Luciferase Assay for MYC Promoter Regulation

The luciferase reporter vector incorporating fragments of the MYC promoter into the pGL4 vector (Promega) was utilized [16 (link)]. The CRISPR activation system (plenti-dCas9-VP64, plenti-MS2-p65-HSF1, and pE1-U6-gRNA-MS2 [28 (link)] of the individual candidate genes) or pT3.5 overexpression vectors were transfected to HEK293T cells expressing the luciferase reporter vector pRL Renilla using the Lipofectamine 3000 reagent (Invitrogen). Cells were collected 48 h after transduction. Luciferase activity was measured by following the protocol of the dual-luciferase reporter assay system (Promega) and Lumat LB9507 (Perkin Elmer). The luciferase activity values were standardized with the luciferase activity value of pE1-h-HPRT.

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Tanaka Y., Kambayashi H., Yamamoto A., Onishi I., Sugita K., Matsumura M., Ishibashi S., Ikeda M., Yamamoto K., Kitagawa M, & Kurata M. (2022). Efficient Identification of the MYC Regulator with the Use of the CRISPR Library and Context-Matched Database Screenings. International Journal of Molecular Sciences, 23(14), 7723.

Publication 2022

pGL4 vector Lipofectamine 3000 reagent dual-luciferase reporter assay system Lumat LB9507

Assay Cells Crispr Genes Lipofectamine Luciferase Pgl4 Promega Renilla Vector

Corresponding Organization : Tokyo Medical and Dental University

Other organizations : Tokyo Medical University

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Variable analysis

independent variables

CRISPR activation system (plenti-dCas9-VP64, plenti-MS2-p65-HSF1, and pE1-U6-gRNA-MS2 [28 (link)] of the individual candidate genes)
PT3.5 overexpression vectors

dependent variables

Luciferase activity

control variables

HEK293T cells expressing the luciferase reporter vector pRL Renilla
Lipofectamine 3000 reagent (Invitrogen)
Luciferase activity value of pE1-h-HPRT

positive controls

PE1-h-HPRT

Annotations

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