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Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis
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Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Published on: December 12, 2019

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

Julia Maier1, Salma Elmenofi1, Alexander Taschauer1

  • 1Laboratory of MacroMolecular Cancer Therapeutics (MMCT), Center of Pharmaceutical Sciences, Department of Pharmaceutical Chemistry, University of Vienna, Althanstrasse, Vienna, Austria.

Plos One
|December 21, 2019
PubMed

Insights

This study introduces novel reporter systems to track Wnt, Hedgehog, and Notch signaling pathways. These systems are valuable tools for cancer research and drug discovery, enabling sensitive detection and pathway crosstalk analysis.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Oncology

Background:

  • Signaling pathways like Wnt, Hedgehog, and Notch are crucial in development and frequently altered in cancer.
  • Targeting these deregulated pathways is a key strategy for novel anticancer drug discovery.
  • Sensitive and specific methods are needed to monitor pathway activity in research and drug screening.

Purpose of the Study:

  • To develop and validate novel reporter systems for tracking Wnt, Hedgehog, and Notch signaling pathway activity.
  • To enable sensitive detection of pathway activation using luciferase and fluorescent reporters.
  • To create multi-gene vectors for simultaneous monitoring of multiple pathways and their interactions.

Main Methods:

  • Utilized specific promoter elements to drive expression of sensitive luciferase (firefly, Gaussia, NanoLuc) and fluorescent (mTurqouise2, tdTomato, iRFP720) reporter genes.
  • Validated pathway specificity through co-transfection with pathway-activating genes.
  • Constructed multi-gene plasmids, including the 3P-Luc vector, for simultaneous pathway detection and crosstalk analysis.

Main Results:

  • Achieved high sensitivity in tracking pathway activity using selected luciferase and fluorescent reporter proteins.
  • Demonstrated significant pathway response to induction, confirming reporter system specificity.
  • Successfully employed a multi-gene vector (3P-Luc) to reveal crosstalk between Wnt, Hedgehog, and Notch pathways without substrate cross-reactivity.
  • Confirmed the system's utility in preliminary drug screening studies with synthetic compounds.

Conclusions:

  • Developed sensitive and specific reporter systems for monitoring Wnt, Hedgehog, and Notch signaling pathways.
  • The multi-gene vector approach effectively allows for the study of pathway crosstalk, crucial for understanding cancer biology.
  • These reporter systems represent a valuable platform for future anticancer drug discovery and screening efforts.

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