中国生物工程杂志

Objective: Construct hepatic-specific knockout mice model of OTUB1, the important deubiquitinase of ovarian tumor domain(OTU) protease superfamily, preliminarily analysis the phenotype of hepatic-specific OTUB1 knockout mice model and explore the physiological function of OTUB1 in liver metabolism.Methods:A mouse model of conventionally disrupting OTUB1 gene in liver using Cre/Loxp system was generate. The obtained OTUB1 ^fl/fl transgenic mice were crossed with Alb-Cre mice and PCR was used to identify the genotype of its offspring. Furthermore, liver-specific OTUB1 knockout mice were obtained by self-crossing the offspring and PCR was used to identify the genotype. At the same time, OTUB1 protein expression level was detected in tissues and organs of adult mice, include liver and other major organs, from hepatic-specific OTUB1-knockout (HCKO) mice and the control group (control, NC) littermate mice, and Western blot were used to detected and evaluated OTUB1 protein levels. The data indicated whether the hepatic-specific OTUB1 knockout mouse model was successfully constructed. Once comfirmed OTUB1 was truly mutant expression in the liver of HCKO mice, histopathological examination was performed and analyzed the morphology of liver, stomach and spleen, which was analyzed whether there was any spontaneous pathological change existed. In addition, the main biochemical indicators of the liver were detected and analyzed by serum to reflect liver lipid metabolism function in HCKO mice. Moreover, the level of blood glucose metabolism control was recorded and compared between HCKO mice and NC littermate mice through the Glucose Tolerance Test (GTT). Results:The genomic sequencing and Western blot analysis showed that OTUB1 was significantly deleted only in the liver of HCKO mice, but the protein expression level of OTUB1 in other tissues was unchanged at all, where the Alb-Cre transgene is not expressed, such as the kidney, spleen, fat and muscle, which proved that the hepatic-specific OTUB1 knockout mouse model was successfully constructed. Genotyping the offspring of OTUB1 hepatic-conditional knockout mice showed its were born normally. Also, these HCKO mice stayed healthy, without spontaneous histopathological abnormalities in embryonic development. Moreover, the total cholesterol levels in biochemical indicators were significantly lower in HCKO mice less than in NC mice, indicating that the OTUB1 affects liver lipid metabolism level to a certain extent. In glucose tolerance test, the blood glucose level of HCKO mice decreased rapidly after reaching its highest level, suggesting that the homeostasis of liver blood glucose depended on the regulation of OTUB1.Conclusions:The hepatic-specific OTUB1 knockout mouse model was successfully established by Cre/Loxp technology strategy, which are essential for research deubiquitinase OTUB1 in physiological condition,as well as provide an important animal model for studying the physiological functions and regulatory mechanisms of OTUB1 in the liver.