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 especially paramount for its main driver mutation is a fusion transcription factor that binds different motifs than its wild-type constituents. Here, we describe the process of 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 sequences of interest from genomic DNA into a firefly luciferase-containing plasmid, transfecting Ewing sarcoma cells with plasmids and measuring luciferase expression by luminescence. The entire procedure can be completed in 14 days.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2021 |
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| Erschienen: |
2021 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:2226 |
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| Enthalten in: |
Methods in molecular biology (Clifton, N.J.) - 2226(2021) vom: 16., Seite 139-149 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Hölting, Tilman L B [VerfasserIn] |
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| Links: |
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| Themen: |
Enhancer activity |
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| Anmerkungen: |
Date Completed 01.04.2021 Date Revised 01.04.2021 published: Print Citation Status MEDLINE |
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| doi: |
10.1007/978-1-0716-1020-6_10 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM318911906 |
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| 520 | |a 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 especially paramount for its main driver mutation is a fusion transcription factor that binds different motifs than its wild-type constituents. Here, we describe the process of 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 sequences of interest from genomic DNA into a firefly luciferase-containing plasmid, transfecting Ewing sarcoma cells with plasmids and measuring luciferase expression by luminescence. The entire procedure can be completed in 14 days | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Enhancer activity | |
| 650 | 4 | |a Ewing sarcoma | |
| 650 | 4 | |a Fusion-driven sarcoma | |
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| 650 | 4 | |a Luciferase assay | |
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| 650 | 4 | |a Reporter assays | |
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