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As the function of autophagy becomes evident in a number of diseases, includingcancer and infection, it is crucial to construct macrophage cell lines with stableexpression of the microtubule-associated protein light chain 3 (GFP-LC3). In thisstudy, a mouse LC3 open-reading frame was amplified by RT-PCR, and cloned intothe pEGFP-C1 plasmid for expression of the GFP-LC3 fusion protein. The recombinantplasmid was transfected into RAW264.7 cells using Lipofectamine 2000 reagent andstably transfected clones were selected by G418 screening. Autophagic puncta formationwas observed by fluorescense microscopy. Additionally, we found that starvationtreatment induced a significant increase in the number of autophagosomes, whilewortmannin treatment significantly repressed the formation of autophagosomes.This study indicated that the RAW264.7 cell line stably expressing GFP-LC3 isavailable for use in a GFP-LC3 puncta formation assay, and may contribute to basicinvestigations of autophagic function or drug screening targeted at autophagy.

A method for the establishment of a cell line with stable expression of the GFP-LC3 reporter protein