Luciferase Reporter Assay in Drosophila and Mammalian Tissue Culture Cells

Chi Yun1, Ramanuj DasGupta2

1 Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, Skirball Institute, NYU RNAi Core, New York, New York, 2 Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, NYU Cancer Institute, New York, New York
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch130149
Online Posting Date:  March, 2014
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Abstract

Luciferase reporter gene assays are one of the most common methods for monitoring gene activity. Because of their sensitivity, dynamic range, and lack of endogenous activity, luciferase assays have been particularly useful for functional genomics in cell‐based assays, such as RNAi screening. This unit describes delivery of two luciferase reporters with other nucleic acids (siRNA/dsRNA), measurement of the dual luciferase activities, and analysis of data generated. The systematic query of gene function (RNAi) combined with the advances in luminescent technology have made it possible to design powerful whole genome screens to address diverse and significant biological questions. Curr. Protoc. Mol. Biol. 6:7‐23. © 2014 by John Wiley & Sons, Inc.

Keywords: luciferase; reporter gene assay; high‐throughput screening; RNAi

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Reverse Transfection of Clone 8 Cells in 384‐Well Plates
  • Alternate Protocol 1: Reverse Transfection of HEK293T Cells in 384‐Well Plates
  • Basic Protocol 2: Measuring Firefly and Renilla Luciferase Activities in Drosophila and Mammalian Tissue Culture Cells
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Reverse Transfection of Clone 8 Cells in 384‐Well Plates

  Materials
  • dsRNAs of interest (∼0.016 to 0.050 µg/µl dsRNA in water)
  • Drosophila clone 8 cells
  • Shields and Sang M3 insect medium (Sigma, cat. no. S3652)
  • Firefly luciferase reporter DNA (0.1 µg/µl stock) (e.g., Promega pGL3/4 plasmid)
  • Renilla luciferase normalization DNA (0.1 µg/µl stock) (e.g., Promega pRL plasmid)
  • Inducer DNA (0.1 µg/µl stock) (optional)
  • Effectene transfection reagent (Qiaqen, cat. no. 1054250) containing:
    • EC buffer
    • Enhancer
  • Compounds from small‐molecule libraries, optional
  • 384‐well white, solid‐bottom plates (e.g., Corning, cat. no. 3570)
  • 20‐ and 200‐µl pipets and tips
  • Adhesive foil (e.g., Corning, cat. no. 6570)
  • Cell scraper (e.g., BD Falcon, cat. no. 353086)
  • 10‐ml serological pipets
  • 15‐ml conical tube (e.g., Falcon)
  • Benchtop centrifuge (e.g., Eppendorf centrifuge 5810)
  • Hemacytometer
  • 1.5‐ml microcentrifuge tubes
  • Vortex mixer
  • Multichannel pipet (capable of 15‐ to 40‐µl transfers)
  • Reagent reservoirs (e.g., Corning, cat. no. 4870)
  • Benchtop centrifuge equipped with conical and plate adapters (e.g., Eppendorf centrifuge 5810 with swing‐bucket rotor A‐4‐81‐MTP/Flex)
  • 25°C incubator with wet paper towels (humidified chamber)

Alternate Protocol 1: Reverse Transfection of HEK293T Cells in 384‐Well Plates

  Materials
  • HEK 293T cells (ATCC # CRL‐3216)
  • siRNAs (Ambion/Life Technologies, Dharmacon/ThermoFisher, Sigma, Qiagen)
  • Phosphate‐buffered saline (PBS; Life Technologies, cat. no. 10010‐023)
  • Trypsin (Life Technologies, cat. no. R001‐100)
  • Dulbecco's modified Eagle medium (DMEM; Life Technologies, cat. No 11965167)
  • Fetal bovine serum (FBS; Life Technologies, cat.no.16000044)
  • Penicillin‐streptomycin (Life Technologies, cat. no. 15140148)
  • Lipofectamine 2000 transfection reagent (Life Technologies, cat. no. 11668‐019)
  • Opti‐MEM 1 Reduced Serum Medium (Life Technologies, cat. no. 31985‐062)
  • Firefly luciferase reporter DNA (0.1 µg/µl stock; e.g., Promega pGL3/4 plasmid)
  • Renilla luciferase normalization DNA (0.1 µg/µl stock; e.g., Promega pRL plasmid)
  • Compounds from small‐molecule libraries (optional)
  • 384‐well white, solid‐bottom plates (e.g., Corning, cat. no. 3570)
  • 20‐ and 200‐µl pipets and tips
  • Adhesive foil (e.g., Corning, cat. no. 6570)
  • 37°C incubator with 5% CO 2
  • 10‐ml serological pipets
  • 15‐ml and 50‐ml conical tube (e.g., Falcon)
  • Hemacytometer
  • 1.5‐ml microcentrifuge tubes
  • Multichannel pipet (capable of 15‐ to 40‐µl transfers)
  • Reagent reservoirs (e.g., Corning, cat. no. 4870)
  • Benchtop centrifuge equipped with conical and plate adapters (e.g., Eppendorf centrifuge 5810 with swinging bucket rotor A‐4‐81‐MTP/Flex)

Basic Protocol 2: Measuring Firefly and Renilla Luciferase Activities in Drosophila and Mammalian Tissue Culture Cells

  Materials
  • Dual‐Glo luciferase assay system (Promega, cat. no. E2920) containing:
    • Stop & Glo substrate reagent
    • Stop & Glo dilution buffer
  • Plate(s) from protocol 1 or protocol 2Alternate Protocol
  • Serum‐free medium (e.g., DMEM; Life Technologies, cat. no. 11965‐167)
  • Dark container (e.g., covered ice bucket)
  • Benchtop centrifuge equipped with plate adapters (e.g., Eppendorf centrifuge 5810 with swing‐bucket rotor A‐4‐81‐MTP/Flex)
  • 24‐channel wand (e.g., V&P Scientific, cat. no. VP 186L‐1) or multichannel pipet
  • Luminometer or plate reader equipped to read luminescence (e.g., EnVision from Perkin Elmer)
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Figures

Videos

Literature Cited

  Birmingham, A., Selfors, L.M., Forster, T., Wrobel, D., Kennedy, C.J., Shanks, E., Santoyo‐Lopez, J., Dunican, D.J., Long, A., Kelleher, D., Smith, Q., Beijersbergen, R.L., Ghazal, P., and Shamu, C.E. 2009. Statistical methods for analysis of high‐throughput RNA interference screens. Nat. Methods 6:569‐575.
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  Castoreno, A.B., Smurnyy, Y., Torres, A.D., Vokes, M.S., Jones, T.R., Carpenter, A.E., and Eggert, U.S. 2010. Small molecules discovered in pathway screen target the Rho pathway in cytokinesis. Nat. Chem. Biol. 6:457‐463.
  Chen, B., Dodge, M.E., Tang, W., Lu, J., Ma, Z., Fan, C.W., Wei, S., Hao, W., Kilgore, J., Williams, N.S., Roth, M.G., Amatruda, J.F., Chen, C., and Lum, L. 2009. Small molecule‐mediated disruption of Wnt‐dependent signaling in tissue regeneration and cancer. Nat. Chem. Biol. 5:100‐107.
  Cherbas, L. and Cherbas, P. 2000. Drosophila cell culture and transformation. In Drosophila Protocols (W. Sullivan, M. Ashburner, and R.S. Hawley, eds.). Cold Spring Harbor Laboratory Press, New York.
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  de Wet, J.R., Wood, K.V., Helinski, D.R., and DeLuca, M. 1985. Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 82:7870‐7873.
  de Wet, J.R., Wood, K.V., DeLuca, M., Helinski, D.R., and Subramani, S. 1987. Firefly luciferase gene: Structure and expression in mammalian cells. Mol. Cell Biol. 7:725‐737.
  Dougherty, D.C. and Sanders, M.M. 2005. Comparison of the responsiveness of the pGL3 and pGL4 luciferase reporter vectors to steroid hormones. Biotechniques 39:203‐207.
  Friedman, A. and Perrimon, N. 2006. A functional RNAi screen for regulators of receptor tyrosine kinase and ERK signalling. Nature 444:230‐234.
  Gonsalves, F.C., Klein, K., Carson, B.B., Katz, S., Ekas, L.A., Evans, S., Nagourney, R., Cardozo, T., Brown, A.M., and DasGupta, R. 2011. An RNAi‐based chemical genetic screen identifies three small‐molecule inhibitors of the Wnt/wingless signaling pathway. Proc. Natl. Acad. Sci. U.S.A. 108:5954‐5963.
  Gould, S.G., Keller, G.A., and Subramani, S. 1987. Identification of a peroxisomal targeting signal at the carboxy terminus of firefly luciferase. J. Cell Biol. 105:2923‐2931.
  Keller, G.A., Gould, S., Deluca, M., and Subramani, S. 1987. Firefly luciferase is targeted to peroxisomes in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 84:3264‐3268.
  Kingston, R.E. 2001. Stable transfer of genes into mammalian cells. Curr. Protoc. Immunol. 12:10.17.1‐10.17.6.
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  Korinek, V., Barker, N., Willert, K., Molenaar, M., Roose, J., Wagenaar, G., Markman, M., Lamers, W., Destree, O., and Clevers, H. 1998b. Two members of the Tcf family implicated in Wnt/beta‐catenin signaling during embryogenesis in the mouse. Mol. Cell Biol. 18:1248‐1256.
  Leclerc, G.M., Boockfor, F.R., Faught, W.J., and Frawley, L.S. 2000. Development of a destabilized firefly luciferase enzyme for measurement of gene expression. Biotechniques 29:590‐591, 594‐596, 598 passim.
  Nybakken, K., Vokes, S.A., Lin, T.Y., McMahon, A.P., and Perrimon, N. 2005. A genome‐wide RNA interference screen in Drosophila melanogaster cells for new components of the Hh signaling pathway. Nat. Genet. 37:1323‐1332.
  Ramadan, N., Flockhart, I., Booker, M., Perrimon, N., and Mathey‐Prevot, B. 2007. Design and implementation of high‐throughput RNAi screens in cultured Drosophila cells. Nat. Protoc. 2:2245‐2264.
  Rudnicki, S. and Johnston, S. 2009. Overview of liquid handling instrumentation for high‐throughput screening applications. Curr. Protoc. Chem. Biol. 1:43‐54.
  Solberg, N., Machon, O., and Krauss, S. 2012. Characterization and functional analysis of the 5'‐flanking promoter region of the mouse Tcf3 gene. Mol. Cell Biochem. 360:289‐299.
  Thorne, N., Auld, D.S., and Inglese, J. 2010. Apparent activity in high‐throughput screening: origins of compound‐dependent assay interference. Curr. Opin. Chem. Biol. 14:315‐324.
  Warzecha, C.C., Sato, T.K., Nabet, B., Hogenesch, J.B., and Carstens, R.P. 2009. ESRP1 and ESRP2 are epithelial cell‐type‐specific regulators of FGFR2 splicing. Mol. Cell 33:591‐601.
  Wood, K.V., de Wet, J.R., Dewji, N., DeLuca, M. 1984. Synthesis of active firefly luciferase by in vitro translation of RNA obtained from adult lanterns. Biochem. Biophys. Res. Commun. 124:592‐596.
  Zhang, X.H.D., Yang, X.C., Chung, N.J., Gates, A., Stec, E., Kunapuli, P., Holder, D.J., Ferrer, M., and Espeseth, A.S. 2006. Robust statistical methods for hit selection in RNA interference high‐throughput screening experiments. Pharmacogenomics 7:299–309.
  Zhao, H., Lin, W., Kumthip, K., Cheng, D., Fusco, D.N., Hofmann, O., Jilg, N., Tai, A.W., Goto, K., Zhang, L., Hide, W., Jang, J.Y., Peng, L.F., and Chung, R.T. 2012. A functional genomic screen reveals novel host genes that mediate interferon‐alpha's effects against hepatitis C virus. J. Hepatol. 56:326‐333.
Key References
  Gonsalves, F.C. and DasGupta, R. 2008. High‐throughput RNAi screen in Drosophila. Methods Mol. Biol. 469:163‐184.
  This review describes the advantages and limitations of high‐throughput RNAi screening in Drosophila, using a screen for novel modifiers of the Wnt/wg signaling pathway as an example.
  Xhang, X.D. 2011. Optimal High‐Throughput Screening: Practical Experimental Design and Data Analysis for Genome‐Scale RNAi Research. Cambridge University Press, Cambridge.
  This book provides a comprehensive discussion of data analysis for high‐throughput RNAi screening.
  Mohr, S., Bakal, C., and Perrimon, N. 2010. Genomic screening with RNAi: Results and challenges. Annu. Rev. Biochem. 79:37‐64.
  This chapter is an excellent review of high throughput RNAi screening and provides a useful summary table of results from genome‐scale cell‐based high‐throughput screens in Drosophila and mammalian cells.
Internet Resources
  http://www.flyrnai.org/DRSC‐TOO.html
  This site links to the On‐Line Tool Box from the Drosophila RNAi Screening Center at Harvard Medical School.
  http://www.flyrnai.org/DRSC‐PRS.html
  This links to protocols for dsRNA synthesis from the Drosophila RNAi Screening Center at Harvard Medical School.
  http://miare.sourceforge.net/HomePage
  This link for the Minimum Information About an RNAi Experiment provides information about proposed reporting guidelines for data exchange and comparison between datasets.
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