Topflash plasmid

Manufactured by Addgene

The TOPFlash plasmid is a reporter construct used to measure the activity of the Wnt signaling pathway. It contains multiple copies of the TCF/LEF transcriptional response element upstream of a minimal promoter driving the expression of the firefly luciferase gene.

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Lab products found in correlation

Fbs dmem medium, by Thermo Fisher Scientific (1 mentions) Varioskan flash, by Thermo Fisher Scientific (1 mentions) Wnt3a, by Thermo Fisher Scientific (1 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions) Dual luciferase reporter assay system, by Promega (1 mentions)

Spelling variants of this product

TOPFlash (64 citations) M50 Super 8x TOPFlash plasmid (3 citations) TOPFlash or FOPFlash plasmids (2 citations) M50 Super 8× TOPFlash plasmid (2 citations) TOPFlash luciferase reporter plasmid (2 citations) TOPflash reporter plasmid (2 citations)

3 protocols using topflash plasmid

Human iPSC Wnt Signaling Assay

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Human iPSCs were cultured in RPMI/B27 differentiation culture, and HEK293 cells cultured in 10% FBS‐DMEM medium (Gibco, Gaithersburg, MD). Cells were transfected with TOPflash plasmid (Addgene, Cambridge, MA) and renila plasmid as internal reference. After 24 hours, CHIR, CHIR plus rapamycin, rapamycin, and mouse Wnt3a (100 ng/mL) were added to the medium. After an additional 24 hours, the luciferase activities were measured by a laser‐scanning Varioskan Flash (Thermo Fisher).

Qiu X., Liu Y., Zhang Y., Guan Y., Jia Q., Wang C., Liang H., Li Y., Yang H., Qin Y., Huang S., Zhao X, & Jing Q. (2017). Rapamycin and CHIR99021 Coordinate Robust Cardiomyocyte Differentiation From Human Pluripotent Stem Cells Via Reducing p53‐Dependent Apoptosis. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 6(10), e005295.

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NF-κB and Wnt Pathway Activation Assay

Cited 1 time

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1×10⁵ cells were seeded in 12-well cell culture plates, and transfected with a total of 1.6 μg of plasmids including reporter, expression and pCMV-β-galactosidase plasmids using Lipofectamine 2000 (14 (link)). NF-κB-luc (Igκ2-IFN-luc wt or mut) was a kind gift of Dr. Joel L. Pomerantz (15 (link)). The TOPFlash plasmid (16 (link)) was purchased from Addgene.

Sadik H., Korangath P., Nguyen N., Gyorffy B., Kumar R., Hedayati M., Teo W.W., Park S., Panday H., Munoz T.G., Menyhart O., Shah N., Pandita R.K., Chang J.C., DeWeese T., Chang H.Y., Pandita T.K, & Sukumar S. (2016). HOXC10 expression supports the development of chemotherapy resistance by fine tuning DNA repair in breast cancer cells. Cancer research, 76(15), 4443-4456.

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Wnt Pathway Activation in Muscle Stem Cells

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After overnight treatment with PFI-2 (5 µM) , expanding MuSCs were transfected with TOPFlash plasmid (Addgene #12456) using Lipofectamine 3000 (Life Technologies) following the manufacturer’s instructions. MuSCs were then treated with Wnt3a (Peprotech) overnight at indicated concentrations before being lysed and Firefly/Renilla luciferase activity quantified by luminescence using a Dual-Luciferase® Reporter Assay System (Promega) following the manufacturer’s instructions.

Judson R.N., Quarta M., Oudhoff M.J., Soliman H., Yi L., Chang C.K., Lou G., Werff R.V., Cait A., Hamer M., Blonigan J., Paine P., Doan L.T., Groppa E., He W., Su L., Zhang R.H., Xu P., Eisner C., Low M., Barta I., Lewis C.A., Zaph C., Karimi M.M., Rando T.A, & Rossi F.M. (2018). Inhibition of methyltransferase Setd7 allows the in vitro expansion of myogenic stem cells with improved therapeutic potential. Cell stem cell, 22(2), 177-190.e7.

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