Analysis of Regulatory DNA Sequences by Dual-Luciferase Reporter Assays in Ewing Sarcoma

Reporter gene assays allow for examining the influence of regulatory DNA sequences on the transcription of target genes. In Ewing sarcoma, the study of these DNA sequences is paramount because the disease's main driver mutation is a fusion transcription factor that binds distinct motifs compared with its wild-type constituents. Here, we describe a workflow for analyzing the enhancer activity of regulatory DNA sequences using transfection-based dual-luciferase reporter assays in Ewing sarcoma cell lines. To this end, we provide a protocol for cloning genomic sequences into a firefly luciferase-containing plasmid, co-transfecting Ewing sarcoma cells with firefly reporter and Renilla control plasmids and measuring luciferase expression by luminescence. The entire procedure can be completed in 14 days.