Tian Wenhong, Wang Gang, Luo Shuntao, Dong Xiaoyan, Fu Xinyang, Tan Wenjie, Wu Xiaobing
Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, National Laboratory of Molecular Virology and Genetic Engineering, Beijing 100052, China.
Sheng Wu Gong Cheng Xue Bao. 2009 Oct;25(10):1552-7.
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 secreted form as reported. Live bioluminescence imaging of mice hydrodynamic injected pAAV2neo-Gluc showed whole body distribution, while the pAAV2neo-Fluc primarily located in the liver. Then we injected different doses of pAAV2neo-Gluc into mice by tail-vein hydrodynamic injection, took minor amount of blood from mice tails at different time points and measured the luciferase activity to investigate dynamic changes of Gluc expression and secretion in vivo. The results suggested that the time courses of Gluc expression were highly consistent among each dose groups. The luciferase activity in blood could be detected as early as 2 h after injection, reached the peak at about 10 h and gradually decreased from then on. The expression level of Glue was positively correlated with the dose of injected plasmid DNA. To further detect the assay sensitivity of the ex vivo Gluc measurement method, we investigated three additional groups of mice injected with lower doses of 0.001 microg, 0.01 microg and 0.1 microg pAAV2neo-Gluc respectively. Results revealed that activity of Gluc in blood could be detected even at dose as low as 0.001 microg DNA, suggesting the assay sensitivity was extremely high. In conclusion, a real-time ex vivo detection method of dynamically monitoring of gene expression in vivo by hydrodynamic injection can be a valuable means for the study of gene expression regulation in vivo.
我们选择了分泌型荧光素酶基因高斯荧光素酶（Gluc）作为报告基因，以实时检测和动态监测流体动力学注射基因的表达。首先，我们构建了表达载体pAAV2neo-Gluc。然后用pAAV2neo-Gluc转染Huh7和HepG2细胞，并检测上清液和细胞裂解物中Gluc的活性。结果显示，上清液中的Gluc活性比细胞裂解物中的高约100倍，表明所表达的Gluc主要以分泌形式存在，与报道一致。对流体动力学注射pAAV2neo-Gluc的小鼠进行活体生物发光成像显示其全身分布，而pAAV2neo-Fluc主要位于肝脏。然后通过尾静脉流体动力学注射向小鼠注射不同剂量的pAAV2neo-Gluc，在不同时间点从小鼠尾部采集少量血液并测量荧光素酶活性，以研究体内Gluc表达和分泌的动态变化。结果表明，各剂量组中Gluc表达的时间进程高度一致。注射后2小时即可在血液中检测到荧光素酶活性，约10小时达到峰值，随后逐渐下降。Gluc的表达水平与注射的质粒DNA剂量呈正相关。为了进一步检测体外Gluc测量方法的检测灵敏度，我们另外研究了分别注射低剂量0.001微克、0.01微克和0.1微克pAAV2neo-Gluc的三组小鼠。结果显示，即使DNA剂量低至0.001微克，血液中Gluc的活性也能被检测到，表明该检测方法的灵敏度极高。总之，一种通过流体动力学注射动态监测体内基因表达的实时体外检测方法，可为体内基因表达调控的研究提供有价值的手段。
