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

Real time ex vivo detection and dynamic monitoring of in vivo expression of secreted luciferase gene injected by hydrodynamic method

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、50 μg每只的pAAV2neo-Gluc DNA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2 h即可检测到Gluc表达, 10 h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0.1 μg/每只)时Gluc体内表达情况。结果发现注射剂量低至0.001 mg每只小鼠都能在血中检测到Gluc表达, 这一结果提示了Gluc检测的高度灵敏性。本研究首次报道了以Gluc为报告基因体外实时动态监测水动力注射基因的表达规律, 为动态分析水动力注射基因的体内表达调控及功能研究提供了新的有效手段。

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 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 Gluc 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 μg, 0.01 μg and 0.1 μg pAAV2neo-Gluc respectively. Results revealed that activity of Gluc in blood could be detected even at dose as low as 0.001 μg 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.