[Real time ex vivo detection and dynamic monitoring of in vivo expression of secreted luciferase gene injected by hydrodynamic method].
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.
Tian
Wenhong
W
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.
Wang
Gang
G
Luo
Shuntao
S
Dong
Xiaoyan
X
Fu
Xinyang
X
Tan
Wenjie
W
Wu
Xiaobing
X
D000818:Animals; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000235:genetics; D015316:Genetic Therapy; D056945:Hep G2 Cells; D006801:Humans; D057446:Hydrodynamics; D007275:Injections, Intravenous; D008099:Liver / Q000378:metabolism; D008156:Luciferases / Q000008:administration & dosage / Q000096:biosynthesis / Q000235:genetics* / Q000493:pharmacokinetics*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D014162:Transfection
D008156:Luciferases