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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

To identify novel regulators of transcription factors, we developed an approach to screen arrayed lentiviral or retroviral RNAi libraries using a dual-luciferase-based transcriptional reporter assay. This approach offers a quick and relatively inexpensive way to screen hundreds of candidates in a single experiment.

Abstract

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for anti-cancer therapies. However, directly targeting transcription factors is often difficult and can cause adverse side effects if the transcription factor is necessary in one or more adult tissues. Identifying upstream regulators that aberrantly activate transcription factors in cancer cells offers a more feasible alternative, particularly if these proteins are easy to drug. Here, we describe a protocol that can be used to combine arrayed medium-scale lentiviral libraries and a dual-luciferase-based transcriptional reporter assay to identify novel regulators of transcription factors in cancer cells. Our approach offers a quick, easy, and inexpensive way to test hundreds of genes in a single experiment. To demonstrate the use of this approach, we performed a screen of an arrayed lentiviral RNAi library containing several regulators of Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), two transcriptional co-activators that are the downstream effectors of the Hippo pathway. However, this approach could be modified to screen for regulators of virtually any transcription factor or co-factor and could also be used to screen CRISPR/CAS9, cDNA, or ORF libraries.

Introduction

The purpose of this assay is to use viral libraries to identify regulators of transcription factors in a relatively quick and inexpensive manner. Aberrant transcriptional activity is associated with cancer and metastasis1,2,3,4,5,6, so targeting transcription factors in cancer cells is a promising therapeutic approach. However, transcription factors are often difficult to target pharmacologically7 and many are required for normal cellular function i....

Protocol

NOTE: A schematic summary of this protocol is shown in Figure 1.

1. Lentiviral vector library preparation

NOTE: The demonstrated screen used an arrayed shRNA library purchased as glycerol stocks in 96-well plates, but libraries can also be assembled manually based on a list of candidates. Appropriate controls should be considered and included in any library. This includes a non-targeting control shRNA (shNTC), a control shRNA targeting th.......

Representative Results

Our YAP/TAZ-TEAD reporter construct (pGL3-5xMCAT (SV)-492,14,15) contains a minimal SV-49 promoter with 5 repeats of the canonical TEAD binding element (MCAT)15 driving the firefly luciferase gene (Figure 1). It is co-transfected into cells along with the PRL-TK control vector (Promega), which expresses Renilla luciferase from the con.......

Discussion

In this study, we demonstrate an approach for medium-throughput screening of arrayed viral libraries in combination with a dual-luciferase-based transcriptional reporter assay that can be used to identify and test novel regulators of transcription factors. It is critical to characterize and optimize the reporter system for each cell line prior to any screen. Experiments should be done to confirm that the reporter is responsive to altered activity of the transcription factor being investigated and the magnitude of change .......

Acknowledgements

We would like to thank Emily Norton and Mikaelan Cucciarre-Stuligross for assisting in the preparation of shRNA vectors. This work was supported in part by a Susan G. Komen Career Catalyst Grant that awarded to J.M.L. (#CCR17477184).

....

Materials

NameCompanyCatalog NumberComments
2.0 ml 96-well deep well polypropylene plateUSA Scientific1896-2000For bacterial mini-prep
Trypsin - 2.50%Gibco15090-046Component of trypsin-EDTA
96 well flat bottom white assay plateCorning3922For dual-luciferase assay
Ampicillin - 100 mg/mlSigma-Aldrich45-10835242001-EAFor bacterial mini-prep
Bacto-tryptone - powderSigma-Aldrich95039Component of LB broth
Dual-luciferase reporter assay system, which include LAR II reagent (reagent A), Stop & Glo substrate (reagent B substrate) and Stop & Glo buffer (reagent B buffer) - KitPromegaE1960For dual-luciferase assay
Dulbecco's phosphate buffered saline w/o calcium, magnesium and phenol red - 9.6 g/LHimediaTS1006For PBS
EDTA - 0.5 MVWR97061-406Component of trypsin-EDTA
Ethanol - 100%Pharmco-AAPER111000200For bacterial mini-prep
Foetal Bovine Serum - 100%VWR97068-085Component of complete growth media
Hexadimethrine bromide (Polybrene) - 8 mg/mlSigma-Aldrich45-H9268For virus infection
HyClone DMEM/High glucose - 4 mM L-Glutamine; 4500 mg/L glucose; sodium pyruvateGE Healthcare life sciencesSH30243.01Component of complete growth media
I3-P/i3 Multi-Mode Microplate/EAMolecular devicesFor dual-luciferase assay
L-Glutamine - 200 mMGibco25030-081Component of complete growth media
Lipofectamine 3000 (Transfection Reagent 2) - 100%Life technologiesL3000008For transfections
Molecular Biology Water - 100%VWR02-0201-0500For dilution of shRNA vector for virus packaging
NaCl - powderBDHBDH9286Component of LB broth
NanoDrop One Microvolume UV-Vis SpectrophotometerThermo scientificFor measuring vector DNA concentration
Opti-MEM (Transfection Buffer) - 100%Gibco31985-062For transfections
Penicillin Streptomycin - 10,000 Unit/ml (Penicillin); 10,000 µg/ml (Streptomycin)Gibco15140-122Component of complete growth media
PureLink Quick Plasmid Miniprep Kit - KitThermo Fisher ScientificK210010For bacterial mini-prep
Puromycin - 2.5 mg/mlSigma-Aldrich45-P7255For antibiotic selection after infection
TC20 automated cell counterBio-RadFor cell counting
X-tremeGENE 9 DNA transfection reagent (Transfection Reagent 1) - 100%Roche6365787001For virus packaging
Yeast extract - powderVWRJ850Component of LB broth
P3000 (Transfection Reagent 3) - 100%Life technologiesL3000008For transfections

References

  1. Chen, K. S., Lim, J. W. C., Richards, L. J., Bunt, J. The convergent roles of the nuclear factor I transcription factors in development and cancer. Cancer Letters. 410, 124-138 (2017).
  2. Lamar, J. M., et al.

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