Abstract
Elucidating gene function is heavily reliant on the ability to modulate gene expression in biological model systems. Although transient expression systems can provide useful information about the biological outcome resulting from short-term gene overexpression or silencing, methods providing stable integration of desired expression constructs (cDNA or RNA interference) are often preferred for functional studies. To this end, lentiviral vectors offer the ability to deliver long-term and regulated gene expression to mammalian cells, including the expression of gene targeting small hairpin RNAs (shRNAmirs). Unfortunately, constructing vectors containing the desired combination of cDNAs, markers, and shRNAmirs can be cumbersome and time-consuming if using traditional sequence based restriction enzyme and ligation-dependent methods. Here we describe the use of a recombination based Gateway cloning strategy to rapidly and efficiently produce recombinant lentiviral vectors for the expression of one or more cDNAs with or without simultaneous shRNAmir expression. Additionally, we describe a luciferase-based approach to rapidly triage shRNAs for knockdown efficacy and specificity without the need to create stable shRNAmir expressing cells.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Vannucci L, Lai M, Chiuppesi F et al (2013) Viral vectors: a look back and ahead on gene transfer technology. New Microbiol 36(1):1–22
Sakuma T, Barry MA, Ikeda Y (2012) Lentiviral vectors: basic to translational. Biochem J 443(3):603–618. doi:10.1042/BJ20120146, BJ20120146 [pii]
Palu G, Parolin C, Takeuchi Y et al (2000) Progress with retroviral gene vectors. Rev Med Virol 10(3):185–202, doi:10.1002/(SICI)1099-1654(200005/06)10:3<185::AID-RMV285>3.0.CO;2-8 [pii]
Naldini L, Blomer U, Gallay P et al (1996) In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272(5259):263–267
Fewell GD, Schmitt K (2006) Vector-based RNAi approaches for stable, inducible and genome-wide screens. Drug Discov Today 11(21–22):975–982. doi:10.1016/j.drudis.2006.09.008, S1359-6446(06)00369-2 [pii]
Aslanidis C, de Jong PJ (1990) Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res 18(20):6069–6074
Berrow NS, Alderton D, Sainsbury S et al (2007) A versatile ligation-independent cloning method suitable for high-throughput expression screening applications. Nucleic Acids Res 35(6), e45. doi:10.1093/nar/gkm047, gkm047 [pii]
Li C, Evans RM (1997) Ligation independent cloning irrespective of restriction site compatibility. Nucleic Acids Res 25(20):4165–4166, doi:gka645 [pii]
Li MZ, Elledge SJ (2012) SLIC: a method for sequence- and ligation-independent cloning. Methods Mol Biol 852:51–59. doi:10.1007/978-1-61779-564-0_5
Shuman S (1994) Novel approach to molecular cloning and polynucleotide synthesis using vaccinia DNA topoisomerase. J Biol Chem 269(51):32678–32684
Yang J, Zhang Z, Zhang XA et al (2010) A ligation-independent cloning method using nicking DNA endonuclease. Biotechniques 49(5):817–821. doi:10.2144/000113520, 000113520 [pii]
Hartley JL, Temple GF, Brasch MA (2000) DNA cloning using in vitro site-specific recombination. Genome Res 10(11):1788–1795
Nash HA, Mizuuchi K, Enquist LW et al (1981) Strand exchange in lambda integrative recombination: genetics, biochemistry, and models. Cold Spring Harb Symp Quant Biol 45(Pt 1):417–428
Bernard P, Couturier M (1991) The 41 carboxy-terminal residues of the miniF plasmid CcdA protein are sufficient to antagonize the killer activity of the CcdB protein. Mol Gen Genet 226(1–2):297–304
Sasaki Y, Sone T, Yoshida S et al (2004) Evidence for high specificity and efficiency of multiple recombination signals in mixed DNA cloning by the Multisite Gateway system. J Biotechnol 107(3):233–243, doi:S0168165603002657 [pii]
Cheo DL, Titus SA, Byrd DR et al (2004) Concerted assembly and cloning of multiple DNA segments using in vitro site-specific recombination: functional analysis of multi-segment expression clones. Genome Res 14(10):2111–2120. doi:10.1101/gr.2512204, 14/10b/2111 [pii]
Xia H, Mao Q, Paulson HL et al (2002) siRNA-mediated gene silencing in vitro and in vivo. Nat Biotechnol 20(10):1006–1010. doi:10.1038/nbt739, nbt739 [pii]
Yu JY, 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(9):6047–6052. doi:10.1073/pnas.092143499, 092143499 [pii]
Rao DD, Vorhies JS, Senzer N et al (2009) siRNA vs. shRNA: similarities and differences. Adv Drug Deliv Rev 61(9):746–759. doi:10.1016/j.addr.2009.04.004, S0169-409X(09)00096-9 [pii]
Manjunath N, Wu H, Subramanya S et al (2009) Lentiviral delivery of short hairpin RNAs. Adv Drug Deliv Rev 61(9):732–745. doi:10.1016/j.addr.2009.03.004, S0169-409X(09)00060-X [pii]
Brummelkamp TR, Bernards R, Agami R (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science 296(5567):550–553. doi:10.1126/science.1068999, 1068999 [pii]
Paddison PJ, Caudy AA, Bernstein E et al (2002) Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev 16(8):948–958. doi:10.1101/gad.981002
Grimm D, Streetz KL, Jopling CL et al (2006) Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature 441(7092):537–541. doi:10.1038/nature04791, nature04791 [pii]
Yi R, Doehle BP, Qin Y et al (2005) Overexpression of exportin 5 enhances RNA interference mediated by short hairpin RNAs and microRNAs. RNA 11(2):220–226. doi:10.1261/rna.7233305, rna.7233305 [pii]
Boudreau RL, Monteys AM, Davidson BL (2008) Minimizing variables among hairpin-based RNAi vectors reveals the potency of shRNAs. RNA 14(9):1834–1844. doi:10.1261/rna.1062908, rna.1062908 [pii]
An DS, Qin FX, Auyeung VC et al (2006) Optimization and functional effects of stable short hairpin RNA expression in primary human lymphocytes via lentiviral vectors. Mol Ther 14(4):494–504. doi:10.1016/j.ymthe.2006.05.015, S1525-0016(06)00213-9 [pii]
Wiznerowicz M, Szulc J, Trono D (2006) Tuning silence: conditional systems for RNA interference. Nat Methods 3(9):682–688. doi:10.1038/nmeth914, nmeth914 [pii]
Dickins RA, Hemann MT, Zilfou JT et al (2005) Probing tumor phenotypes using stable and regulated synthetic microRNA precursors. Nat Genet 37(11):1289–1295. doi:10.1038/Ng1651
Stegmeier F, Hu G, Rickles RJ et al (2005) A lentiviral microRNA-based system for single-copy polymerase II-regulated RNA interference in mammalian cells. Proc Natl Acad Sci U S A 102(37):13212–13217. doi:10.1073/pnas.0506306102, 0506306102 [pii]
Zeng Y, Cullen BR (2005) Efficient processing of primary microRNA hairpins by Drosha requires flanking nonstructured RNA sequences. J Biol Chem 280(30):27595–27603. doi:10.1074/jbc.M504714200, M504714200 [pii]
Xia XG, Zhou H, Xu Z (2006) Multiple shRNAs expressed by an inducible pol II promoter can knock down the expression of multiple target genes. Biotechniques 41(1):64–68, doi:000112198 [pii]
Taxman DJ, Livingstone LR, Zhang J et al (2006) Criteria for effective design, construction, and gene knockdown by shRNA vectors. BMC Biotechnol 6:7. doi:10.1186/1472-6750-6-7, 1472-6750-6-7 [pii]
Bassik MC, Lebbink RJ, Churchman LS et al (2009) Rapid creation and quantitative monitoring of high coverage shRNA libraries. Nat Methods 6(6):443–445. doi:10.1038/nmeth.1330, nmeth.1330 [pii]
Li L, Lin X, Khvorova A et al (2007) Defining the optimal parameters for hairpin-based knockdown constructs. RNA 13(10):1765–1774. doi:10.1261/rna.599107, rna.599107 [pii]
Fellmann C, Zuber J, McJunkin K et al (2011) Functional identification of optimized RNAi triggers using a massively parallel sensor assay. Mol Cell 41(6):733–746. doi:10.1016/j.molcel.2011.02.008, S1097-2765(11)00091-8 [pii]
Geiling B, Vandal G, Posner AR et al (2013) A modular lentiviral and retroviral construction system to rapidly generate vectors for gene expression and gene knockdown in vitro and in vivo. PLoS One 8(10), e76279. doi:10.1371/journal.pone.0076279, PONE-D-13-22537 [pii]
Chang K, Elledge SJ, Hannon GJ (2006) Lessons from Nature: microRNA-based shRNA libraries. Nat Methods 3(9):707–714. doi:10.1038/nmeth923, nmeth923 [pii]
Dow LE, Premsrirut PK, Zuber J et al (2012) A pipeline for the generation of shRNA transgenic mice. Nat Protoc 7(2):374–393. doi:10.1038/nprot.2011.446, nprot.2011.446 [pii]
Li LH, Sen A, Murphy SP et al (1999) Apoptosis induced by DNA uptake limits transfection efficiency. Exp Cell Res 253(2):541–550. doi:10.1006/excr.1999.4666, S0014-4827(99)94666-9 [pii]
Hampf M, Gossen M (2006) A protocol for combined Photinus and Renilla luciferase quantification compatible with protein assays. Anal Biochem 356(1):94–99. doi:10.1016/j.ab.2006.04.046, S0003-2697(06)00307-1 [pii]
Dyer BW, Ferrer FA, Klinedinst DK et al (2000) A noncommercial dual luciferase enzyme assay system for reporter gene analysis. Anal Biochem 282(1):158–161. doi:10.1006/abio.2000.4605, S0003-2697(00)94605-0 [pii]
Kwan KM, Fujimoto E, Grabher C et al (2007) The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs. Dev Dyn 236(11):3088–3099. doi:10.1002/dvdy.21343
Acknowledgements
This work was supported by the Canadian Institutes of Health Research [operating grant MOP-97925 to DD, Graduate scholarship to AdB]; the Canadian Cancer Society [CCSRI# 2011-700876] and the Canadian Cancer Society [CRS #19087]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
de Bruyns, A., Geiling, B., Dankort, D. (2016). Construction of Modular Lentiviral Vectors for Effective Gene Expression and Knockdown. In: Federico, M. (eds) Lentiviral Vectors and Exosomes as Gene and Protein Delivery Tools. Methods in Molecular Biology, vol 1448. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3753-0_1
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3753-0_1
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3751-6
Online ISBN: 978-1-4939-3753-0
eBook Packages: Springer Protocols