Establishment of canine macrophages stably expressing GFP-tagged canine LC3 protein for effectively detecting autophagy
Introduction
Autophagy, a self-digestion process, involves the formation of isolated growing double membrane that sequesters/recognizes intracellular aggregated proteins, damaged organelles as well as inviding pathogens and promotes their clearance through a lysosome-dependent path [1,2]. Cells under starvation utilize autophagy to dispose and recycle unessential and/or misfolded proteins to generate necessary nutrients (e.g amino acids) for essential processes [3]. Another function of autophagy is to remove harmful proteins derived either from self or invading pathogens [4]. Furthermore, autophagy also plays a role during development and negatively affects aging [5]. A comprehensive analysis of autophagy function is thus of great importance, both physiologically and pharmacologically. Consistent with this notion, defects in autophagy contribute to the pathogenesis of many major human diseases, including cancerous [6,7] autoimmune, degenerative [8] and infectious diseases [[9], [10], [11]].
The microtubule-associated protein light chain 3 (LC3) protein is an autophagosomal membrane marker that is usually dispersed throughout the cytoplasm [12]. The cytoplasmic form of LC3 (LC3-I) is conjugated to phosphatidylethanolamine to produce the LC3-phosphatidylethanolamine conjugate (LC3-II), which is recruited to autophagosomal membranes upon starvation [13,14] lack of growth factors [15] or immune factors [16] and certain intracellular pathogens [4,17,18] and is involved in the formation of autophagosomes. Since LC3-II is involved in the formation of autophagosomal membranes, the green fluorescent protein (GFP)-fused LC3 gene transduced cells was widely used to detect the autophagosomes formation by observing GFP puncta formation. Although many GFP-tagged LC3 fusion proteins from the different species, including human [19], mouse [20] and other animals [[21], [22], [23]] were generated and widely used, canine version of GFP-tagged canine LC3 (GFP-cLC3) protein has not been constructed. In this study, canine LC3 gene was first amplified by RT-PCR and its GFP-tagged fusion gene was constructed and transduced into canine macrophages mediated by lentivirus vector. After puromycin selection, the genetically engineered canine macrophages that stably express GFP-cLC3 fusion protein were constructed. Upon starvation and paramysin stimulation, autophagy was induced as shown by the formation of GFP puncta in canine macrophages.
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Plasmids, cells and bacteria
Lentivirus plasmid vectors including cloning vector pFLRcmv-Ypet-puro and packaging vectors (psPAX2 and pMD2G) were gifts from Dr. Jinxin Wang (School of Medicine, University of California, San Diego, USA). The pEGFP-C1 plasmid containing GFP gene was from Clontech Laboratories (Palo Alto, CA, USA) and pcDNA3.1A plasmid, a eukaryotic expression vector, was from Invitrogen (Carlsbad, CA, USA). Human 293T cells, canine DH82 macrophages and Madin-Darby canine kidney (MDCK) cells were from American
Molecular cloning and sequence analysis of cLC3 cDNA
So far, the cLC3 cDNA has not been cloned although its predicted sequence is available in GenBank (XM_858761). To confirm its cDNA sequence and compare it with that of other animals, we have cloned cLC3 cDNA from MDCK cells by RT-PCR (Fig. 1A). Sequence analysis showed that the cLC3 cDNA was completely consistent with that of the prediction in GenBank (Fig. 1B). By comparing the cLC3 cDNA sequence with that of other animals, it can be observed that the LC3 genes from all species are very
Discussion
In this study, we generated canine macrophage cell line stably expressing GFP-cLC3 fusion protein by lentivirus-mediated gene transduction, and verified the autophagosome formation in these cells upon starvation and rapamycin treatment. This study thus provided a useful tool for canine autophagy detection.
Autophagy is a multistep complex process involving initation, nucleation, elongation and closure, and fusion with lysosome [24,25]. Many autophagy-related proteins (Atgs) have been identified
Funding
This study was supported by funding from the National Key Reseach Project, China (No. 2016YFD0501002) and the Research Project of Hebei University Science and Technology, Hebei, China (QN2016189).
CRediT authorship contribution statement
Dan Cui: Methodology, Investigation, Data curation, Writing - original draft. Shanshan Huo: Methodology, Investigation. Xing Wang: Methodology, Investigation. Zhiqiang Zheng: Investigation. Yonghong Zhang: Investigation. Jianlou Zhang: Investigation, Supervision, Funding acquisition. Fei Zhong: Conceptualization, Supervision, Writing - review & editing, Funding acquisition, Project administration.
Declaration of competing interest
The authors declared no conflict of interest.
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