Visualizing bulk autophagy in vivo by tagging endogenous LC3B

Area of Science:

• Cell Biology
• Molecular Biology
• Genetics

Background:

• Macroautophagy (autophagy) is vital for cellular and organismal health.
• Accurate measurement of autophagy flux in vivo is crucial for research.
• Existing methods often rely on overexpressed autophagy probes.

Purpose of the Study:

• To develop a novel knock-in mouse model for studying autophagy flux.
• To enable convenient and high-resolution measurement of autophagic structures and flux.
• To investigate autophagy dynamics in the brain and reproductive system.

Main Methods:

• Developed a knock-in mouse model (tfLC3-KI) by inserting a tandem fluorescent tag into the native Map1lc3b gene locus.
• Expressed mRFP-eGFP-LC3B in tfLC3-KI mice for visualizing autophagic structures and flux.
• Compared tfLC3-KI mice with CAG-tfLC3 mice (overexpressing the probe) and mapped autophagy in the reproductive system.

Main Results:

• tfLC3-KI mice exhibit optimal expression of mRFP-eGFP-LC3B for single-cell resolution analysis.
• The model allows for convenient measurement of autophagy flux both in vivo and in primary cell cultures.
• Demonstrated the utility of tfLC3-KI mice for mapping basal autophagy activity in the reproductive system.

Conclusions:

• The tfLC3-KI mouse model is valuable for investigating autophagy flux in vivo.
• Tagging endogenous proteins provides a feasible method to visualize autophagic structures and flux.
• This approach supports research in both bulk and selective autophagy.