· 1659 · Zhang YL et al / Acta Pharmacol Sin 2004 Dec; 25 (12): 1659-1665 ©2004, Acta Pharmacologica Sinica Chinese Pharmacological Society Shanghai Institute of Materia Medica Chinese Academy of Sciences http://www.ChinaPhar.com 1 Project supported by the grants from National High Technol- ogy Research and Development Program of China (863, No 108- 08-08-03, 2002AA214201). 3 Correspondence to Prof Huan-ran TAN. Phn 86-10-8280-2004. E-mail tanlab@sun.bjmu.edu.cn Received 2004-04-23 Accepted 2004-08-05 Establishment of liver specific glucokinase gene knockout mice: a new animal model for screening anti-diabetic drugs 1 Ya-li ZHANG, Xiao-hong TAN 2 , Mei-fang XIAO, Hui LI, Yi-qing Mao, Xiao YANG 2 , Huan-ran TAN 3 Department of Pharmacology, School of Basic Medical Science, Peking University Health Science Center, Beijing 100083; 2 Institute of Biotechnology Academy of Military Medical Science, Beijing 100071, China KEY WORDS animal disease model; non insulin dependent diabetes mellitus; type 2 diabetes mellitus; glucokinase; knockout mice ABSTRACT AIM: To characterize the liver-specific role of glucokinase in maintaining glucose homeostasis and to create an animal model for diabetes. METHODS: 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. RESULTS: 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. CONCLUSION: 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 knock- out mouse is an ideal animal model for MODY2, and it also can be applied for screening anti-diabetic drugs. INTRODUCTION Diabetes, characterized by hyperglycemia due to defects in insulin production, insulin action, or both, is a common, serious worldwide health issue. There are two major types of diabetes, type 1 (insulin-dependent diabetes mellitus, IDDM), and type 2 (non-insulin-de- pendent diabetes mellitus, NIDDM). Type 1 diabetes results from insulin deficiency caused by autoimmune destruction of pancreatic β cells, and generally deve- lops in the young. Type 2 diabetes is a multifactorial disease, depending on a complex interaction between many genetic and environment factors, and generally develops in adults [1] . However, among diabetes, a mi- nority patients result from a single gene mutation that leads to either β-cell dysfunction or, less frequently in-