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Application of Mito-Priming to Generate BCL-2 Addicted Cells

  • Jonathan Lopez
  • Stephen W. G. Tait
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1877)

Abstract

The majority of apoptotic stimuli trigger cell death through the mitochondrial pathway of apoptosis. Invariably, mitochondrial apoptosis requires engagement of mitochondrial outer membrane permeabilization or MOMP to initiate cell death. We have developed a new method, called mito-priming, that allows for rapid and synchronous induction of mitochondrial apoptosis in an on-target manner. Mito-priming uses coexpression of pro- and antiapoptotic Bcl-2 proteins to render cells sensitive to the addition of Bcl-2 targeting BH3-mimetic drugs. This chapter describes how to design mito-priming constructs and apply them to generate mito-primed lines. Second, we describe how to validate cell death sensitivity of mito-primed lines using different methods. Finally, we describe how to generate MOMP-resistant cell lines, using CRISPR-Cas9 mediated deletion of BAX and BAK. Facilitating the investigation of mitochondrial apoptosis, mito-priming provides a clean, robust way to induce mitochondrial apoptosis both in vitro and in vivo.

Key words

Apoptosis Mitochondria MOMP BCL-2 BH3-only BH3 mimetic mito-priming 

Notes

Acknowledgment

This work was funded by Cancer Research UK (C40872/A20145) and Fondation ARC pour la Recherche sur le Cancer and Hospices Civils de Lyon.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.University of Lyon, Cancer Research Centre of Lyon (CRCL), UMR INSERM 1052 CNRS 5286, Léon Bérard CentreLyonFrance
  2. 2.Department of Biochemistry and Molecular BiologyHospices Civils de Lyon, Lyon Sud University HospitalPierre-BéniteFrance
  3. 3.Cancer Research UK Beatson InstituteGlasgowUK
  4. 4.Institute of Cancer SciencesUniversity of GlasgowGlasgowUK

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