动物造模,基因敲除 z-bio 2021-11-10 693

　　Objective: To construct and identify macrophage conditional Atg5 gene knockout mice, and provide an animal model for studying the role of macrophage autophagy in the pathogenesis of kidney disease.

　　Method: Cross the introduced LysM-Cre mice with Atg5flox/+ mice and self-bred Atg5flox/+ mice to obtain the progeny mice with the genotypes Atg5flox/+Cre+/- and Atg5flox/floxCre-/- respectively; The offspring mice of the above two genotypes were crossed to obtain macrophage conditional Atg5 knockout mice (Atg5flox/floxCre+/-, Atg5-/-) and control mice (Atg5flox/floxCre-/-, Atg5+ /+). Extract the genomic DNA from mouse tail tissue, after PCR amplification and agarose gel electrophoresis, determine the mouse genotype at the DNA level; extract the mouse bone marrow-derived macrophage RNA and protein, and use gene sequencing and Western blot technology to test Knockout effect of Atg5 gene.

　　Result: The macrophage conditional Atg5 gene knockout mouse model was successfully established, and the mouse was alive and fertile. At the gene and protein level, the macrophage Atg5 gene was successfully knocked out; and compared with Atg5 mice, Atg5 mice had a lower autophagy basal level (p62 was significantly increased, and LC3II was significantly reduced). The stimulus cannot be restored to the normal basic level.

　　Conclusion: Using the Cre/loxp system, this study successfully constructed and identified conditional macrophage Atg5 gene knockout mice, which provides a research platform for studying the role of macrophage autophagy in the pathogenesis of kidney disease at the animal level.