Dec 07, 2024

Public workspacePeroxisome proliferator activated receptor gamma reporter gene assay

DOI
https://dx.doi.org/10.17504/protocols.io.yxmvm9x19l3p/v1
  • Clementine Garoche1,
  • Jorke H. Kamstra2,
  • Barbara Kubíčková3,
  • Bruce Blumburg4,
  • Sebastian Hoffmann5,
  • Miriam Jacobs3,
  • Patrick Balaguer1
  • 1INSERM;
  • 2Utrecht University;
  • 3UK Health Security Agency;
  • 4University of California Irvine;
  • 5seh consulting + services
Jorke H. Kamstra
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DOI: https://dx.doi.org/10.17504/protocols.io.yxmvm9x19l3p/v1
Protocol Citation: Clementine Garoche, Jorke H. Kamstra, Barbara Kubíčková, Bruce Blumburg, Sebastian Hoffmann, Miriam Jacobs, Patrick Balaguer 2024. Peroxisome proliferator activated receptor gamma reporter gene assay. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm9x19l3p/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 15, 2024
Last Modified: December 07, 2024
Protocol Integer ID: 112160
Keywords: PPAR gamma transactivation, in vitro, metabolism disruption, relative hpparγ activation by test chemical, relative hpparγ activation, hpparγ cell line, receptor gamma reporter gene, hpparγ, peroxisome proliferator, chimeric pparγ, unliganded chimeric pparγ, luciferase activity, blocking of the hpparγ, pparγ, substrate of luciferase, expression of luciferase, reporter gene technique, measuring cell luminescence, assay, cell luminescence, luciferase, concentration of the effector ligand, test chemical, effector ligand, hg5ln
Funders Acknowledgements:
European Commission
Grant ID: 825489
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Abstract
The HG5LN-hPPARγ  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 hPPARγ (antagonism) caused by a ligand. Unliganded chimeric PPARγ binds to GAL4RE and transactivates luciferase at a basal level. Once a chemical ligand binds to chimeric PPARγ, 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. 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 hPPARγ activation by test chemicals provided here utilises the HG5LN-hPPARγ cell line.
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SOP_HG5LN-hPPARg.pdf
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