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  2. Visualizing bulk autophagy in vivo by tagging endogenous LC3B

Visualizing bulk autophagy in vivo by tagging endogenous LC3B

  • Autophagy. 2025 Feb 14:1-17. doi: 10.1080/15548627.2025.2457910.
Xiukui Gao 1 Yue Xiong 1 Hangbin Ma 2 Hao Zhou 2 Wei Liu 1 Qiming Sun 1 3
Affiliations

Affiliations

  • 1 Department of Respiratory and Critical Care Medicine, Center for Metabolism Research, The Fourth Affiliated Hospital of Zhejiang University School of Medicine and International School of Medicine, International Institutes of Medicine, Zhejiang University, Yiwu, China.
  • 2 Department of Urology, The Fourth Affiliated Hospital of Zhejiang University School of Medicine and International School of Medicine, International Institutes of Medicine, Zhejiang University, Yiwu, China.
  • 3 Department of Biochemistry, and Department of Cardiology of Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
Abstract

Macroautophagy/Autophagy plays a crucial role in maintaining cellular and organismal health, making the measurement of Autophagy flux in vivo essential for its study. Current tools often depend on the overexpression of Autophagy probes. In this study, we developed a knock-in mouse model, termed tfLC3-KI, by inserting a tandem fluorescent tag coding sequence into the native Map1lc3b gene locus. We found that tfLC3-KI mice exhibit optimal expression of mRFP-eGFP-LC3B, allowing for convenient measurement of autophagic structures and flux at single-cell resolution, both in vivo and in primary cell cultures. Additionally, we compared Autophagy in neurons and glial cells across various brain regions between tfLC3-KI mice and CAG-tfLC3 mice, the latter overexpressing the probe under the strong CMV promoter. Finally, we used tfLC3-KI mice to map the spatial and temporal dynamics of basal Autophagy activity in the reproductive system. Our findings highlight the value of the tfLC3-KI mouse model for investigating Autophagy flux in vivo and demonstrate the feasibility of tagging endogenous proteins to visualize autophagic structures and flux in both bulk and selective Autophagy research in vivo.Abbreviation: BafA1: bafilomycin A1; CQ: chloroquine; EBSS: Earle's balanced salt solution; Es: elongating spermatids; HPF: hippocampalformation; HY: hypothalamus; LCs: leydig cells; OLF: olfactory areas; PepA: pepstatin A; Rs: round spermatids; SCs: sertoli cells; Spc: spermatocytes; Spg: spermatogonia; tfLC3: tandem fluorescently tagged mRFP-eGFP-LC3; TH: thalamus.

Keywords

Autophagy flux; endogenous LC3B; in vivo; knock-in; tandem fluorescence protein.

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