| Summary: | 碩士 === 靜宜大學 === 食品營養學系 === 104 === Increasing in diabetes toward younger people worldwide attracted most of scientist’s concerns. The liver plays an important role to keep the circulating blood sugar levels steady and constant. When the glucose level rises, the liver utilizes cell membrane’s Glucose transporter 2 to transport excessive glucose to cells, and then (glucokinase, GLK) facilitates phosphorylation of glucose to Glouse-6-phosphate. Glycogen is then transferred via Glycogenesis process. It is stored or glycolyzed. There are 4 major types of diabetes: type I, due to the failure of production of insulin, type II, due to the deficiency of insulin or insulin resistance, other specific types of diabetes, and gestational diabetes, showing elevated blood glucose levels during pregnancy. If GLK gene expression can be activated immediately as glucose level rises, the blood sugar level could be declined. Therefore, one key factor in blood sugar control and development of dietary supplements is to activate hepatic glucokinase promoter and its gene expression. In the past, the development of dietary supplements for blood sugar control and their functional analysis was experimented with diabetic mice to evaluate their efficacy. However, because of the high cost and time consuming in animal experiments as well as the great regard of animal ethics, an alternative to construct a cell mode for blood sugar control is of high necessity and worthwhile.
In this study, the GLK gene was intended to replace with gene of enhanced green fluorescent protein (EGFP) by homologous recombination technique to construct a cell reporting system for GLK regulation and expression. Furthermore, this system can be used to screen the effective element to activate GLK, however, it has, thus far. not been constructed yet before the end of investigation career of my master degree. In the future, the construction of EGFP reporting system will not only help to screen the effective GLK activating elements, but will also help to monitor the GLK activation by the fluorescent expression and its intensity during the cell culture. This can replace some post cell culturing methods, such as real-time quantitative PCR, Western blot, and GLK activation testing, which are expensive, time-consuming and without a simultaneous monitor function.
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