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.
Keywords: Apoptosis; BCL-2; BH3 mimetic mito-priming; BH3-only; MOMP; Mitochondria.