Construction of membrane reporter gene for noninvasive imaging of gene expression in vivo

• Main Authors: Jhih-Hong Lin, 林志鴻
• Other Authors: Tiang-Lu Cheng
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/96283248737995634860

碩士 === 高雄醫學大學 === 醫藥暨應用化學系碩士班 === 93 === The most important target of gene therapy is to express the therapeutic genes in specific tissues or organs. Hence the researchers developed reporter genes to monitor the expression locations of therapeutic genes. This paper relates developing a novel reporter gene to proceed in vivo noninvasive imaging for the same purpose. We had constructed membrane form mouse β-glucuronidase(Mem-βGlu) as a reporter gene. The imaging probe (fluorescein di-β-D-glucuronide, FDGlcU) will be catalyzed by β-glucuronidase enzyme activity, and then we can employ suitable imaging apparatus to monitor the expression site of this reporter gene. The Mem-βGlu gene is persistently expressed in CT26 cells by retrovirus infection. We confirmed that the Mem-βGlu enzyme is expressed on the cell membrane by flow cytometry. And we proved that the Mem-βGlu enzyme retains activity to catalyze imaging probe. After that in vivo imaging was exercised in Mem-βGlu expressed tumor implanted mouse with i.v. injection of imaging probes. Significant fluorescence is detected at the location of Mem-βGlu expressed tumor in short time but not the site of control tumor. Similar results were also shown in in vitro and in vivo experiments of HCC36 cells infected with adenovirus carrying Mem-βGlu gene. According to these results, we knew the noninvasive imaging system based on Mem-βGlu enzyme is workable. This reporter gene system could be used to test the tissue specificity of therapeutic genes'' expression or transmitting genes by kinds of gene therapy vectors. It will enable the related researches of gene therapy to be more convenient in future.