 suppresses cell death and is required for normal fat metabolism. Curr Biol 2003; 13(9):790–5.PubMedCrossRefGoogle Scholar
  71. 71.
    Sempere LF, Sokol NS, Dubrovsky EB et al. Temporal regulation of microRNA expression in Drosophila melanogaster mediated by hormonal signals and broad-Complex gene activity. Dev Biol 2003; 259(1):9–18.PubMedCrossRefGoogle Scholar
  72. 72.
    Calin GA, Dumitru CD, Shimizu M et al. Frequent deletions and down-regulation of micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci USA 2002; 99(24):15524–9.PubMedCrossRefGoogle Scholar
  73. 73.
    Seitz H, Youngson N, Lin SP et al. Imprinted microRNA genes transcribed antisense to a reciprocally imprinted retrotransposon-like gene. Nat Genet 2003.Google Scholar
  74. 74.
    Clemens MJ, Elia A. The double-stranded RNA-dependent protein kinase PKR: Structure and function. J Interferon Cytokine Res 1997; 17(9):503–24.PubMedCrossRefGoogle Scholar
  75. 75.
    Minks MA, West DK, Benvin S et al. Structural requirements of double-stranded RNA for the activation of 2′,5′-oligo(A) polymerase and protein kinase of interferon-treated HeLa cells. J Biol Chem 1979; 254(20):10180–3.PubMedGoogle Scholar
  76. 76.
    Manche L, Green SR, Schmedt C et al. Interactions between double-stranded RNA regulators and the protein kinase DAI. Mol Cell Biol 1992; 12(11):5238–48.PubMedGoogle Scholar
  77. 77.
    Caplen NJ, Parrish S, Imani F et al. Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc Natl Acad Sci USA 2001; 98(17):9742–9747.PubMedCrossRefGoogle Scholar
  78. 78.
    Elbashir SM, Harborth J, Lendeckel W et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001; 411(6836):494–8.PubMedCrossRefGoogle Scholar
  79. 79.
    Billy E, Brondani V, Zhang H et al. Specific interference with gene expression induced by long, double-stranded RNA in mouse embryonal teratocarcinoma cell lines. Proc Natl Acad Sci USA 2001; 98(25):14428–33.PubMedCrossRefGoogle Scholar
  80. 80.
    Paddison PJ, Caudy AA, Hannon GJ. Stable suppression of gene expression by RNAi in mammalian cells. Proc Natl Acad Sci USA 2002; 99(3):1443–8.PubMedCrossRefGoogle Scholar
  81. 81.
    Hutvagner G, Zamore PD. A microRNA in a multiple-turnover RNAi enzyme complex. Science 2002; 297(5589):2056–60.PubMedCrossRefGoogle Scholar
  82. 82.
    Doi N, Zenno S, Ueda R et al. Short-interfering-RNA-mediated gene silencing in mammalian cells requires dicer and eif2c translation initiation factors. Curr Biol 2003; 13(1):41–6.PubMedCrossRefGoogle Scholar
  83. 83.
    Elbashir SM, Harborth J, Weber K et al. Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods 2002; 26(2):199–213.PubMedCrossRefGoogle Scholar
  84. 84.
    Chiu YL, Rana TM. RNAi in human cells: Basic structural and functional features of small interfering RNA. Mol Cell 2002; 10(3):549–61.PubMedCrossRefGoogle Scholar
  85. 85.
    Harborth J, Elbashir SM, Vandenburgh K et al. Sequence, chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing. Antisense Nucleic Acid Drug Dev 2003; 13(2):83–105.PubMedCrossRefGoogle Scholar
  86. 86.
    Holen T, Amarzguioui M, Wiiger MT et al. Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor. Nucleic Acids Res 2002; 30(8):1757–66.PubMedCrossRefGoogle Scholar
  87. 87.
    Lee NS, Dohjima T, Bauer G et al. Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat Biotechnol 2002; 20(5):500–5.PubMedGoogle Scholar
  88. 88.
    Semizarov D, Frost L, Sarthy A et al. Specificity of short interfering RNA determined through gene expression signatures. Proc Natl Acad Sci USA 2003; 100(11):6347–52.PubMedCrossRefGoogle Scholar
  89. 89.
    Jackson AL, Bartz SR, Schelter J et al. Expression profiling reveals off-target gene regulation by RNAi. Nat Biotechnol 2003; 21(6):635–7.PubMedCrossRefGoogle Scholar
  90. 90.
    Doench JG, Petersen CP, Sharp PA. siRNAs can function as miRNAs. Genes Dev 2003; 17(4):438–42.PubMedCrossRefGoogle Scholar
  91. 91.
    Chi JT, Chang HY, Wang NN et al. Genomewide view of gene silencing by small interfering RNAs. Proc Natl Acad Sci USA 2003; 100(11):6343–6.PubMedCrossRefGoogle Scholar
  92. 92.
    Amarzguioui M, Holen T, Babaie E et al. Tolerance for mutations and chemical modifications in a siRNA. Nucleic Acids Res 2003; 31(2):589–95.PubMedCrossRefGoogle Scholar
  93. 93.
    Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science 2002; 296(5567):550–3.PubMedCrossRefGoogle Scholar
  94. 94.
    Brummelkamp TR, Bernards R, Agami R. Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell 2002; 2(3):243–7.PubMedCrossRefGoogle Scholar
  95. 95.
    Miller VM, Xia H, Marrs GL et al. Allele-specific silencing of dominant disease genes. Proc Natl Acad Sci USA 2003; 100(12):7195–200.PubMedCrossRefGoogle Scholar
  96. 96.
    Kisielow M, Kleiner S, Nagasawa M et al. Isoform-specific knockdown and expression of adaptor protein ShcA using small interfering RNA. Biochem J 2002; 363 (Pt l):1–5.PubMedCrossRefGoogle Scholar
  97. 97.
    Stein P, Svoboda P, Schultz RM. Transgenic RNAi in mouse oocytes: A simple and fast approach to study gene function. Dev Biol 2003; 256(1):188–94.CrossRefGoogle Scholar
  98. 98.
    Tabara H, Grishok A, Mello CC. RNAi in C. elegans: Soaking in the genome sequence. Science 1998; 282(5388):430–1.PubMedCrossRefGoogle Scholar
  99. 99.
    Timmons L, Fire A. Specific interference by ingested dsRNA. Nature 1998; 395(6705):854.PubMedCrossRefGoogle Scholar
  100. 100.
    Kamath RS, Martinez-Campos M, Zipperlen P et al. Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans. Genome Biol 2000; 2(1).Google Scholar
  101. 101.
    Clemens JC, Worby CA, Simonson-Leff N et al. Use of double-stranded RNA interference in drosophila cell lines to dissect signal transduction pathways. Proc Natl Acad Sci USA 2000; 97(12):6499–503.PubMedCrossRefGoogle Scholar
  102. 102.
    Caplen NJ, Fleenor J, Fire A et al. dsRNA-mediated gene silencing in cultured Drosophila cells: A tissue culture model for the analysis of RNA interference. Gene 2000; 252(1–2):95–105.PubMedCrossRefGoogle Scholar
  103. 103.
    Kennerdell JR, Carthew RW. Heritable gene silencing in Drosophila using double-stranded RNA. Nat Biotechnol 2000; 18(8):896–898.PubMedCrossRefGoogle Scholar
  104. 104.
    Donze O, Picard D. RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase. Nucleic Acids Res 2002; 30(10):e46.PubMedCrossRefGoogle Scholar
  105. 105.
    Yu JY, DeRuiter SL, Turner DL. RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci USA 2002; 99(9):6047–52.PubMedCrossRefGoogle Scholar
  106. 106.
    Calegari F, Haubensak W, Yang D et al. Tissue-specific RNA interference in postimplantation mouse embryos with endoribonuclease-prepared short interfering RNA. Proc Natl Acad Sci USA 2002; 99(22):14236–40.PubMedCrossRefGoogle Scholar
  107. 107.
    Yang D, Buchholz F, Huang Z et al. Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells. Proc Natl Acad Sci USA 2002; 99(15):9942–7.PubMedCrossRefGoogle Scholar
  108. 108.
    Myers JW, Jones JT, Meyer T et al. Recombinant dicer efficiently converts large dsRNAs into siRNAs suitable for gene silencing. Nat Biotechnol 2003; 21(3):324–8.PubMedCrossRefGoogle Scholar
  109. 109.
    Kawasaki H, Suyama E, Iyo M et al. siRNAs generated by recombinant human Dicer induce specific and significant but target site-independent gene silencing in human cells. Nucleic Acids Res 2003; 31(3):981–7.PubMedCrossRefGoogle Scholar
  110. 110.
    Paddison PJ, Caudy AA, Bernstein E et al. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev 2002; 16(8):948–58.PubMedCrossRefGoogle Scholar
  111. 111.
    Castanotto D, Li H, Rossi JJ. Functional siRNA expression from transfected PCR products. Rna 2002; 8(11):1454–60.PubMedCrossRefGoogle Scholar
  112. 112.
    Miyagishi M, Taira K. U6 promoter driven siRNAs with four uridine 3′ overhangs efficiently suppress targeted gene expression in mammalian cells. Nat Biotechnol 2002; 20(5):497–500.PubMedCrossRefGoogle Scholar
  113. 113.
    Sui G, Soohoo C, Affar el B et al. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci USA 2002; 99(8):5515–20.PubMedCrossRefGoogle Scholar
  114. 114.
    Paul CP, Good PD, Winer I et al. Effective expression of small interfering RNA in human cells. Nat Biotechnol 2002; 20(5):505–8.PubMedCrossRefGoogle Scholar
  115. 115.
    Kawasaki H, Taira K. Short hairpin type of dsRNAs that are controlled by tRNA(Val) promoter significantly induce RNAi-mediated gene silencing in the cytoplasm of human cells. Nucleic Acids Res 2003; 31(2):700–7.PubMedCrossRefGoogle Scholar
  116. 116.
    Paul CP, Good PD, Li SX et al. Localized expression of small RNA inhibitors in human cells. Mol Ther 2003; 7(2):237–47.PubMedCrossRefGoogle Scholar
  117. 117.
    Barton GM, Medzhitov R. Retroviral delivery of small interfering RNA into primary cells. Proc Natl Acad Sci USA 2002; 99(23):14943–5.PubMedCrossRefGoogle Scholar
  118. 118.
    Devroe E, Silver PA. Retrovirus-delivered siRNA. BMC Biotechnol 2002; 2(1):15.PubMedCrossRefGoogle Scholar
  119. 119.
    Xia H, Mao Q, Paulson HL et al. siRNA-mediated gene silencing in vitro and in vivo. Nat Biotechnol 2002; 20(10):1006–10.PubMedCrossRefGoogle Scholar
  120. 120.
    McManus MT, Petersen CP, Haines BB et al. Gene silencing using micro-RNA designed hairpins. Rna 2002; 8(6):842–50.PubMedCrossRefGoogle Scholar
  121. 121.
    Bridge AJ, Pebernard S, Ducraux A et al. Induction of an interferon response by RNAi vectors in mammalian cells. Nat Genet 2003.Google Scholar
  122. 122.
    Fraser AG, Kamath RS, Zipperlen P et al. Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature 2000; 408(6810):325–330.PubMedCrossRefGoogle Scholar
  123. 123.
    Gonczy P, Echeverri G, Oegema K et al. Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III. Nature 2000; 408(6810):331–336.PubMedCrossRefGoogle Scholar
  124. 124.
    Piano F, Schetterdagger AJ, Mangone M et al. RNAi analysis of genes expressed in the ovary of Caenorhabditis elegans. Curr Biol 2000; 10(24):1619–1622.PubMedCrossRefGoogle Scholar
  125. 125.
    Piano F, Schetter AJ, Morton DG et al. Gene clustering based on rnai phenotypes of ovary-enriched genes in C. elegans. Curr Biol 2002; 12(22):1959–64.PubMedCrossRefGoogle Scholar
  126. 126.
    Lee SS, Lee RY, Fraser AG et al. A systematic RNAi screen identifies a critical role for mitochondria in C. elegans longevity. Nat Genet 2003; 33(1):40–8.PubMedCrossRefGoogle Scholar
  127. 127.
    Kamath RS, Fraser AG, Dong Y et al. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 2003; 421(6920):231–7.PubMedCrossRefGoogle Scholar
  128. 128.
    Ashrafi K, Chang FY, Watts JL et al. Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes. Nature 2003; 421(6920):268–72.PubMedCrossRefGoogle Scholar
  129. 129.
    Pothof J, Van Haaften G, Thijssen K et al. Identification of genes that protect the C. elegans genome against mutations by genome-wide RNAi. Genes Dev 2003; 17(4):443–8.PubMedCrossRefGoogle Scholar
  130. 130.
    Lum L, Yao S, Mozer B et al. Identification of hedgehog pathway components by RNAi in Drosophila cultured cells. Science 2003; 299(5615):2039–45.PubMedCrossRefGoogle Scholar
  131. 131.
    Vickers TA, Koo S, Bennett CF et al. Efficient reduction of target RNAs by small interfering RNA and RNase h-dependent antisense agents a comparative analysis. J Biol Chem 2003; 278(9):7108–18.PubMedCrossRefGoogle Scholar
  132. 132.
    Aoki Y, Cioca D, Oidaira H et al. RNA interference may be more potent than antisense RNA in human cancer cell lines. Clin Exp Pharmacol Physiol 2003; 30(1–2):96–102.PubMedCrossRefGoogle Scholar
  133. 133.
    Miyagishi M, Hayashi M, Taira K. Comparison of the suppressive effects of antisense oligonucleotides and siRNAs directed against the same targets in mammalian cells. Antisense Nucleic Acid Drug Dev 2003; 13(1):1–7.PubMedCrossRefGoogle Scholar
  134. 134.
    Stucke VM, Sillje HH, Arnaud L et al. Human Mps1 kinase is required for the spindle assembly checkpoint but not for centrosome duplication. Embo J 2002; 21(7):1723–32.PubMedCrossRefGoogle Scholar
  135. 135.
    Mailand N, Lukas C, Kaiser BK et al. Deregulated human Cdc14A phosphatase disrupts centrosome separation and chromosome segregation. Nat Cell Biol 2002; 4(4):318–22.CrossRefGoogle Scholar
  136. 136.
    Harborth J, Elbashir SM, Bechert K et al. Identification of essential genes in cultured mammalian cells using small interfering RNAs. J Cell Sci 2001; 114 (Pt 24):4557–65.PubMedGoogle Scholar
  137. 137.
    Hasuwa H, Kaseda K, Einarsdottir T et al. Small interfering RNA and gene silencing in transgenic mice and rats. FEBS Lett 2002; 532(1–2):227–30.PubMedCrossRefGoogle Scholar
  138. 138.
    Tiscornia G, Singer O, Ikawa M et al. A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA. Proc Natl Acad Sci USA 2003; 100(4):1844–8.PubMedCrossRefGoogle Scholar
  139. 139.
    Carmell MA, Zhang L, Conklin DS et al. Germline transmission of RNAi in mice. Nat Struct Biol 2003.Google Scholar
  140. 140.
    Rubinson DA, Dillon CP, Kwiatkowski AV et al. Corrigendum: A lentivirus-based system to silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet 2003; 34(2):231.Google Scholar
  141. 141.
    Kunath T, Gish G, Lickert H et al. Transgenic RNA interference in ES cell-derived embryos recapitulates a genetic null phenotype. Nat Biotechnol 2003; 21(5):559–61.PubMedCrossRefGoogle Scholar
  142. 142.
    Hemann MT, Fridman JS, Zilfou JT et al. An epiallelic series of p53 hypomorphs created by stable RNAi produced distinct tumor phenotypes in vivo. Nature Genetics 3 February 2003; 10:1038/ng1091.Google Scholar
  143. 143.
    Paddison PJ, Hannon GJ. RNA interference: The new somatic cell genetics? Cancer Cell 2002; 2(1):17–23.PubMedCrossRefGoogle Scholar
  144. 144.
    Frankish H. Consortium uses RNAi to uncover genes’ function. Lancet 2003; 361(9357):584.PubMedCrossRefGoogle Scholar
  145. 145.
    McCaffrey AP, Meuse L, Pham TT et al. RNA interference in adult mice. Nature 2002; 418(6893):38–9.PubMedCrossRefGoogle Scholar
  146. 146.
    Lewis DL, Hagstrom JE, Loomis AG et al. Efficient delivery of siRNA for inhibition of gene expression in postnatal mice. Nat Genet 2002; 32(1):107–8.PubMedCrossRefGoogle Scholar
  147. 147.
    Zender L, Hutker S, Liedtke C et al. Caspase 8 small interfering RNA prevents acute liver failure in mice. Proc Natl Acad Sci USA 2003.Google Scholar
  148. 148.
    Wilda M, Fuchs U, Wossmann W et al. Killing of leukemic cells with a BCR/ABL fusion gene by RNA interference (RNAi). Oncogene 2002; 21(37):5716–24.PubMedCrossRefGoogle Scholar
  149. 149.
    Scherr M, Battmer K, Winkler T et al. Specific inhibition of bcr-abl gene expression by small interfering RNA. Blood 2003; 101(4):1566–9.PubMedCrossRefGoogle Scholar
  150. 150.
    Zhang L, Yang N, Mohamed-Hadley A et al. Vector-based RNAi, a novel tool for isoform-specific knock-down of VEGF and anti-angiogenesis gene therapy of cancer. Biochem Biophys Res Commun 2003; 303(4):1169–78.PubMedCrossRefGoogle Scholar
  151. 151.
    Nagy P, Arndt-Jovin DJ, Jovin TM. Small interfering RNAs suppress the expression of endogenous and GFP-fused epidermal growth factor receptor (erbB1) and induce apoptosis in erbB1-overexpressing cells. Exp Cell Res 2003; 285(1):39–49.PubMedCrossRefGoogle Scholar
  152. 152.
    Wu H, Hait WN, Yang JM. Small interfering RNA-induced suppression of MDR1 (P-glycoprotein) restores sensitivity to multidrug-resistant cancer cells. Cancer Res 2003; 63(7):1515–9.PubMedGoogle Scholar
  153. 153.
    Novina CD, Murray MF, Dykxhoorn DM et al. siRNA-directed inhibition of HIV-1 infection. Nat Med 2002; 8(7):681–6.PubMedGoogle Scholar
  154. 154.
    Martinez MA, Gutierrez A, Armand-Ugon M et al. Suppression of chemokine receptor expression by RNA interference allows for inhibition of HIV-1 replication. Aids 2002; 16(18):2385–90.PubMedCrossRefGoogle Scholar
  155. 155.
    Qin XF, An DS, Chen IS et al. Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5. Proc Natl Acad Sci USA 2003; 100(1):183–8.PubMedCrossRefGoogle Scholar
  156. 156.
    Song E, Lee SK, Dykxhoorn DM et al. Sustained small interfering RNA-mediated human immunodeficiency virus type 1 inhibition in primary macrophages. J Virol 2003; 77(13):7174–81.PubMedCrossRefGoogle Scholar
  157. 157.
    Capodici J, Kariko K, Weissman D. Inhibition of HIV-1 infection by small interfering RNA-mediated RNA interference. J Immunol 2002; 169(9):5196–201.PubMedGoogle Scholar
  158. 158.
    Park WS, Miyano-Kurosaki N, Hayafune M et al. Prevention of HIV-1 infection in human peripheral blood mononuclear cells by specific RNA interference. Nucleic Acids Res 2002; 30(22):4830–5.PubMedCrossRefGoogle Scholar
  159. 159.
    Coburn GA, Cullen BR. Potent and specific inhibition of human immunodeficiency virus type 1 replication by RNA interference. J Virol 2002; 76(18):9225–31.PubMedCrossRefGoogle Scholar
  160. 160.
    Jacque JM, Triques K, Stevenson M. Modulation of HIV-1 replication by RNA interference. Nature 2002; 418(6896):435–8.PubMedCrossRefGoogle Scholar
  161. 161.
    Kapadia SB, Brideau-Andersen A, Chisari FV. Interference of hepatitis C virus RNA replication by short interfering RNAs. Proc Natl Acad Sci USA 2003; 100(4):2014–8.PubMedCrossRefGoogle Scholar
  162. 162.
    Randall G, Grakoui A, Rice CM. Clearance of replicating hepatitis C virus replicon RNAs in cell culture by small interfering RNAs. Proc Natl Acad Sci USA 2003; 100(1):235–40.PubMedCrossRefGoogle Scholar
  163. 163.
    Wilson JA, Jayasena S, Khvorova A et al. RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells. Proc Natl Acad Sci USA 2003; 100(5):2783–2788.PubMedCrossRefGoogle Scholar
  164. 164.
    Seo MY, Abrignani S, Houghton M et al. Small interfering RNA-mediated inhibition of hepatitis C virus replication in the human hepatoma cell line Huh-7. J Virol 2003; 77(1):810–2.PubMedCrossRefGoogle Scholar
  165. 165.
    Gitlin L, Karelsky S, Andino R. Short interfering RNA confers intracellular antiviral immunity in human cells. Nature 2002; 418(6896):430–4.PubMedCrossRefGoogle Scholar
  166. 166.
    Bitko V, Barik S. Phenotypic silencing of cytoplasmic genes using sequence-specific double-stranded short interfering RNA and its application in the reverse genetics of wild type negative-strand RNA viruses. BMC Microbiol 2001; 1(1):34.PubMedCrossRefGoogle Scholar
  167. 167.
    Ge Q, McManus MT, Nguyen T et al. RNA interference of influenza virus production by directly targeting mRNA for degradation and indirectly inhibiting all viral RNA transcription. Proc Natl Acad Sci USA 2003; 100(5):2718–23.PubMedCrossRefGoogle Scholar
  168. 168.
    Jia Q, Sun R. Inhibition of gammaherpesvirus replication by RNA interference. J Virol 2003; 77(5):3301–6.PubMedCrossRefGoogle Scholar
  169. 169.
    Jiang M, Milner J. Selective silencing of viral gene expression in HPV-positive human cervical carcinoma cells treated with siRNA, a primer of RNA interference. Oncogene 2002; 21(39):6041–8.PubMedCrossRefGoogle Scholar
  170. 170.
    Caplen NJ, Taylor JP, Statham VS et al. Rescue of polyglutamine-mediated cytotoxicity by double-stranded RNA-mediated RNA interference. Hum Mol Genet 2002; 11(2):175–84.PubMedCrossRefGoogle Scholar
  171. 171.
    Gonzalez-Alegre P, Miller VM, Davidson BL et al. Toward therapy for DYT1 dystonia: Allele-specific silencing of mutant TorsinA. Ann Neurol 2003; 53(6):781–7.PubMedCrossRefGoogle Scholar
  172. 172.
    Song E, Lee SK, Wang J et al. RNA interference targeting Fas protects mice from fulminant hepatitis. Nat Med 2003; 9(3):347–51.PubMedCrossRefGoogle Scholar
  173. 173.
    Mousses S, Caplen NJ et al. RNAi microarray analysis in cultured mammalian cells. Genome Res 2003; 13:2341–7.PubMedCrossRefGoogle Scholar

Copyright information

© and Kluwer Academic / Plenum Publishers 2006

Authors and Affiliations

  • David O. Azorsa
    • 1
  • Spyro Mousses
    • 1
  • Natasha J. Caplen
    • 2
  1. 1.Translational Genomics Research Institute (TGen)GaithersburgUSA
  2. 2.Medical Genetics Branch National Human Genome Research InstituteNational Institutes of HealthBethesdaUSA

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