Luminescent and fluorescent triple reporter plasmid constructs for Wnt, Hedgehog and Notch pathway


Autoři: Julia Maier aff001;  Salma Elmenofi aff001;  Alexander Taschauer aff001;  Martina Anton aff002;  Haider Sami aff001;  Manfred Ogris aff001
Působiště autorů: Laboratory of MacroMolecular Cancer Therapeutics (MMCT), Center of Pharmaceutical Sciences, Department of Pharmaceutical Chemistry, University of Vienna, Althanstrasse, Vienna, Austria aff001;  Institutes of Molecular Immunology and Experimental Oncology, Klinikum rechts der Isar, Technische Universität München, Munich, Germany aff002
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article
doi: 10.1371/journal.pone.0226570

Souhrn

Tracking the activity of signalling pathways is a fundamental method for basic science, as well as in cancer- and pharmaceutical research. The developmental pathways Wnt, Hedgehog and Notch are frequently deregulated in cancers and represent a valuable target for the discovery of novel anticancer compounds. Here we present reporter systems for tracking activity of these pathways by using specific promoter elements driving the expression of either sensitive luciferases or fluorescent proteins. A high level of sensitivity was obtained using the luciferase reporter genes for firefly (FLuc), secreted Gaussia (GLuc) and synthetic NanoLuc (NLuc). As fluorescent reporter proteins, mTurqouise2, tdTomato and iRFP720 were chosen. Specificity of pathway activity was validated by co-transfection with pathway activating genes, showing significant response to induction. In addition, multi-gene plasmids were cloned, allowing the detection of all three pathways by one vector. By using the multi-gene vector 3P-Luc (wnt-NLuc, hedgehog-FLuc, Notch-GLuc), we could unambiguously demonstrate the crosstalk between pathways, while excluding cross reactivity of luciferase substrates. First studies with synthetic compounds confirmed the applicability of the system for future drug screening approaches.

Klíčová slova:

293T cells – Hedgehog signaling – Luciferase – Notch signaling – Plasmid construction – Polymerase chain reaction – Transfection – Wnt signaling cascade


Zdroje

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