Figure 4.
Generation of ER-TRG knock-in mice and assessment of ER-phagy in primary cells derived from the ER-phagy reporter mouse. (A) Schematic representation of the gene targeting strategy illustrating the insertion of ssRFP-GFP-KDEL into the H11 locus to create the ER-TRG mouse model via CRISPR-Cas9 gene editing. The CAG promoter (CMV immediate enhancer/β-actin) and the Kozak sequence (a key eukaryotic mRNA translation initiation site) are highlighted. (B) Whole-mount pictures of E14.5 ER-TRG (+/−) embryos showing the transgenic RFP and GFP fluorescence. Scale bar: 1 mm. (C) Confocal microscopy images showing RFP-GFP fluorescence colocalization with lysosomes in mouse embryonic fibroblast (MEF) cells under mTOR inhibition. MEF cells, isolated from E13.5 ER-TRG (+/−) embryos, were treated with 100 nM Torin1 for 4 h. LysoTracker, a cell-permeable lysosomal marker, was used to identify and trace acidic organelles in live cells. Lysosomes were also marked by LAMP1. Enhanced images demonstrate RFP-only signals colocalizing with lysosomes. The pink arrowheads indicate the ER-phagy sites. Scale bar: 5 μm. (D–F) Confocal microscopy images of RFP and GFP fluorescence in cultured primary cells. Sensory neurons, isolated from dorsal root ganglia (DRG) of E14.5 ER-TRG (+/−) embryos (D), were marked by Tuj1, a neuronal marker. Bone marrow-derived macrophages (BMDMs) (E) and hepatocytes (F) were isolated from adult ER-TRG (+/−) mice. The cells were examined for expression and subcellular localization of the fluorescent proteins. Scale bar: 10 μm. (G–I) Immunoblot analysis showing the expression of ssRFP-GFP-KDEL in primary cells isolated from transgenic mice. Cell extracts from wild-type and ER-TRG transgenic mice were subjected to immunoblotting with specific antibodies. In MEF cells, BMDMs, and hepatocytes, the full-length RFP-GFP was detected in both heterozygotes and homozygotes. Full-length RFP-GFP and RFP fragments were identified using an anti-RFP antibody. The presence of RFP fragments indicates ER-phagy activity, representing the ER-phagy product. GAPDH and Tubulin were used as internal controls. “−” denotes WT, “+” represents heterozygotes, and “++” indicates homozygotes. See also Fig. S2. Source data are available for this figure: SourceData F4.

Generation of ER-TRG knock-in mice and assessment of ER-phagy in primary cells derived from the ER-phagy reporter mouse. (A) Schematic representation of the gene targeting strategy illustrating the insertion of ssRFP-GFP-KDEL into the H11 locus to create the ER-TRG mouse model via CRISPR-Cas9 gene editing. The CAG promoter (CMV immediate enhancer/β-actin) and the Kozak sequence (a key eukaryotic mRNA translation initiation site) are highlighted. (B) Whole-mount pictures of E14.5 ER-TRG (+/−) embryos showing the transgenic RFP and GFP fluorescence. Scale bar: 1 mm. (C) Confocal microscopy images showing RFP-GFP fluorescence colocalization with lysosomes in mouse embryonic fibroblast (MEF) cells under mTOR inhibition. MEF cells, isolated from E13.5 ER-TRG (+/−) embryos, were treated with 100 nM Torin1 for 4 h. LysoTracker, a cell-permeable lysosomal marker, was used to identify and trace acidic organelles in live cells. Lysosomes were also marked by LAMP1. Enhanced images demonstrate RFP-only signals colocalizing with lysosomes. The pink arrowheads indicate the ER-phagy sites. Scale bar: 5 μm. (D–F) Confocal microscopy images of RFP and GFP fluorescence in cultured primary cells. Sensory neurons, isolated from dorsal root ganglia (DRG) of E14.5 ER-TRG (+/−) embryos (D), were marked by Tuj1, a neuronal marker. Bone marrow-derived macrophages (BMDMs) (E) and hepatocytes (F) were isolated from adult ER-TRG (+/−) mice. The cells were examined for expression and subcellular localization of the fluorescent proteins. Scale bar: 10 μm. (G–I) Immunoblot analysis showing the expression of ssRFP-GFP-KDEL in primary cells isolated from transgenic mice. Cell extracts from wild-type and ER-TRG transgenic mice were subjected to immunoblotting with specific antibodies. In MEF cells, BMDMs, and hepatocytes, the full-length RFP-GFP was detected in both heterozygotes and homozygotes. Full-length RFP-GFP and RFP fragments were identified using an anti-RFP antibody. The presence of RFP fragments indicates ER-phagy activity, representing the ER-phagy product. GAPDH and Tubulin were used as internal controls. “−” denotes WT, “+” represents heterozygotes, and “++” indicates homozygotes. See also Fig. S2. Source data are available for this figure: SourceData F4.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal