Titel
Luminescent and fluorescent triple reporter plasmid constructs for Wnt, Hedgehog and Notch pathway
Autor*in
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Abstract
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.
Stichwort
Notch signalingLuciferasePlasmid constructionTransfection293T cellsHedgehog signalingWnt signaling cascadePolymerase chain reaction
Objekt-Typ
Sprache
Englisch [eng]
Persistent identifier
Erschienen in
Titel
PLOS ONE
Band
14
Ausgabe
12
ISSN
1932-6203
Erscheinungsdatum
2019
Publication
Public Library of Science (PLoS)
Projekt
Kod / Identifikator
uni:docs
Fördergeber
Erscheinungsdatum
2019
Zugänglichkeit
Rechte
Rechteangabe
© 2019 Maier et al
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