巨噬细胞条件性Atg5基因敲除小鼠的构建及鉴定

Construction and identification of macrophage-conditional Atg5-knockout mice

目的构建并鉴定巨噬细胞条件性Atg5基因敲除小鼠,为研究巨噬细胞自噬在肾脏疾病发病机制中的作用提供动物模型。方法将引进LysM-Cre小鼠与Atg5^flox/+小鼠进行杂交、Atg5^flox/+小鼠自交,分别获得基因型为Atg5^flox/+Cre^+/-和Atg5^flox/flox Cre^-/-的子代小鼠;将上述两种基因型的子代小鼠杂交,得到巨噬细胞条件性Atg5基因敲除小鼠(Atg5^flox/flox Cre^+/-,Atg5^-/-)和对照小鼠(Atg5^flox/flox Cre^-/-,Atg5^+/+)。提取小鼠鼠尾组织基因组DNA,经PCR扩增及琼脂糖凝胶电泳后,在DNA水平判断小鼠基因型;提取小鼠骨髓来源巨噬细胞RNA和蛋白,利用基因测序和Western blot技术检验Atg5基因的敲除效果。结果成功建立巨噬细胞条件性Atg5基因敲除小鼠模型,该小鼠存活并且可育。在基因及蛋白水平上,巨噬细胞Atg5基因敲除成功;且与Atg5^+/+小鼠相比,Atg5^-/-小鼠自噬基础水平低下(p62明显增多,LC3Ⅱ明显减少),对于自噬激活剂雷帕霉素刺激不能恢复到正常基础水平。结论利用Cre/loxp系统,本研究成功构建并鉴定出条件性巨噬细胞Atg5基因敲除小鼠,为在动物水平研究巨噬细胞自噬在肾脏疾病发病机制中的作用提供研究平台。

Objective To construct and identify macrophage-conditional Atg5-knockout mice to provide an animal model for studying the role of macrophage autophagy in the pathogenesis of renal diseases.Methods LysM-Cre mice were hybridized with Atg5^flox/+mice,and Atg5^flox/+mice were self-crossbred to obtain progeny mice with Atg5^flox/+Cre^+/-and Atg5^flox/flox Cre^-/-.The progeny mice of the above two genotypes were then hybridized to obtain macrophage-conditional Atg5 gene-knockout mice(Atg5^flox/flox Cre^+/-,Atg5^-/-)and control mice(Atg5^flox/flox Cre^-/-,Atg5^+/+).The phenotypes of the mice were determined via electrophoresis of DNA that had been extracted from mouse tail tissue and amplified via PCR.Mouse bone marrow-derived macrophage RNA and protein were extracted,and Atg5 gene expression was tested via sequencing and Western blotting.Results A macrophage-conditional Atg5-knockout mouse model was established;these mice survived and were fertile.The macrophage Atg5 gene was successfully knocked out at both the gene and protein levels.The basic macrophage autophagy level in the Atg5^-/-mice was much lower than that in the Atg5^+/+mice(p62 was significantly increased,and LC3Ⅱwas significantly reduced)and could not be restored to the basic level without deleting Atg5 after stimulation with the autophagy activator rapamycin.Conclusions Macrophage-conditional Atg5-knockout mice are successfully constructed and identified via the Cre/loxp system,providing a research platform for studying the role of macrophage autophagy in renal disease pathogenesis at the animal level.