Abstract
Transgene-based reporter gene assays have been used for discovery of inhibitors targeting vital gene transcription. In traditional assays, the reporter gene is commonly fused with a cloned promoter and integrated into a random genomic location. This has been widely applied but significantly dampened by disadvantages, including incomplete cis-acting elements, the influence of foreign epigenetic environments, and generation of false hits that disrupt the luciferase reporter activity. Therefore, there is a need to develop novel strategies for developing in situ reporter assays closely mimicking endogenous gene expression without disrupting its function. By employing the CRISPR-Cas9 system, we developed an effective in situ coincidence reporter system with a selection marker in the endogenous locus of ATAD3A, which provides a means of screening for transcription-targeted lead compounds with high confidence.
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References
Chau N-M, Rogers P, Aherne W, Carroll V, Collins I, McDonald E et al (2005) Identification of novel small molecule inhibitors of hypoxia-inducible factor-1 that differentially block hypoxia-inducible factor-1 activity and hypoxia-inducible factor-1α induction in response to hypoxic stress and growth factors. Cancer Res 65(11):4918–4928
Lang L, Ding H-F, Chen X, Sun S-Y, Liu G, Yan C (2015) Internal ribosome entry site-based bicistronic in situ reporter assays for discovery of transcription-targeted lead compounds. Chem Biol 22(7):957–964
Dansithong W, Paul S, Scoles DR, Pulst SM, Huynh DP (2015) Generation of SNCA cell models using zinc finger nuclease (ZFN) technology for efficient high-throughput drug screening. PLoS One 10(8):e0136930. https://doi.org/10.1371/journal.pone.0136930
Hasson SA, Fogel AI, Wang C, MacArthur R, Guha R, Heman-Ackah S et al (2015) Chemogenomic profiling of endogenous PARK2 expression using a genome-edited coincidence reporter. ACS Chem Biol 10(5):1188–1197
Yang W, Zhang S, Zhang Y, Huang X (2017) A novel strategy to dissect endogenous gene transcriptional regulation in live cells. Biochem Biophys Res Commun 487(3):573–579
Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8(11):2281–2308
Auld DS, Southall NT, Jadhav A, Johnson RL, Diller DJ, Simeonov A et al (2008) Characterization of chemical libraries for luciferase inhibitory activity. J Med Chem 51(8):2372–2386
Cheng KC, Inglese J (2012) A coincidence reporter-gene system for high-throughput screening. Nat Methods 9(10):937. https://doi.org/10.1038/nmeth.2170
Schuck BW, MacArthur R, Inglese J (2017) Quantitative high-throughput screening using a coincidence reporter biocircuit. Curr Protoc Neurosci 79:5.32.1–5.32.27. https://doi.org/10.1002/cpns.27
Teng Y, Ren X, Li H, Shull A, Kim J, Cowell JK (2016) Mitochondrial ATAD3A combines with GRP78 to regulate the WASF3 metastasis-promoting protein. Oncogene 35(3):333–343
Dickerson T, Jauregui CE, Teng Y (2017) Friend or foe? Mitochondria as a pharmacological target in cancer treatment. Future Med Chem 9(18):2197–2210
Fang H-Y, Chang C-L, Hsu S-H, Huang C-Y, Chiang S-F, Chiou S-H et al (2010) ATPase family AAA domain-containing 3A is a novel anti-apoptotic factor in lung adenocarcinoma cells. J Cell Sci 123(Pt 7):1171–1180
Acknowledgments
This research was supported by NIH grant R03DE028387Â and R01DE028351 (to Y.T.).
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Lang, L., Teng, Y. (2020). Using Genome-Editing Tools to Develop a Novel In Situ Coincidence Reporter Assay for Screening ATAD3A Transcriptional Inhibitors. In: Guest, P. (eds) Clinical and Preclinical Models for Maximizing Healthspan. Methods in Molecular Biology, vol 2138. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0471-7_8
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DOI: https://doi.org/10.1007/978-1-0716-0471-7_8
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