siRNA Design pp 205-232 | Cite as

Short Hairpin RNA-Mediated Gene Silencing

Part of the Methods in Molecular Biology book series (MIMB, volume 942)


Since the first application of RNA interference (RNAi) in mammalian cells, the expression of short hairpin RNAs (shRNAs) for targeted gene silencing has become a benchmark technology. Using plasmid and viral vectoring systems, the transcription of shRNA precursors that are effectively processed by the RNAi pathway can lead to potent gene knockdown. The past decade has seen continual advancement and improvement to the various strategies that can be used for shRNA delivery, and the use of shRNAs for clinical applications is well underway. Driving these developments has been the many benefits afforded by shRNA technologies, including the stable integration of expression constructs for long-term expression, infection of difficult-to-target cell lines and tissues using viral vectors, and the temporal control of shRNA transcription by inducible promoters. The use of different effector molecule formats, promoters, and vector types, has meant that experiments can be tailored to target specific cell types and minimize cellular toxicities. Through the application of combinatorial RNAi (co-RNAi), multiple shRNA delivery strategies can improve gene knockdown, permit multiple transcripts to be targeted simultaneously, and curtail the emergence of viral escape mutants. This chapter reviews the history, cellular processing, and various applications of shRNAs in mammalian systems, including options for effector molecule design, vector and promoter types, and methods for multiple shRNA delivery.

Key words

RNA interference Short hairpin RNA microRNA Combinatorial RNAi 


  1. 1.
    Brummelkamp TR, Bernards R, Agami R (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science 296:550–553. doi:10.1126/science.1068999 Google Scholar
  2. 2.
    Yu J-Y, DeRuiter SL, Turner DL (2002) RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci U S A 99:6047–6052. doi:10.1073/pnas.092143499 Google Scholar
  3. 3.
    Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, Conklin DS (2002) Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev 16:948–958. doi:10.1101/gad.981002 Google Scholar
  4. 4.
    Paul CP, Good PD, Winer I, Engelke DR (2002) Effective expression of small interfering RNA in human cells. Nat Biotechnol 20:505–508. doi:10.1038/nbt0502-505 Google Scholar
  5. 5.
    Ter Brake O, t’Hooft K, Liu YP, Centlivre M, von Eije KJ, Berkhout B (2008) Lentiviral vector design for multiple shRNA expression and durable HIV-1 inhibition. Mol Ther 16:557–564. doi:10.1038/ Google Scholar
  6. 6.
    Siolas D, Lerner C, Burchard J, Ge W, Linsley PS, Paddison PJ, Hannon GJ, Cleary MA (2005) Synthetic shRNAs as potent RNAi triggers. Nat Biotechnol 23:227–231. doi:10.1038/nbt1052 Google Scholar
  7. 7.
    McAnuff MA, Rettig GR, Rice KG (2007) Potency of siRNA versus shRNA mediated knockdown in vivo. J Pharm Sci 96:2922–2930. doi:10.1002/jps.20968 Google Scholar
  8. 8.
    Klinghoffer RA, Magnus J, Schelter J, Mehaffey M, Coleman C, Cleary MA (2010) Reduced seed region-based off-target activity with lentivirus-mediated RNAi. RNA 16:879–884. doi:10.1261/rna.1977810 Google Scholar
  9. 9.
    Lee RC, Ambros V (2001) An extensive class of small RNAs in Caenorhabditis elegans. Science 294:862–864. doi:10.1126/science.1065329 Google Scholar
  10. 10.
    Lagos-Quintana M, Rauhut R, Yalcin A, Meyer J, Lendeckel W, Tuschl T (2002) Identification of tissue-specific microRNAs from mouse. Curr Biol 12:735–739Google Scholar
  11. 11.
    Cullen BR (2004) Transcription and processing of human microRNA precursors. Mol Cell 16:861–865. doi:10.1016/j.molcel.2004.12.002 Google Scholar
  12. 12.
    Bernstein E, Caudy AA, Hammond SM, Hannon GJ (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409:363–366. doi:10.1038/35053110 Google Scholar
  13. 13.
    Catalanotto C, Azzalin G, Macino G, Cogoni C (2000) Gene silencing in worms and fungi. Nature 404:245. doi:10.1038/35005169 Google Scholar
  14. 14.
    Tabara H, Sarkissian M, Kelly WG, Fleenor J, Grishok A, Timmons L, Fire A, Mello CC (1999) The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell 99:123–132Google Scholar
  15. 15.
    Elbashir SM, Lendeckel W, Tuschl T (2001) RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev 15:188–200Google Scholar
  16. 16.
    Hammond SM, Bernstein E, Beach D, Hannon GJ (2000) An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404:293–296. doi:10.1038/35005107 Google Scholar
  17. 17.
    Nykänen A, Haley B, Zamore PD (2001) ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 107:309–321Google Scholar
  18. 18.
    Martinez J, Tuschl T (2004) RISC is a 5′ phosphomonoester-producing RNA endonuclease. Genes Dev 18:975–980. doi:10.1101/gad.1187904 Google Scholar
  19. 19.
    Schwarz DS, Tomari Y, Zamore PD (2004) The RNA-induced silencing complex is a Mg2+-dependent endonuclease. Curr Biol 14:787–791. doi:10.1016/j.cub.2004.03.008 Google Scholar
  20. 20.
    Doench JG, Petersen CP, Sharp PA (2003) siRNAs can function as miRNAs. Genes Dev 17:438–442. doi:10.1101/gad.1064703 Google Scholar
  21. 21.
    Ge Q, Ilves H, Dallas A, Kumar P, Shorenstein J, Kazakov SA, Johnston BH (2010) Minimal-length short hairpin RNAs: the relationship of structure and RNAi activity. RNA 16:106–117. doi:10.1261/rna.1894510 Google Scholar
  22. 22.
    Rao DD, Maples PB, Senzer N, Kumar P, Wang Z, Pappen BO, Yu Y, Haddock C, Jay C, Phadke AP et al (2010) Enhanced target gene knockdown by a bifunctional shRNA: a novel approach of RNA interference. Cancer Gene Ther 17:780–791. doi:10.1038/cgt.2010.35 Google Scholar
  23. 23.
    Phadke AP, Jay CM, Wang Z, Chen S, Liu S, Haddock C, Kumar P, Pappen BO, Rao DD, Templeton NS et al (2011) In vivo safety and antitumor efficacy of bifunctional small hairpin RNAs specific for the human Stathmin 1 oncoprotein. DNA Cell Biol 30:715–726. doi:10.1089/dna.2011.1240 Google Scholar
  24. 24.
    Taxman DJ, Livingstone LR, Zhang J, Conti BJ, Iocca HA, Williams KL, Lich JD, Ting JP-Y, Reed W (2006) Criteria for effective design, construction, and gene knockdown by shRNA vectors. BMC Biotechnol 6:7. doi:10.1186/1472-6750-6-7 Google Scholar
  25. 25.
    Li L, Lin X, Khvorova A, Fesik SW, Shen Y (2007) Defining the optimal parameters for hairpin-based knockdown constructs. RNA 13:1765–1774. doi:10.1261/rna.599107 Google Scholar
  26. 26.
    Kim D-H, Behlke MA, Rose SD, Chang M-S, Choi S, Rossi JJ (2005) Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nat Biotechnol 23:222–226. doi:10.1038/nbt1051 Google Scholar
  27. 27.
    Rose SD, Kim D-H, Amarzguioui M, Heidel JD, Collingwood MA, Davis ME, Rossi JJ, Behlke MA (2005) Functional polarity is introduced by Dicer processing of short substrate RNAs. Nucleic Acids Res 33:4140–4156. doi:10.1093/nar/gki732 Google Scholar
  28. 28.
    Yu J-Y, Taylor J, DeRuiter SL, Vojtek AB, Turner DL (2003) Simultaneous inhibition of GSK3alpha and GSK3beta using hairpin siRNA expression vectors. Mol Ther 7:228–236Google Scholar
  29. 29.
    Miyagishi M, Sumimoto H, Miyoshi H, Kawakami Y, Taira K (2004) Optimization of an siRNA-expression system with an improved hairpin and its significant suppressive effects in mammalian cells. J Gene Med 6:715–723. doi:10.1002/jgm.556 Google Scholar
  30. 30.
    Mcintyre GJ, Yu Y-H, Lomas M, Fanning GC (2011) The effects of stem length and core placement on shRNA activity. BMC Mol Biol 12:34. doi:10.1186/1471-2199-12-34 Google Scholar
  31. 31.
    Vlassov AV, Korba B, Farrar K, Mukerjee S, Seyhan AA, Ilves H, Kaspar RL, Leake D, Kazakov SA, Johnston BH (2007) shRNAs targeting hepatitis C: effects of sequence and structural features, and comparison with siRNA. Oligonucleotides 17:223–236. doi:10.1089/oli.2006.0069 Google Scholar
  32. 32.
    Terasawa K, Shimizu K, Tsujimoto G (2011) Synthetic pre-miRNA-based shRNA as potent RNAi triggers. J Nucleic Acids 2011:131579. doi:10.4061/2011/131579 Google Scholar
  33. 33.
    Ge Q, Dallas A, Ilves H, Shorenstein J, Behlke MA, Johnston BH (2010) Effects of chemical modification on the potency, serum stability, and immunostimulatory properties of short shRNAs. RNA 16:118–130. doi:10.1261/rna.1901810 Google Scholar
  34. 34.
    Lee NS, Dohjima T, Bauer G, Li H, Li M-J, Ehsani A, Salvaterra P, Rossi J (2002) Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat Biotechnol 20:500–505. doi:10.1038/nbt0502-500 Google Scholar
  35. 35.
    Mcintyre GJ, Fanning GC (2006) Design and cloning strategies for constructing shRNA expression vectors. BMC Biotechnol 6:1. doi:10.1186/1472-6750-6-1 Google Scholar
  36. 36.
    Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T (2001) Identification of novel genes coding for small expressed RNAs. Science 294:853–858. doi:10.1126/science.1064921 Google Scholar
  37. 37.
    Kawasaki H, Taira K (2003) 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 31:700–707Google Scholar
  38. 38.
    Carmona S, Ely A, Crowther C, Moolla N, Salazar FH, Marion PL, Ferry N, Weinberg MS, Arbuthnot P (2006) Effective inhibition of HBV replication in vivo by anti-HBx short hairpin RNAs. Mol Ther 13:411–421. doi:10.1016/j.ymthe.2005.10.013 Google Scholar
  39. 39.
    Boden D (2004) Enhanced gene silencing of HIV-1 specific siRNA using microRNA designed hairpins. Nucleic Acids Res 32:1154–1158. doi:10.1093/nar/gkh278 Google Scholar
  40. 40.
    Lambeth LS, Zhao Y, Smith LP, Kgosana L, Nair V (2009) Targeting Marek’s disease virus by RNA interference delivered from a herpesvirus vaccine. Vaccine 27:298–306. doi:10.1016/j.vaccine.2008.10.023 Google Scholar
  41. 41.
    Hinton TM, Wise TG, Cottee PA, Doran TJ (2008) Native microRNA loop sequences can improve short hairpin RNA processing for virus gene silencing in animal cells. J RNAi Gene Silencing 4:295–301Google Scholar
  42. 42.
    Chung K-H, Hart CC, Al-Bassam S, Avery A, Taylor J, Patel PD, Vojtek AB, Turner DL (2006) Polycistronic RNA polymerase II expression vectors for RNA interference based on BIC/miR-155. Nucleic Acids Res 34:e53. doi:10.1093/nar/gkl143 Google Scholar
  43. 43.
    Zhou H, Xia X-G, Xu Z (2005) An RNA polymerase II construct synthesizes short-hairpin RNA with a quantitative indicator and mediates highly efficient RNAi. Nucleic Acids Res 33:e62. doi:10.1093/nar/gni061 Google Scholar
  44. 44.
    Silva JM, Li MZ, Chang K, Ge W, Golding MC, Rickles RJ, Siolas D, Hu G, Paddison PJ, Schlabach MR et al (2005) Second-generation shRNA libraries covering the mouse and human genomes. Nat Genet 37:1281–1288. doi:10.1038/ng1650 Google Scholar
  45. 45.
    Dickins RA, Hemann MT, Zilfou JT, Simpson DR, Ibarra I, Hannon GJ, Lowe SW (2005) Probing tumor phenotypes using stable and regulated synthetic microRNA precursors. Nat Genet 37:1289–1295. doi:10.1038/ng1651 Google Scholar
  46. 46.
    Boudreau RL, Monteys AM, Davidson BL (2008) Minimizing variables among hairpin-based RNAi vectors reveals the potency of shRNAs. RNA 14:1834–1844. doi:10.1261/rna.1062908 Google Scholar
  47. 47.
    Grimm D, Streetz KL, Jopling CL, Storm TA, Pandey K, Davis CR, Marion P, Salazar F, Kay MA (2006) Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature 441:537–541. doi:10.1038/nature04791 Google Scholar
  48. 48.
    Ehlert EM, Eggers R, Niclou SP, Verhaagen J (2010) Cellular toxicity following application of adeno-associated viral vector-mediated RNA interference in the nervous system. BMC Neurosci 11:20. doi:10.1186/1471-2202-11-20 Google Scholar
  49. 49.
    Martin JN, Wolken N, Brown T, Dauer WT, Ehrlich ME, Gonzalez-Alegre P (2011) Lethal toxicity caused by expression of shRNA in the mouse striatum: implications for therapeutic design. Gene Ther 18:666–673. doi:10.1038/gt.2011.10 Google Scholar
  50. 50.
    McBride JL, Boudreau RL, Harper SQ, Staber PD, Monteys AM, Martins I, Gilmore BL, Burstein H, Peluso RW, Polisky B et al (2008) Artificial miRNAs mitigate shRNA-mediated toxicity in the brain: implications for the therapeutic development of RNAi. Proc Natl Acad Sci U S A 105:5868–5873. doi:10.1073/pnas.0801775105 Google Scholar
  51. 51.
    Boden D, Pusch O, Lee F, Tucker L, Shank PR, Ramratnam B (2003) Promoter choice affects the potency of HIV-1 specific RNA interference. Nucleic Acids Res 31:5033–5038Google Scholar
  52. 52.
    Rao MK, Wilkinson MF (2006) Tissue-specific and cell type-specific RNA interference in vivo. Nat Protoc 1:1494–1501. doi:10.1038/nprot.2006.260 Google Scholar
  53. 53.
    Nielsen TT, Marion IV, Hasholt L, Lundberg C (2009) Neuron-specific RNA interference using lentiviral vectors. J Gene Med 11:559–569. doi:10.1002/jgm.1333 Google Scholar
  54. 54.
    Dong K, Wang R, Wang X, Lin F, Shen J-J, Gao P, Zhang H-Z (2009) Tumor-specific RNAi targeting eIF4E suppresses tumor growth, induces apoptosis and enhances cisplatin cytotoxicity in human breast carcinoma cells. Breast Cancer Res Treat 113:443–456. doi:10.1007/s10549-008-9956-x Google Scholar
  55. 55.
    Hernandez N (2001) Small nuclear RNA genes: a model system to study fundamental mechanisms of transcription. J Biol Chem 276:26733–26736. doi:10.1074/jbc.R100032200 Google Scholar
  56. 56.
    Lee Y, Kim M, Han J, Yeom K-H, Lee S, Baek SH, Kim VN (2004) MicroRNA genes are transcribed by RNA polymerase II. EMBO J 23:4051–4060. doi:10.1038/sj.emboj.7600385 Google Scholar
  57. 57.
    Paule MR, White RJ (2000) Survey and summary: transcription by RNA polymerases I and III. Nucleic Acids Res 28:1283–1298Google Scholar
  58. 58.
    Czauderna F, Santel A, Hinz M, Fechtner M, Durieux B, Fisch G, Leenders F, Arnold W, Giese K, Klippel A et al (2003) Inducible shRNA expression for application in a prostate cancer mouse model. Nucleic Acids Res 31:e127Google Scholar
  59. 59.
    Kobayashi S, Higuchi T, Anzai K (2005) Application of the BC1 RNA gene promoter for short hairpin RNA expression in cultured neuronal cells. Biochem Biophys Res Commun 334:1305–1309. doi:10.1016/j.bbrc.2005.07.033 Google Scholar
  60. 60.
    Scherer LJ, Frank R, Rossi JJ (2007) Optimization and characterization of tRNA-shRNA expression constructs. Nucleic Acids Res 35:2620–2628. doi:10.1093/nar/gkm103 Google Scholar
  61. 61.
    Gunnery S, Ma Y, Mathews MB (1999) Termination sequence requirements vary among genes transcribed by RNA polymerase III. J Mol Biol 286:745–757. doi:10.1006/jmbi.1998.2518 Google Scholar
  62. 62.
    Murphy S, Tripodi M, Melli M (1986) A sequence upstream from the coding region is required for the transcription of the 7SK RNA genes. Nucleic Acids Res 14:9243–9260Google Scholar
  63. 63.
    Das G, Henning D, Wright D, Reddy R (1988) Upstream regulatory elements are necessary and sufficient for transcription of a U6 RNA gene by RNA polymerase III. EMBO J 7:503–512Google Scholar
  64. 64.
    Kunkel GR, Pederson T (1988) Upstream elements required for efficient transcription of a human U6 RNA gene resemble those of U1 and U2 genes even though a different polymerase is used. Genes Dev 2:196–204Google Scholar
  65. 65.
    Schramm L, Hernandez N (2002) Recruitment of RNA polymerase III to its target promoters. Genes Dev 16:2593–2620. doi:10.1101/gad.1018902 Google Scholar
  66. 66.
    Valadkhan S (2005) snRNAs as the catalysts of pre-mRNA splicing. Curr Opin Chem Biol 9:603–608. doi:10.1016/j.cbpa.2005.10.008 Google Scholar
  67. 67.
    Hayashi K (1981) Organization of sequences related to U6 RNA in the human genome. Nucleic Acids Res 9:3379–3388Google Scholar
  68. 68.
    Domitrovich AM, Kunkel GR (2003) Multiple, dispersed human U6 small nuclear RNA genes with varied transcriptional efficiencies. Nucleic Acids Res 31:2344–2352Google Scholar
  69. 69.
    Lambeth LS, Moore RJ, Muralitharan M, Dalrymple BP, Mcwilliam S, Doran TJ (2005) Characterisation and application of a bovine U6 promoter for expression of short hairpin RNAs. BMC Biotechnol 5:13. doi:10.1186/1472-6750-5-13 Google Scholar
  70. 70.
    Lambeth LS, Wise TG, Moore RJ, Muralitharan MS, Doran TJ (2006) Comparison of bovine RNA polymerase III promoters for short hairpin RNA expression. Anim Genet 37:369–372. doi:10.1111/j.1365-2052.2006.01468.x Google Scholar
  71. 71.
    Kudo T, Sutou S (2005) Usage of putative chicken U6 promoters for vector-based RNA interference. J Reprod Dev 51:411–417Google Scholar
  72. 72.
    Wise TG, Schafer DJ, Lambeth LS, Tyack SG, Bruce MP, Moore RJ, Doran TJ (2007) Characterization and comparison of chicken U6 promoters for the expression of short hairpin RNAs. Anim Biotechnol 18:153–162. doi:10.1080/10495390600867515 Google Scholar
  73. 73.
    Zenke K, Kim KH (2008) Novel fugu U6 promoter driven shRNA expression vector for efficient vector based RNAi in fish cell lines. Biochem Biophys Res Commun 371:480–483. doi:10.1016/j.bbrc.2008.04.116 Google Scholar
  74. 74.
    Boonanuntanasarn S, Panyim S, Yoshizaki G (2009) Usage of putative zebrafish U6 promoters to express shRNA in Nile tilapia and shrimp cell extracts. Transgenic Res 18:323–325. doi:10.1007/s11248-009-9249-0 Google Scholar
  75. 75.
    Hu S, Ni W, Hazi W, Zhang H, Zhang N, Meng R, Chen C (2011) Cloning and functional analysis of sheep U6 promoters. Anim Biotechnol 22:170–174. doi:10.1080/10495398.2011.580669 Google Scholar
  76. 76.
    Chuang C-K, Lee K-H, Fan C-T, Su Y-S (2009) Porcine type III RNA polymerase III promoters for short hairpin RNA expression. Anim Biotechnol 20:34–39. doi:10.1080/10495390802603064 Google Scholar
  77. 77.
    Myslinski E, Amé JC, Krol A, Carbon P (2001) An unusually compact external promoter for RNA polymerase III transcription of the human H1RNA gene. Nucleic Acids Res 29:2502–2509Google Scholar
  78. 78.
    Koper-Emde D, Herrmann L, Sandrock B, Benecke B-J (2011) RNA interference by small hairpin RNAs synthesised under control of the human 7S K RNA promoter. Biol Chem 385:791–794. doi:10.1515/BC.2004.103 Google Scholar
  79. 79.
    Bannister SC, Wise TG, Cahill DM, Doran TJ (2007) Comparison of chicken 7SK and U6 RNA polymerase III promoters for short hairpin RNA expression. BMC Biotechnol 7:79. doi:10.1186/1472-6750-7-79 Google Scholar
  80. 80.
    Cummins D, Doran TJ, Tyack S, Purcell D, Hammond J (2008) Identification and characterisation of the porcine 7SK RNA polymerase III promoter for short hairpin RNA expression. J RNAi Gene Silencing 4:289–294Google Scholar
  81. 81.
    Mäkinen PI, Koponen JK, Kärkkäinen A-M, Malm TM, Pulkkinen KH, Koistinaho J, Turunen MP, Ylä-Herttuala S (2006) Stable RNA interference: comparison of U6 and H1 promoters in endothelial cells and in mouse brain. J Gene Med 8:433–441. doi:10.1002/jgm.860 Google Scholar
  82. 82.
    An DS, Qin FX-F, Auyeung VC, Mao SH, Kung SKP, Baltimore D, Chen ISY (2006) Optimization and functional effects of stable short hairpin RNA expression in primary human lymphocytes via lentiviral vectors. Mol Ther 14:494–504. doi:10.1016/j.ymthe.2006.05.015 Google Scholar
  83. 83.
    Farris AD, Gross JK, Hanas JS, Harley JB (1996) Genes for murine Y1 and Y3 Ro RNAs have class 3 RNA polymerase III promoter structures and are unlinked on mouse chromosome 6. Gene 174:35–42Google Scholar
  84. 84.
    Grimm D, Wang L, Lee JS, Schürmann N, Gu S, Börner K, Storm TA, Kay MA (2010) Argonaute proteins are key determinants of RNAi efficacy, toxicity, and persistence in the adult mouse liver. J Clin Invest 120:3106–3119. doi:10.1172/JCI43565 Google Scholar
  85. 85.
    Rumi M, Ishihara S, Aziz M, Kazumori H, Ishimura N, Yuki T, Kadota C, Kadowaki Y, Kinoshita Y (2006) RNA polymerase II mediated transcription from the polymerase III promoters in short hairpin RNA expression vector. Biochem Biophys Res Commun 339:540–547. doi:10.1016/j.bbrc.2005.11.037 Google Scholar
  86. 86.
    Galli G, Hofstetter H, Birnstiel ML (1981) Two conserved sequence blocks within eukaryotic tRNA genes are major promoter elements. Nature 294:626–631Google Scholar
  87. 87.
    Xia X-G, Zhou H, Samper E, Melov S, Xu Z (2006) Pol II-expressed shRNA knocks down Sod2 gene expression and causes phenotypes of the gene knockout in mice. PLoS Genet 2:e10. doi:10.1371/journal.pgen.0020010 Google Scholar
  88. 88.
    Takahashi Y, Yamaoka K, Nishikawa M, Takakura Y (2009) Quantitative and temporal analysis of gene silencing in tumor cells induced by small interfering RNA or short hairpin RNA expressed from plasmid vectors. J Pharm Sci 98:74–80. doi:10.1002/jps.21398 Google Scholar
  89. 89.
    Dyer V, Ely A, Bloom K, Weinberg M, Arbuthnot P (2010) tRNA Lys3 promoter cassettes that efficiently express RNAi-activating antihepatitis B virus short hairpin RNAs. Biochem Biophys Res Commun 398:640–646. doi:10.1016/j.bbrc.2010.06.122 Google Scholar
  90. 90.
    Weiwei M, Zhenhua X, Feng L, Hang N, Yuyang J (2009) A significant increase of RNAi efficiency in human cells by the CMV enhancer with a tRNAlys promoter. J Biomed Biotechnol 2009:514287. doi:10.1155/2009/514287 Google Scholar
  91. 91.
    Xia H, Mao Q, Paulson HL, Davidson BL (2002) siRNA-mediated gene silencing in vitro and in vivo. Nat Biotechnol 20:1006–1010. doi:10.1038/nbt739 Google Scholar
  92. 92.
    Zeng Y, Wagner EJ, Cullen BR (2002) Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol Cell 9:1327–1333Google Scholar
  93. 93.
    Denti MA, Rosa A, Sthandier O, De Angelis FG, Bozzoni I (2004) A new vector, based on the PolII promoter of the U1 snRNA gene, for the expression of siRNAs in mammalian cells. Mol Ther 10:191–199Google Scholar
  94. 94.
    Huang M, Jia F-J, Yan Y-C, Guo L-H, Li Y-P (2006) Transactivated minimal E1b promoter is capable of driving the expression of short hairpin RNA. J Virol Methods 134:48–54. doi:10.1016/j.jviromet.2005.11.016 Google Scholar
  95. 95.
    Konstantinova P, De Vries W, Haasnoot J, Ter Brake O, De Haan P, Berkhout B (2006) Inhibition of human immunodeficiency virus type 1 by RNA interference using long-hairpin RNA. Gene Ther 13:1403–1413. doi:10.1038/ Google Scholar
  96. 96.
    Liu YP, Haasnoot J, Ter Brake O, Berkhout B, Konstantinova P (2008) Inhibition of HIV-1 by multiple siRNAs expressed from a single microRNA polycistron. Nucleic Acids Res 36:2811–2824. doi:10.1093/nar/gkn109 Google Scholar
  97. 97.
    Chen SC-Y, Stern P, Guo Z, Chen J (2011) Expression of multiple artificial microRNAs from a chicken miRNA126-based lentiviral vector. PLoS One 6:e22437. doi:10.1371/journal.pone.0022437 Google Scholar
  98. 98.
    Dong K, Wang R, Wang X, Lin F, Shen J-J, Gao P, Zhang H-Z (2008) Tumor-specific RNAi targeting eIF4E suppresses tumor growth, induces apoptosis and enhances cisplatin cytotoxicity in human breast carcinoma cells. Breast Cancer Res Treat 113:443–456. doi:10.1007/s10549-008-9956-x Google Scholar
  99. 99.
    Giering JC, Grimm D, Storm TA, Kay MA (2008) Expression of shRNA from a tissue-specific pol II promoter is an effective and safe RNAi therapeutic. Mol Ther 16:1630–1636. doi:10.1038/mt.2008.144 Google Scholar
  100. 100.
    Zhu Z, Zheng T, Lee CG, Homer RJ, Elias JA (2002) Tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modeling. Semin Cell Dev Biol 13:121–128Google Scholar
  101. 101.
    van de Wetering M, Oving I, Muncan V, Pon Fong MT, Brantjes H, van Leenen D, Holstege FCP, Brummelkamp TR, Agami R, Clevers H (2003) Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector. EMBO Rep 4:609–615. doi:10.1038/sj.embor.embor865 Google Scholar
  102. 102.
    Ventura A, Meissner A, Dillon CP, McManus M, Sharp PA, Van Parijs L, Jaenisch R, Jacks T (2004) Cre-lox-regulated conditional RNA interference from transgenes. Proc Natl Acad Sci U S A 101:10380–10385. doi:10.1073/pnas.0403954101 Google Scholar
  103. 103.
    Szulc J, Wiznerowicz M, Sauvain M-O, Trono D, Aebischer P (2006) A versatile tool for conditional gene expression and knockdown. Nat Meth 3:109–116. doi:10.1038/nmeth846 Google Scholar
  104. 104.
    Herold MJ, van den Brandt J, Seibler J, Reichardt HM (2008) Inducible and reversible gene silencing by stable integration of an shRNA-encoding lentivirus in transgenic rats. Proc Natl Acad Sci U S A 105:18507–18512. doi:10.1073/pnas.0806213105 Google Scholar
  105. 105.
    No D, Yao TP, Evans RM (1996) Ecdysone-inducible gene expression in mammalian cells and transgenic mice. Proc Natl Acad Sci U S A 93:3346–3351Google Scholar
  106. 106.
    Gupta S, Schoer RA, Egan JE, Hannon GJ, Mittal V (2004) Inducible, reversible, and stable RNA interference in mammalian cells. Proc Natl Acad Sci U S A 101:1927–1932. doi:10.1073/pnas.0306111101 Google Scholar
  107. 107.
    Rangasamy D, Tremethick DJ, Greaves IK (2008) Gene knockdown by ecdysone-based inducible RNAi in stable mammalian cell lines. Nat Protoc 3:79–88. doi:10.1038/nprot.2007.456 Google Scholar
  108. 108.
    Orban PC, Chui D, Marth JD (1992) Tissue- and site-specific DNA recombination in transgenic mice. Proc Natl Acad Sci U S A 89:6861–6865Google Scholar
  109. 109.
    McCaffrey AP, Meuse L, Pham T-TT, Conklin DS, Hannon GJ, Kay MA (2002) RNA interference in adult mice. Nature 418:38–39. doi:10.1038/418038a Google Scholar
  110. 110.
    McCaffrey AP, Nakai H, Pandey K, Huang Z, Salazar FH, Xu H, Wieland SF, Marion PL, Kay MA (2003) Inhibition of hepatitis B virus in mice by RNA interference. Nat Biotechnol 21:639–644. doi:10.1038/nbt824 Google Scholar
  111. 111.
    Brummelkamp TR, Bernards R, Agami R (2002) Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell 2:243–247Google Scholar
  112. 112.
    Devroe E, Silver PA (2002) Retrovirus-delivered siRNA. BMC Biotechnol 2:15Google Scholar
  113. 113.
    Bromberg-White JL, Webb CP, Patacsil VS, Miranti CK, Williams BO, Holmen SL (2004) Delivery of short hairpin RNA sequences by using a replication-competent avian retroviral vector. J Virol 78:4914–4916Google Scholar
  114. 114.
    Hughes SH, Greenhouse JJ, Petropoulos CJ, Sutrave P (1987) Adaptor plasmids simplify the insertion of foreign DNA into helper-independent retroviral vectors. J Virol 61:3004–3012Google Scholar
  115. 115.
    Déglon N, Tseng JL, Bensadoun JC, Zurn AD, Arsenijevic Y, Pereira de Almeida L, Zufferey R, Trono D, Aebischer P (2000) Self-inactivating lentiviral vectors with enhanced transgene expression as potential gene transfer system in Parkinson’s disease. Hum Gene Ther 11:179–190. doi:10.1089/10430340050016256 Google Scholar
  116. 116.
    Abbas-Terki T, Blanco-Bose W, Déglon N, Pralong W, Aebischer P (2002) Lentiviral-mediated RNA interference. Hum Gene Ther 13:2197–2201. doi:10.1089/104303402320987888 Google Scholar
  117. 117.
    Rubinson DA, Dillon CP, Kwiatkowski AV, Sievers C, Yang L, Kopinja J, Rooney DL, Zhang M, Ihrig MM, McManus MT et al (2003) A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet 33:401–406. doi:10.1038/ng1117 Google Scholar
  118. 118.
    Cartier N, Hacein-Bey-Abina S, Bartholomae CC, Veres G, Schmidt M, Kutschera I, Vidaud M, Abel U, Dal-Cortivo L, Caccavelli L et al (2009) Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy. Science 326:818–823. doi:10.1126/science.1171242 Google Scholar
  119. 119.
    Berkhout B (2009) Toward a durable anti-HIV gene therapy based on RNA interference. Ann N Y Acad Sci 1175:3–14. doi:10.1111/j.1749-6632.2009.04972.x Google Scholar
  120. 120.
    Tomar RS, Matta H, Chaudhary PM (2003) Use of adeno-associated viral vector for delivery of small interfering RNA. Oncogene 22:5712–5715. doi:10.1038/sj.onc.1206733 Google Scholar
  121. 121.
    Büning H, Perabo L, Coutelle O, Quadt Humme S, Hallek M (2008) Recent developments in adeno-associated virus vector technology. J Gene Med 10:717–733. doi:10.1002/jgm.1205 Google Scholar
  122. 122.
    Saydam O, Glauser DL, Heid I, Turkeri G, Hilbe M, Jacobs AH, Ackermann M, Fraefel C (2005) Herpes simplex virus 1 amplicon vector-mediated siRNA targeting epidermal growth factor receptor inhibits growth of human glioma cells in vivo. Mol Ther 12:803–812. doi:10.1016/j.ymthe.2005.07.534 Google Scholar
  123. 123.
    Hong C-S, Goins WF, Goss JR, Burton EA, Glorioso JC (2006) Herpes simplex virus RNAi and neprilysin gene transfer vectors reduce accumulation of Alzheimer’s disease-related amyloid-beta peptide in vivo. Gene Ther 13:1068–1079. doi:10.1038/ Google Scholar
  124. 124.
    Nicholson LJ, Philippe M, Paine AJ, Mann DA, Dolphin CT (2005) RNA interference mediated in human primary cells via recombinant baculoviral vectors. Mol Ther 11:638–644. doi:10.1016/j.ymthe.2004.12.010 Google Scholar
  125. 125.
    Castanotto D, Sakurai K, Lingeman R, Li H, Shively L, Aagaard L, Soifer H, Gatignol A, Riggs A, Rossi JJ (2007) Combinatorial delivery of small interfering RNAs reduces RNAi efficacy by selective incorporation into RISC. Nucleic Acids Res 35:5154–5164. doi:10.1093/nar/gkm543 Google Scholar
  126. 126.
    Grimm D, Kay MA (2007) Combinatorial RNAi: a winning strategy for the race against evolving targets? Mol Ther 15:878–888. doi:10.1038/ Google Scholar
  127. 127.
    Boden D, Pusch O, Lee F, Tucker L, Ramratnam B (2003) Human immunodeficiency virus type 1 escape from RNA interference. J Virol 77:11531–11535Google Scholar
  128. 128.
    Liu YP, von Eije KJ, Schopman NCT, Westerink J-T, Ter Brake O, Haasnoot J, Berkhout B (2009) Combinatorial RNAi against HIV-1 using extended short hairpin RNAs. Mol Ther 17:1712–1723. doi:10.1038/mt.2009.176 Google Scholar
  129. 129.
    Liu YP, Haasnoot J, Berkhout B (2007) Design of extended short hairpin RNAs for HIV-1 inhibition. Nucleic Acids Res 35:5683–5693. doi:10.1093/nar/gkm596 Google Scholar
  130. 130.
    Saayman S, Barichievy S, Capovilla A, Morris KV, Arbuthnot P, Weinberg MS (2008) The efficacy of generating three independent anti-HIV-1 siRNAs from a single U6 RNA Pol III-expressed long hairpin RNA. PLoS One 3:e2602. doi:10.1371/journal.pone.0002602 Google Scholar
  131. 131.
    Sano M, Li H, Nakanishi M, Rossi JJ (2008) Expression of long anti-HIV-1 hairpin RNAs for the generation of multiple siRNAs: advantages and limitations. Mol Ther 16:170–177. doi:10.1038/ Google Scholar
  132. 132.
    Henry SD, van der Wegen P, Metselaar HJ, Tilanus HW, Scholte BJ, van der Laan LJW (2006) Simultaneous targeting of HCV replication and viral binding with a single lentiviral vector containing multiple RNA interference expression cassettes. Mol Ther 14:485–493. doi:10.1016/j.ymthe.2006.04.012 Google Scholar
  133. 133.
    Anderson J, Akkina R (2005) HIV-1 resistance conferred by siRNA cosuppression of CXCR4 and CCR5 coreceptors by a bispecific lentiviral vector. AIDS Res Ther 2:1. doi:10.1186/1742-6405-2-1 Google Scholar
  134. 134.
    Hinton TM, Doran TJ (2008) Inhibition of chicken anaemia virus replication using multiple short-hairpin RNAs. Antiviral Res 80:143–149. doi:10.1016/j.antiviral.2008.05.009 Google Scholar
  135. 135.
    Ter Brake O, Konstantinova P, Ceylan M, Berkhout B (2006) Silencing of HIV-1 with RNA interference: a multiple shRNA approach. Mol Ther 14:883–892. doi:0.1016/j.ymthe.2006.07.007 Google Scholar
  136. 136.
    Song J, Giang A, Lu Y, Pang S, Chiu R (2008) Multiple shRNA expressing vector enhances efficiency of gene silencing. BMB Rep 41:358–362Google Scholar
  137. 137.
    Gou D, Weng T, Wang Y, Wang Z, Zhang H, Gao L, Chen Z, Wang P, Liu L (2007) A novel approach for the construction of multiple shRNA expression vectors. J Gene Med 9:751–763. doi:10.1002/jgm.1080 Google Scholar
  138. 138.
    Cheng TL, Teng CF, Tsai WH, Yeh CW, Wu MP, Hsu HC, Hung CF, Chang WT (2009) Multitarget therapy of malignant cancers by the head-to-tail tandem array multiple shRNAs expression system. Cancer Gene Ther 16:516–531. doi:10.1038/cgt.2008.102 Google Scholar
  139. 139.
    Gonzalez S, Castanotto D, Li H, Olivares S, Jensen MC, Forman SJ, Rossi JJ, Cooper LJN (2005) Amplification of RNAi-targeting HLA mRNAs. Mol Ther 11:811–818. doi:10.1016/j.ymthe.2004.12.023 Google Scholar
  140. 140.
    Mcintyre GJ, Groneman JL, Tran A, Applegate TL (2008) An infinitely expandable cloning strategy plus repeat-proof PCR for working with multiple shRNA. PLoS One 3:e3827. doi:10.1371/journal.pone.0003827.t001 Google Scholar
  141. 141.
    Lambeth LS, Van Hateren NJ, Wilson SA, Nair V (2010) A direct comparison of strategies for combinatorial RNA interference. BMC Mol Biol 11:77. doi:10.1186/1471-2199-11-77 Google Scholar
  142. 142.
    Akashi H, Miyagishi M, Yokota T, Watanabe T, Hino T, Nishina K, Kohara M, Taira K (2005) Escape from the interferon response associated with RNA interference using vectors that encode long modified hairpin-RNA. Mol Biosyst 1:382–390. doi:10.1039/b510159j Google Scholar
  143. 143.
    Barichievy S, Saayman S, Von Eije KJ, Morris KV, Arbuthnot P, Weinberg MS (2007) The inhibitory efficacy of RNA POL III-expressed long hairpin RNAs targeted to untranslated regions of the HIV-1 5′ long terminal repeat. Oligonucleotides 17:419–431. doi:10.1089/oli.2007.0095 Google Scholar
  144. 144.
    Weinberg MS, Ely A, Barichievy S, Crowther C, Mufamadi S, Carmona S, Arbuthnot P (2007) Specific inhibition of HBV replication in vitro and in vivo with expressed long hairpin RNA. Mol Ther 15:534–541. doi:10.1038/ Google Scholar
  145. 145.
    Konstantinova P, Ter Brake O, Haasnoot J, De Haan P, Berkhout B (2007) Trans-inhibition of HIV-1 by a long hairpin RNA expressed within the viral genome. Retrovirology 4:15. doi:10.1186/1742-4690-4-15 Google Scholar
  146. 146.
    Sun D, Melegari M, Sridhar S, Rogler CE, Zhu L (2006) Multi-miRNA hairpin method that improves gene knockdown efficiency and provides linked multi-gene knockdown. Biotechniques 41:59–63Google Scholar
  147. 147.
    Zhu X, Santat LA, Chang MS, Liu J, Zavzavadjian JR, Wall EA, Kivork C, Simon MI, Fraser ID (2007) A versatile approach to multiple gene RNA interference using microRNA-based short hairpin RNAs. BMC Mol Biol 8:98. doi:10.1186/1471-2199-8-98 Google Scholar
  148. 148.
    Das RM, Van Hateren NJ, Howell GR, Farrell ER, Bangs FK, Porteous VC, Manning EM, McGrew MJ, Ohyama K, Sacco MA et al (2006) A robust system for RNA interference in the chicken using a modified microRNA operon. Dev Biol 294:554–563. doi:10.1016/j.ydbio.2006.02.020 Google Scholar
  149. 149.
    Aagaard LA, Zhang J, von Eije KJ, Li H, Saetrom P, Amarzguioui M, Rossi JJ (2008) Engineering1 and optimization of the miR-106b cluster for ectopic expression of multiplexed anti-HIV RNAs. Gene Ther 15:1536–1549. doi:10.1038/gt.2008.147 Google Scholar
  150. 150.
    Yang X, Haurigot V, Zhou S, Luo G, Couto LB (2010) Inhibition of hepatitis C virus replication using adeno-associated virus vector delivery of an exogenous anti-hepatitis C virus microRNA cluster. Hepatology 52:1877–1887. doi:10.1002/hep.23908 Google Scholar
  151. 151.
    Bartel DP, Chen C-Z (2004) Micromanagers of gene expression: the potentially widespread influence of metazoan microRNAs. Nat Rev Genet 5:396–400. doi:10.1038/nrg1328 Google Scholar
  152. 152.
    Mcintyre GJ, Arndt AJ, Gillespie KM, Mak WM, Fanning GC (2011) A comparison of multiple shRNA expression methods for combinatorial RNAi. Genet Vaccines Ther 9:9. doi:10.1186/1479-0556-9-9 Google Scholar
  153. 153.
    Butler JEF, Kadonaga JT (2002) The RNA polymerase II core promoter: a key component in the regulation of gene expression. Genes Dev 16:2583–2592. doi:10.1101/gad.1026202 Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  1. 1.Murdoch Childrens Research InstituteRoyal Childrens HospitalMelbourneAustralia
  2. 2.Poultry Cooperative Research CentreArmidaleAustralia
  3. 3.Department of PaediatricsUniversity of MelbourneParkvilleAustralia

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