Visualizing bulk autophagy <i>in vivo</i> by tagging endogenous LC3B

Macroautophagy/autophagy plays a crucial role in maintaining cellular and organismal health, making the measurement of autophagy flux <i>in vivo</i> 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 <i>Map1lc3b</i> 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 <i>in vivo</i> 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 <i>in vivo</i> and demonstrate the feasibility of tagging endogenous proteins to visualize autophagic structures and flux in both bulk and selective autophagy research <i>in vivo</i>. <b>Abbreviation</b>: 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.