A Knock-in Reporter for a Novel AR-Targeted Therapy
Abstract
Since castration resistance of prostate cancer is often caused by AR overexpression, down-regulation of AR expression using small molecules is a promising yet untested strategy to combat castration-resistant prostate cancer (CRPC). The main objective of this research isto explore a possibility whether the CRISPR-Cas9 technology, an emerging genome-editing approach, could be applied to develop a reliable high-throughput drug screening platform for the identification of small-molecule AR transcriptional inhibitors to treat CRPC. We demonstrated in this report that the CRISPR-Cas9 system could indeed mediate high-efficient insertion of a selection gene into a site immediately downstream of the AR stop codon in castration-resistant C4-2 and 22Rv1 cells. Replacing the selection gene with a firefly luciferase (FLuc) gene led by an internal ribosomal entry site (IRES) through recombinase-mediated cassette exchange, we have successfully developed a C4-2-based recombinant cell line carrying FLuc in the AR gene locus and thereby bicistronically co-expressing the reporter gene with AR under the control of the endogenous AR promoter and enhancer. While the knock-in reporter is expected to faithfully reproduce chemical responses of the endogenous AR gene, we carried out a pilot screen using the NCI Diversity-set library and demonstrated that the genome-edited prostate cancer cells were useful in identifying small molecules inhibitory for AR expression.
Document Details
- Document Type
- Technical Report
- Publication Date
- May 01, 2016
- Accession Number
- AD1013362
Entities
People
- Chunhong Yan