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| 体外实时动态监测水动力注射分泌型荧光素酶基因的表达 | |
| 引用本文： | 田文洪,王刚,罗顺涛,董小岩,付昕阳,谭文杰,吴小兵.体外实时动态监测水动力注射分泌型荧光素酶基因的表达[J].生物工程学报,2009,25(10):1552-1557. |
| 作者姓名： | 田文洪 王刚 罗顺涛 董小岩 付昕阳 谭文杰 吴小兵 |
| 作者单位： | 1. 中国疾病预防控制中心病毒病预防控制所病毒基因工程国家重点实验室,北京,100052 2. 中国疾病预防控制中心病毒病预防控制所病毒基因工程国家重点实验室,北京,100052;四川大学华西医学院华西医院生物治疗国家重点实验室,成都,610064 3. 中国疾病预防控制中心病毒病预防控制所病毒基因工程国家重点实验室,北京,100052;北京五加和分子医学研究所有限责任公司,北京,100176 |
| 基金项目： | 国家重点基础研究发展计划(973计划)(No.2004CB518806)资助~~ |
| 摘 要： | 为了建立体外实时动态监测转导基因的体内表达,本研究选择分泌型的荧光素酶基因Gluc作为报告基因,对其体内外表达特性和检测方法进行了研究。首先构建了Gluc表达质粒pAAV2neo-Gluc。将pAAV2neo-Gluc转染体外培养的Huh7、HepG2细胞后,细胞培养上清和细胞裂解液中分别检测到Gluc的活性,而上清比细胞中的含量高约100倍。表明表达的Gluc以分泌形式为主。用水动力法经小鼠尾静脉注射pAAV2neo-Gluc质粒,活体成像表明Gluc在小鼠体内呈全身分布,而注射了萤火虫荧光素酶质粒pAAV2neo-Fluc的对照小鼠则主要在肝脏显像。将剂量分别为0.1、1、10、50μg每只的pAAV2neo-GlucDNA用水动力法尾静脉注射小鼠,不同时间点连续尾静脉采血测定其中的Gluc酶活性,观察其Gluc体内表达和分泌的动态变化。结果显示,各剂量组的Gluc表达变化规律高度一致:注射后2h即可检测到Gluc表达,10h后达到高峰,之后逐渐下降;Gluc的表达水平与注射质粒DNA的量呈正相关;为了进一步观察Gluc检测的灵敏性,本研究又比较了注射更低的质粒剂量(包括0.001、0.01和0.1μg每... |
| 关 键 词： | 水动力注射 基因表达 Gluc 监测 ex vivo |
| 收稿时间： | 6/3/2009 12:00:00 AM |
Real time ex vivo detection and dynamic monitoring of in vivo expression of secreted luciferase gene injected by hydrodynamic method |
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| Wenhong Tian,Gang Wang,Shuntao Luo,Xiaoyan Dong,Xinyang Fu,Wenjie Tan and XiaobingWu.Real time ex vivo detection and dynamic monitoring of in vivo expression of secreted luciferase gene injected by hydrodynamic method[J].Chinese Journal of Biotechnology,2009,25(10):1552-1557. | |
| Authors: | Wenhong Tian Gang Wang Shuntao Luo Xiaoyan Dong Xinyang Fu Wenjie Tan and XiaobingWu |
| Institution: | State Basic Research and Development Program of China (973 Program)(No. 2004CB518806).;State Basic Research and Development Program of China (973 Program)(No. 2004CB518806).;State Basic Research and Development Program of China (973 Program)(No. 2004CB518806).;State Basic Research and Development Program of China (973 Program)(No. 2004CB518806).;State Basic Research and Development Program of China (973 Program)(No. 2004CB518806).;State Basic Research and Development Program of China (973 Program)(No. 2004CB518806).;State Basic Research and Development Program of China (973 Program)(No. 2004CB518806). |
| Abstract: | We chose Gaussia luciferase (Gluc), a secreted luciferase gene as reporter to real-time detect and dynamically monitor hydrodynamic injection gene expression. First, we constructed an expression vector pAAV2neo-Gluc. Then Huh7 and HepG2 cells were transfected with pAAV2neo-Gluc and the activity of Gluc in the supernatant and cell lysates were assayed. Results showed that the Gluc activity in the supernatant was about 100 higher than that in cell lysates, indicating the expressed Gluc existing mainly as a se... |
| Keywords: | hydrodynamic injection gene expression Gluc monitoring ex vivo |
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