AMP-activated protein kinase (AMPK) is involved in the development and progression of tumors including hepatocellular carcinoma (HCC). However, studies on AMPK and tumorigenesis were largely based on experiments in vitro or tumor xenografts model. Here, we introduce a liver-specific AMPKα1 knockout mice model, which is achieved by Alb-Cre recombinase system. The expression of AMPKα1 in the liver of AMPKα1-/--Alb-Cre mice is absent. AMPKα1 knockout in the liver does not affect the growth and histological structure of mouse liver. This model provides a favorable tool to the study of the roles of AMPKα1 in liver metabolism or tumorigenesis.
The emergence of regular short repetitive palindromic sequence clusters (CRISPR) and CRISPR- associated proteins 9 (Cas9) gene editing technology has greatly promoted the wide application of genetically modified pigs. Efficient single guide RNA (sgRNA) is the key to the success of gene editing using CRISPR/Cas9 technology. For large animals with a long reproductive cycle, such as pigs, it is necessary to screen out efficient sgRNA in vitro to avoid wasting time and resource costs before animal experiments. In addition, how to efficiently obtain positive gene editing monoclonal cells is a difficult problem to be solved. In this study, a rapid sgRNA screening method targeting the pig genome was established and we rapidly obtained Fah gene edited cells, laying a foundation for the subsequent production of Fah knockout pigs as human hepatocyte bioreactor. At the same time, the method of obtaining monoclonal cells using pattern microarray culture technology was explored.
ObjectiveTo investigate the effect of Gasdermin D(GSDMD) gene knockout on lung injury and reactive oxygen species (ROS)/nod-like receptor protein 3 (NLRP3)/cysteinyl aspartate specific proteinase-1(caspase-1) pyroptosis pathway of pneumonia mice. Methods20 wild-type (WT) male C57BL/6J mice were randomly divided into WT control group and WT model group. 20 GSDMD gene knockout (KO) mice were randomly divided into KO control group and KO model group, each group consisted of 10 animals. The pneumonia model was induced by puncturing the nasal mucosa with a sterile needle and slowly instilling Streptococcus pneumoniae suspension in WT model group and KO model group. Serum and bronchoalveolar lavage fluid (BALF) were collected 7 days after modeling, and the levels of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were detected; the lung tissues were collected and the dry wet ratio (W/D), pathological changes, the levels of malondialdehyde (MDA), total antioxidant capacity (T-AOC), superoxide dismutase (SOD), ROS activity, and the protein expression levels of GSDMD, GSDMD-N, NLRP3, Caspase-1 were detected. ResultsThe lung tissue of WT model group showed cell infiltration with alveolar septal thickening, the ratio of W/d in lung tissue, the levels of IL-1β, IL-6 and TNF-α in serum and BALF, the levels of MDA, ROS, NLRP3 and Caspase-1 in lung tissue were all higher than those in WT control group, the levels of T-AOC and SOD in lung tissue were lower than those in WT control group (P<0.05), and the levels of serum, BALF and lung tissue in KO control group were not significantly different from those in WT control group (P>0.05) There was no expression of GSDMD-N in the lung tissue of KO model group, lung tissue w/d ratio, serum and BALF levels of IL-1β, IL-6, TNF-α, MDA, ROS activity, GSDMD-N, NLRP3 and Caspase-1 protein expression were lower than those in WT model group, the levels of T-AOC and SOD in lung tissue were higher than those in WT Model Group (P<0.05). ConclusionGSDMD gene knockout attenuates lung injury and inhibits ROS/NLRP3/Caspase-1 pathway in pneumonia mice.