In vivo electroporation and ubiquitin promoter -: a protocol for sustained gene expression in the lung
In vivo electroporation and ubiquitin promoter -: a protocol for sustained gene expression in the lung
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DOI：
10.1002/jgm.911
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发表时间：
2006-07-01
影响因子：
3.5
通讯作者：
Schmid, Ralph Alexander
中科院分区：
文献类型：
--
作者：
Gazdhar, Amiq;Bilici, Murat;Schmid, Ralph Alexander
基因治疗的应用需要安全有效的基因转移方法。现有的转基因表达方法存在效率低、时间短等问题。体内电穿孔是近年来发展起来的一种有效的基因转移方法。我们提出了一种涉及电穿孔结合长效启动子系统的基因转移到肺的方案。方法本研究旨在评估电穿孔介导的基因向肺的转移，并分析允许长时间转基因表达的启动子系统。将250 μ l的纯化质粒DNA悬浮水灌注麻醉大鼠左肺，切开左胸，电穿孔暴露左肺。在巨细胞病毒立即早期启动子/增强子（CMV-IEPE）或人多聚素（Ubc）启动子的控制下，表达荧光素酶的质粒pCiKlux和pUblux被使用。采用平板电极，以4个脉冲（200 V/cm, 20 ms, 1 Hz）优化电穿孔条件。这些动物在基因转移后的第40天的不同时间点被处死。采用生物荧光报告成像（BLI）检测和定量基因表达，采用荧光素酶荧光计测定p.Pyralis荧光素酶的相对光单位/毫克蛋白（RLU/mg），采用抗荧光素酶抗体免疫组化。结果CMV-IEPE启动子基因转染后24 h表达量最高（2932 +/- 249.4相对光单位(RLU)/mg总肺蛋白），第3天表达量恢复至基线水平（382 +/- 318 RLU/mg总肺蛋白）；在第5天没有检测到表达，而在Ubc启动子下检测到基因表达到第40天（1989 +/- 710 RLU/mg总肺蛋白），并在第20天达到峰值（2821 +/- 2092 RLU/mg总肺蛋白）。在第1天和第40天，动脉血气（PaO2）、组织学评估和细胞因子测量均显示无明显毒性。结论体内电穿孔是一种安全有效的非病毒基因肺传递工具。如果该方法与长效启动子系统结合使用，可以实现持续的转基因表达。版权所有(c) 2006约翰威利父子有限公司
Background Gene therapy applications require safe and efficient methods for gene transfer. Present methods are restricted by low efficiency and short duration of transgene expression. In vivo electroporation, a physical method of gene transfer, has evolved as an efficient method in recent years. We present a protocol involving electroporation combined with a long-acting promoter system for gene transfer to the lung.Methods The study was designed to evaluate electroporation-mediated gene transfer to the lung and to analyze a promoter system that allows prolonged transgene expression. A volume of 250 mu l of purified plasmid DNA suspended in water was instilled into the left lung of anesthetized rats, followed by left thoracotomy and electroporation of the exposed left lung. Plasmids pCiKlux and pUblux expressing luciferase under the control of the cytomegalovirus immediate-early promoter/enhancer (CMV-IEPE) or human polyubiquitin c (Ubc) promoter were used. Electroporation conditions were optimized with four pulses (200 V/cm, 20 ms at 1 Hz) using flat plate electrodes. The animals were sacrificed at different time points up to day 40, after gene transfer. Gene expression was detected and quantified by bioluminescent reporter imaging (BLI) and relative light units per milligram of protein (RLU/mg) was measured by luminometer for p.Pyralis luciferase and immunohistochemistry, using an anti-luciferase antibody.Results Gene expression with the CMV-IEPE promoter was highest 24 h after gene transfer (2932 +/- 249.4 relative light units (RLU)/mg of total lung protein) and returned to baseline by day 3 (382 +/- 318 RLU/mg of total lung protein); at day 5 no-expression was detected, whereas gene expression under the Ubc promoter was detected up to day 40 (1989 +/- 710 RLU/mg of total lung protein) with a peak at day 20 (2821 +/- 2092 RLU/mg of total lung protein). Arterial blood gas (PaO2), histological assessment and cytokine measurements showed no significant toxicity neither at day 1 nor at day 40.Conclusions These results provide evidence that in vivo electroporation is a safe and effective tool for non-viral gene delivery to the lungs. If this method is used in combination with a long-acting promoter system, sustained transgene expression can be achieved. Copyright (c) 2006 John Wiley & Sons, Ltd.