Zhang Ya-li, Tan Xiao-hong, Xiao Mei-fang, Li Hui, Mao Yi-qing, Yang Xiao, Tan Huan-ran
Department of Pharmacology, School of Basic Medical Science, Peking University Health Science Center, Beijing 100083, China.
Acta Pharmacol Sin. 2004 Dec;25(12):1659-65.
To characterize the liver-specific role of glucokinase in maintaining glucose homeostasis and to create an animal model for diabetes.
We performed hepatocyte-specific gene knockout of glucokinase in mice using Cre-loxP gene targeting strategy. First, two directly repeated loxP sequences were inserted to flank the exon 9 and exon 10 of glucokinase in genomic DNA. To achieve this, linearized targeting vector was electroporated into ES cells. Then G418- and Gancyclovir-double-resistant clones were picked and screened by PCR analysis and the positives identified by PCR were confirmed by Southern blot. A targeted clone was selected for microinjection into C57BL/6J blastocysts and implanted into pseudopregnant FVB recipient. Chimeric mice and their offspring were analyzed by Southern blot. Then by intercrossing the Alb-Cre transgenic mice with mice containing a conditional gk allele, we obtained mice with liver-specific glucokinase gene knockout.
Among 161 double resistant clones 4 were positive to PCR and Southern blot and only one was used for further experiments. Eventually we generated the liver specific glucokinase knockout mice. These mice showed increased glucose level with age and at the age of 6 weeks fasting blood glucose level was significantly higher than control and they also displayed impaired glucose tolerance.
Our studies indicate that hepatic glucokinase plays an important role in glucose homeostasis and its deficiencies contribute to the development of diabetes. The liver glucokinase knockout mouse is an ideal animal model for MODY2, and it also can be applied for screening anti-diabetic drugs.
明确葡萄糖激酶在维持葡萄糖稳态中的肝脏特异性作用，并建立糖尿病动物模型。
我们采用Cre-loxP基因靶向策略在小鼠中进行肝细胞特异性葡萄糖激酶基因敲除。首先，在基因组DNA中，将两个直接重复的loxP序列插入到葡萄糖激酶的外显子9和外显子10两侧。为实现这一目标，将线性化的靶向载体电穿孔导入胚胎干细胞。然后挑选出对G418和更昔洛韦双重耐药的克隆，并通过PCR分析进行筛选，经PCR鉴定为阳性的克隆通过Southern印迹法进行确认。选择一个靶向克隆显微注射到C57BL/6J囊胚中，并植入假孕的FVB受体小鼠体内。通过Southern印迹法分析嵌合小鼠及其后代。然后将Alb-Cre转基因小鼠与含有条件性gk等位基因的小鼠进行杂交，获得肝脏特异性葡萄糖激酶基因敲除的小鼠。
在161个双重耐药克隆中，4个对PCR和Southern印迹呈阳性，仅1个用于进一步实验。最终我们获得了肝脏特异性葡萄糖激酶敲除小鼠。这些小鼠随着年龄增长血糖水平升高，6周龄时空腹血糖水平显著高于对照组，且葡萄糖耐量受损。
我们的研究表明，肝脏葡萄糖激酶在葡萄糖稳态中起重要作用，其缺陷导致糖尿病的发生。肝脏葡萄糖激酶敲除小鼠是青少年发病的成年型糖尿病2型（MODY2）的理想动物模型，也可用于筛选抗糖尿病药物。
