Establishment of liver specific glucokinase gene knockout mice: a new animal model for screening anti-diabetic drugs.
OBJECTIVE
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.


CONCLUSIONS
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.
Zhang
Ya-li
YL
Department of Pharmacology, School of Basic Medical Science, Peking University Health Science Center, Beijing 100083, China.
Tan
Xiao-hong
XH
Xiao
Mei-fang
MF
Li
Hui
H
Mao
Yi-qing
YQ
Yang
Xiao
X
Tan
Huan-ran
HR
D000818:Animals; D001786:Blood Glucose / Q000378:metabolism; D003924:Diabetes Mellitus, Type 2 / Q000378:metabolism*; D004195:Disease Models, Animal; D005941:Glucokinase / Q000378:metabolism*; D006706:Homeostasis; D008099:Liver / Q000201:enzymology*; D051379:Mice; D018345:Mice, Knockout
D001786:Blood Glucose; D005941:Glucokinase