Generating ER-TRG and CA-ER-TRG Knock-in Mice and Quantitative In Vivo Imaging of ER-phagy.

ER-phagy, a selective autophagy process crucial for maintaining cellular homeostasis by targeting the endoplasmic reticulum (ER), has been challenging to study in vivo due to the lack of suitable spatiotemporal quantification tools. Existing methods like electron microscopy, biochemical assays, and in vitro reporters lack resolution, scalability, or physiological relevance. Here, we present a detailed protocol for generating two transgenic mouse models: ER-TRG (constitutively expressing an ER lumen-targeting tandem RFP-GFP tag) and CA-ER-TRG (Cre-recombinase-activated ER-TRG). Additionally, we outline procedures for quantitative imaging of ER-phagy in vivo, covering tissue preparation, confocal microscopy, and signal analysis. This protocol offers a robust and reproducible tool for investigating ER-phagy dynamics across various tissues, developmental stages, and pathophysiological conditions, facilitating both fundamental and translational research. Key features • Enables live, single-cell resolution imaging of ER-phagy dynamics across intact tissues in mice. • Features a Cre-recombinase-activated knock-in model (CA-ER-TRG) for spatiotemporally controlled ER-phagy studies in specific cell types. • Quantifies ER-phagy flux via pH-sensitive RFP-GFP signal ratiometry and lysosomal co-localization in vivo.