Autophagy plays a crucial role in eliminating protein aggregates, damaged organelles and invading pathogens. Genetically engineered cell line stably expressing green fluorescent protein (GFP)-tagged microtubule-associated protein light chain 3 (LC3) is extensively used to test autophagy through observing GFP puncta formation in the cells by fluorescence imaging. However, canine LC3 (cLC3) gene has not been cloned, therefore, GFP-tagged canine LC3 (GFP-cLC3) detection system has not been established. To generate GFP-cLC3 stably expressing canine-derived macrophages, the cLC3 cDNA was first amplified by RT-PCR and inserted into pEGFP-C1 plasmid to create GFP-cLC3 gene fusion. This genetic element was then transducted into canine macrophages mediated by lentivirus vector to generate the canine macrophages stably expressing fusion protein. Results showed that the sequence of cLC3 cloned in this study is highly homologous with other animals (80-95% homology). Phenotypic and functional analysis of these engineered cells revealed that GFP-cLC3 was indeed stably expressed and rapamycin or starvation can effectively induce GFP puncta formation in the cells, indicative of autophagosome formation. These GFP-cLC3-expressing cells may thus be useful to study autophagy in canine.

中文翻译：

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建立稳定表达GFP标签的犬LC3蛋白的犬巨噬细胞，以有效检测自噬。

自噬在消除蛋白质聚集体，受损的细胞器和侵入病原体中起着至关重要的作用。稳定表达绿色荧光蛋白（GFP）标签的微管相关蛋白轻链3（LC3）的基因工程细胞系通过荧光成像观察细胞中GFP点的形成而广泛用于测试自噬。但是，犬LC3（cLC3）基因尚未克隆，因此，尚未建立带有GFP标签的犬LC3（GFP-cLC3）检测系统。为了生成稳定表达犬源巨噬细胞的GFP-cLC3，首先通过RT-PCR扩增cLC3 cDNA，然后将其插入pEGFP-C1质粒中，以创建GFP-cLC3基因融合体。然后将该基因元件转导至由慢病毒载体介导的犬巨噬细胞中，以产生稳定表达融合蛋白的犬巨噬细胞。结果表明，在本研究中克隆的cLC3序列与其他动物高度同源（80-95％同源性）。这些工程细胞的表型和功能分析表明，GFP-cLC3确实稳定表达，雷帕霉素或饥饿可以有效诱导细胞中GFP点的形成，这表明自噬体的形成。这些表达GFP-cLC3的细胞因此可用于研究犬的自噬。