Characterizing ERRFI1 Enhancer Activity

ERRFI1 enhancer element was PCR amplified and cloned upstream of the minimal promoter of the luciferase reporter plasmid pGL4.23luc2/minP (Promega) as described (46 (link), 47 (link)). Point mutations were introduced using the Q5 Site Directed Mutagenesis (NEB) according to the manufacturer’s instructions (48 (link)). The RD or RH4 tumor cells were transfected with a Renilla control plasmid and the respective pGL4.23 enhancer construct using Viafect transfection reagent (Promega). 48 hours after transfection, reporter activity was quantified using the Dual-Luciferase Reporter Assay System (Promega) as described (49 (link)). Enhancer activity is reported as Firefly luciferase activity normalized to Renilla luciferase.

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Milewski D., Shukla S., Gryder B.E., Pradhan A., Donovan J., Sudha P., Vallabh S., Pyros A., Xu Y., Barski A., Szabo S., Turpin B., Pressey J.G., Millay D.P., Khan J., Kalinichenko V.V, & Kalin T.V. (2021). FOXF1 is required for the oncogenic properties of PAX3-FOXO1 in rhabdomyosarcoma.. Oncogene, 40(12), 2182-2199.

Publication 2021

pGL4.23luc2/minP Viafect transfection reagent Dual-Luciferase Reporter Assay System

Assay Cells Enhancer element Firefly luciferase Luciferase Pgl4 Plasmid Point mutations Promega Renilla Renilla luciferase Site directed mutagenesis Transfection Tumor

Corresponding Organization :

Other organizations : Cincinnati Children's Hospital Medical Center, National Cancer Institute, University of Cincinnati Medical Center

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Variable analysis

independent variables

Point mutations introduced using the Q5 Site Directed Mutagenesis (NEB)

dependent variables

Reporter activity quantified using the Dual-Luciferase Reporter Assay System (Promega)

control variables

Renilla control plasmid
PGL4.23 enhancer construct

controls

Renilla control plasmid (positive control)
Untransfected cells (negative control)

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