Dec 05, 2024

Public workspacePeroxisome proliferator activated receptor alpha reporter gene assay

DOI
dx.doi.org/10.17504/protocols.io.q26g7mbo1gwz/v1
  • Clementine Garoche1,
  • Jorke H. Kamstra2,
  • Barbara Kubíčková3,
  • Bruce Blumberg4,
  • Sebastian Hoffmann5,
  • Miriam Jacobs3,
  • Patrick Balaguer1
  • 1INSERM;
  • 2Utrecht University;
  • 3UK Health Security Agency;
  • 4University of California Irvine;
  • 5SEH
Jorke H. Kamstra
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DOI: dx.doi.org/10.17504/protocols.io.q26g7mbo1gwz/v1
Protocol Citation: Clementine Garoche, Jorke H. Kamstra, Barbara Kubíčková, Bruce Blumberg, Sebastian Hoffmann, Miriam Jacobs, Patrick Balaguer 2024. Peroxisome proliferator activated receptor alpha reporter gene assay. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7mbo1gwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 18, 2024
Last Modified: December 05, 2024
Protocol Integer ID: 112279
Funders Acknowledgements:
European Commission
Grant ID: 825489
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Abstract
The HG5LN-hPPARα  assay using a reporter gene technique is an in vitro tool that provides mechanistic data. The assay is used to establish signal activation (agonism) or blocking of the hPPARα (antagonism) caused by a ligand. Unliganded chimeric PPARα binds to GAL4RE and transactivates luciferase at a basal level. Once a chemical ligand binds to chimeric PPARα, together they form heterodimers with RXR and recruit cofactors to modulate luciferase transcription. The expression of luciferase is thus directly linked to the concentration of the effector ligand and to its affinity for PPARα. Once the substrate of luciferase, luciferin, is added to the cells, it is possible to assess transactivation by measuring cell luminescence. Luciferase activity can be evaluated quickly and inexpensively with a number of commercially available test kits. The test system for the assessment of the relative hPPARα activation or inhibition by test chemicals provided here utilises the HG5LN-hPPARα cell line.
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SOP_HG5LN-hPPARa.pdf
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