Application of mito-priming to generate BCL-2 addicted cells

Lopez, J. and Tait, S. W.G. (2019) Application of mito-priming to generate BCL-2 addicted cells. In: Gavathiotis, E. (ed.) BCL-2 Family Proteins. Series: Methods in molecular biology (1877). Humana Press: New York, pp. 45-60. ISBN 9781493988600 (doi: 10.1007/978-1-4939-8861-7_3)

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.

• Item Type: Book Sections
• Keywords: Apoptosis, BCL-2, BH3 mimetic mito-priming, BH3-only, MOMP, mitochondria.
• Status: Published
• Glasgow Author(s) Enlighten ID: Tait, Professor Stephen and Lopez, Dr Jonathan
• Authors: Lopez, J., and Tait, S. W.G.
• College/School: College of Medical Veterinary and Life Sciences > School of Cancer Sciences
• Journal Name: Methods in molecular biology (Clifton, N.J.)
• Publisher: Humana Press
• ISSN: 1940-6029
• ISBN: 9781493988600