Dynamic chromatin organization: Role in development and disease

Кузнецова, Т. Н.; Stunnenberg, Hendrik G.

doi:10.1016/j.biocel.2016.05.006

Cited by 4 publications

(1 citation statement)

References 29 publications

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“…Since the development of luciferase assays that enabled rapid readout of gene expression with a high dynamic range [1], they have been used in a wide variety of screening experiments. When employed in small molecule screening assays, luciferase constructs were often generated with fusions to candidate gene promoters and/or regulatory elements [2,3] While this strategy is informative, it is common for genes to be regulated by multiple enhancers [4], which are often generally dispersed at locations quite far away from the promoter, either upstream, downstream or within introns [5–9]. Given that selection of candidate regulatory elements is often based on incomplete information, it would be advantageous to embed reporter genes within the candidate locus.…”

Section: Introductionmentioning

confidence: 99%

Genome-Edited Cell Lines for High-Throughput Screening

Dranchak

Moran

MacArthur

et al. 2018

Methods in Molecular Biology

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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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