Genome Editing-Enabled HTS Assays Expand Drug Target Pathways for Charcot–Marie–Tooth Disease
| Autor: | Yolanda Santiago, Rajini Srinivasan, Lei Zhang, James Inglese, Rajarshi Guha, Natalia J. Martinez, Ryan MacArthur, John Svaren, John J. Moran, Gregory J. Cost, Sung-Wook Jang, Patricia Dranchak |
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| Jazyk: | angličtina |
| Rok vydání: | 2014 |
| Předmět: |
Genetics
Reporter gene Genome Human High-Throughput Nucleotide Sequencing General Medicine Computational biology Articles Biology Biochemistry Genome editing Transcription (biology) Charcot-Marie-Tooth Disease Peripheral myelin protein 22 Molecular Medicine Humans Human genome Copy-number variation Allele Gene |
| Zdroj: | ACS Chemical Biology |
| ISSN: | 1554-8937 1554-8929 |
| Popis: | Copy number variation resulting in excess PMP22 protein causes the peripheral neuropathy Charcot-Marie-Tooth disease, type 1A. To broadly interrogate chemically sensitive transcriptional pathways controlling PMP22 protein levels, we used the targeting precision of TALEN-mediated genome editing to embed reporters within the genetic locus harboring the Peripheral Myelin Protein 22 (Pmp22) gene. Using a Schwann cell line with constitutively high endogenous levels of Pmp22, we obtained allelic insertion of secreted bioluminescent reporters with sufficient signal to enable a 1536-well assay. Our findings from the quantitative high-throughput screening (qHTS) of several thousand drugs and clinically investigated compounds using this assay design both overlapped and expanded results from a previous assay using a randomly inserted reporter gene controlled by a single regulatory element of the Pmp22 gene. A key difference was the identification of a kinase-controlled inhibitory pathway of Pmp22 transcription revealed by the activity of the Protein kinase C (PKC)-modulator bryostatin. |
| Databáze: | OpenAIRE |
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