Abstract
Measurement of gene expression for high-throughput screening is an increasingly used technique that has been developed for not only gene dosage disorders resulting from disease-associated copy number variations, but also for induction/repression of genes modulating the severity of a disease phenotype. Traditional methods have employed transient or stable transfection of reporter constructs in which a single reporter is driven by selected regulatory elements from the candidate gene. However, individual regulatory elements are inherently unable to capture the integrated regulation of multiple enhancers at the endogenous locus, and random reporter insertion can result in neighborhood effects that impact the physiological responsiveness of the reporter. Therefore, we outline a general method of employing genome editing to insert reporters into the 3′ UTR of a candidate gene, which has been used successfully in our studies of the Pmp22 gene associated with Charcot–Marie–Tooth disease. The method employs genome editing to insert two nonhomologous reporters that maximize the efficiency of identification of biologically active molecules through concordant responses in small molecule screening. We include a number of aspects of the design and construction of these reporter assays that will be applicable to creation of similar assays in a variety of cell types.
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Dranchak, P., Moran, J.J., MacArthur, R., Lopez-Anido, C., Inglese, J., Svaren, J. (2018). Genome-Edited Cell Lines for High-Throughput Screening. In: Damoiseaux, R., Hasson, S. (eds) Reporter Gene Assays. Methods in Molecular Biology, vol 1755. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7724-6_1
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DOI: https://doi.org/10.1007/978-1-4939-7724-6_1
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