Skip to main content
SpringerLink
Log in

Short Hairpin RNA-Mediated Gene Silencing

  • Protocol
  • First Online:
siRNA Design

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

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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/sj.mt.6300382

    Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  9. Lee RC, Ambros V (2001) An extensive class of small RNAs in Caenorhabditis elegans. Science 294:862–864. doi:10.1126/science.1065329

    CAS  Google Scholar 

  10. Lagos-Quintana M, Rauhut R, Yalcin A, Meyer J, Lendeckel W, Tuschl T (2002) Identification of tissue-specific microRNAs from mouse. Curr Biol 12:735–739

    CAS  Google Scholar 

  11. Cullen BR (2004) Transcription and processing of human microRNA precursors. Mol Cell 16:861–865. doi:10.1016/j.molcel.2004.12.002

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  13. Catalanotto C, Azzalin G, Macino G, Cogoni C (2000) Gene silencing in worms and fungi. Nature 404:245. doi:10.1038/35005169

    CAS  Google Scholar 

  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 99:123–132

    CAS  Google Scholar 

  15. Elbashir SM, Lendeckel W, Tuschl T (2001) RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev 15:188–200

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  17. Nykänen A, Haley B, Zamore PD (2001) ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 107:309–321

    Google Scholar 

  18. Martinez J, Tuschl T (2004) RISC is a 5′ phosphomonoester-producing RNA endonuclease. Genes Dev 18:975–980. doi:10.1101/gad.1187904

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  20. Doench JG, Petersen CP, Sharp PA (2003) siRNAs can function as miRNAs. Genes Dev 17:438–442. doi:10.1101/gad.1064703

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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. 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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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 7:228–236

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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. 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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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. 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

    CAS  Google Scholar 

  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 31:700–707

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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 4:295–301

    CAS  Google Scholar 

  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. 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. 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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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. 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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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 31:5033–5038

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  57. Paule MR, White RJ (2000) Survey and summary: transcription by RNA polymerases I and III. Nucleic Acids Res 28:1283–1298

    CAS  Google Scholar 

  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:e127

    Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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 14:9243–9260

    CAS  Google Scholar 

  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 7:503–512

    CAS  Google Scholar 

  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 2:196–204

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  67. Hayashi K (1981) Organization of sequences related to U6 RNA in the human genome. Nucleic Acids Res 9:3379–3388

    CAS  Google Scholar 

  68. Domitrovich AM, Kunkel GR (2003) Multiple, dispersed human U6 small nuclear RNA genes with varied transcriptional efficiencies. Nucleic Acids Res 31:2344–2352

    CAS  Google Scholar 

  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. 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

    CAS  Google Scholar 

  71. Kudo T, Sutou S (2005) Usage of putative chicken U6 promoters for vector-based RNA interference. J Reprod Dev 51:411–417

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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 29:2502–2509

    CAS  Google Scholar 

  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. 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. 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 4:289–294

    CAS  Google Scholar 

  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. 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

    CAS  Google Scholar 

  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 174:35–42

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  86. Galli G, Hofstetter H, Birnstiel ML (1981) Two conserved sequence blocks within eukaryotic tRNA genes are major promoter elements. Nature 294:626–631

    CAS  Google Scholar 

  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. 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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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. 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

    CAS  Google Scholar 

  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 9:1327–1333

    CAS  Google Scholar 

  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 10:191–199

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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/sj.gt.3302786

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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. 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

    CAS  Google Scholar 

  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 13:121–128

    CAS  Google Scholar 

  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. 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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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 93:3346–3351

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  108. Orban PC, Chui D, Marth JD (1992) Tissue- and site-specific DNA recombination in transgenic mice. Proc Natl Acad Sci U S A 89:6861–6865

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  111. Brummelkamp TR, Bernards R, Agami R (2002) Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell 2:243–247

    CAS  Google Scholar 

  112. Devroe E, Silver PA (2002) Retrovirus-delivered siRNA. BMC Biotechnol 2:15

    Google Scholar 

  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 78:4914–4916

    CAS  Google Scholar 

  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 61:3004–3012

    CAS  Google Scholar 

  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. 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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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. 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

    CAS  Google Scholar 

  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/sj.gt.3302719

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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/sj.mt.6300116

    CAS  Google Scholar 

  127. Boden D, Pusch O, Lee F, Tucker L, Ramratnam B (2003) Human immunodeficiency virus type 1 escape from RNA interference. J Virol 77:11531–11535

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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. 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/sj.mt.6300298

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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. 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

    CAS  Google Scholar 

  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. Song J, Giang A, Lu Y, Pang S, Chiu R (2008) Multiple shRNA expressing vector enhances efficiency of gene silencing. BMB Rep 41:358–362

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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. 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. 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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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/sj.mt.6300077

    CAS  Google Scholar 

  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. 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 41:59–63

    CAS  Google Scholar 

  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. 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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

  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

    CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Murdoch Childrens Research Institute, Royal Childrens Hospital, Melbourne, VIC, Australia

    Luke S. Lambeth & Craig A. Smith

  2. Poultry Cooperative Research Centre, Armidale, NSW, Australia

    Luke S. Lambeth & Craig A. Smith

  3. Department of Paediatrics, University of Melbourne, Parkville, VIC, Australia

    Craig A. Smith

Authors
  1. Luke S. Lambeth
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Craig A. Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Luke S. Lambeth .

Editor information

Editors and Affiliations

  1. , Lineberger Comprehensive Cancer Center, University of North Carolina, West Drive 450, Chapel Hill, 27599, North Carolina, USA

    Debra J. Taxman

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Lambeth, L.S., Smith, C.A. (2013). Short Hairpin RNA-Mediated Gene Silencing. In: Taxman, D. (eds) siRNA Design. Methods in Molecular Biology, vol 942. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-119-6_12

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-119-6_12

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-118-9

  • Online ISBN: 978-1-62703-119-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation