CA-ER-TRG mice carry an insertion of a Cre-activated endoplasmic reticulum (ER) lumen-targeting tandem RFP-GFP (CA-ER-TRG) cassette downstream of a floxed STOP cassette inserted into the Igs2 gene. The cassette contains (from 5' to 3') a CAG promoter, a loxP-flanked STOP cassette, and the open reading frame for ssRFP-GFP-KDEL, including a Kozak sequence (GCCACC). ssRFP-GFP-KDEL is a fusion protein that includes tandem mRFP1 and EGFP sequences and the ER retention sequence, KDEL, followed by a polyadenylation signal. In the presence of Cre, the floxed stop cassette is excised, allowing for the expression of the ssRFP-GFP-KDEL cassette. When the dual-fluorescent protein tags are expressed in the ER lumen, they effectively label the ER network without inducing noticeable cytotoxicity. During stressed conditions such as starvation, oncogenic transformation, and tissue injury, ER homeostasis is disrupted, resulting in the accumulation of unfolded proteins in the ER and the subsequent induction of ER stress and ER dysfunction. After stress, redundant or damaged ER membrane structures are eliminated through lysosomal degradation, known as ER-phagy. In this mouse line, the removal of the floxed-STOP cassette leads to expression of the ssRFP-GFP-KDEL, a fusion protein which colocalizes to ER structures. Upon ER-phagy, the RFP- and GFP-labelled ER membrane fragments are transported to the acidic lysosomal environment. The ssRFP-GFP-KDEL protein is then cleaved by lysosomal enzymes to release the RFP and GFP fragments. The fluorescence of GFP is immediately quenched in acidic environments, such as lysosomes, whereas that of mRFP and related fluorescent proteins is relatively stable. In these mice, ER-phagy activity can be measured by relative RFP fragments compared to total RFP-GFP. Homozygous mice are viable and fertile. RFP and GFP are detectable by direct fluorescence, FACS, and western blot. The donating investigator reports no GFP or RFP fluorescence in this mouse line prior to Cre recombination.

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CA-ER-TRG mice carry an insertion of a Cre-activated ER lumen-targeting tandem RFP-GFP (CA-ER-TRG) cassette downstream of a floxed STOP cassette, all inserted into the Igs2 gene. After removal of the floxed-STOP cassette, these mice express tandem mRFP1 and EGFP and the ER retention sequence, KDEL, localized to ER structures. Under ER stress conditions, the GFP signal is quenched in the acidic pH while RFP fluorescence remains. In these mice, ER-phagy activity can be measured by relative free RFP compared to total RFP-GFP.

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