Spatial and temporal control of transgene expression in vivo using a heat-sensitive promoter and MRI-guided focused ultrasound
Spatial and temporal control of transgene expression in vivo using a heat-sensitive promoter and MRI-guided focused ultrasound
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DOI：
10.1002/jgm.345
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发表时间：
2003-04-01
影响因子：
3.5
通讯作者：
Moonen, CTW
中科院分区：
文献类型：
--
作者：
Guilhon, E;Voisin, P;Moonen, CTW
背景在用于诱导和控制基因表达的技术中，基于局部热和热敏启动子的非侵入性物理方法是一种很有前途的替代方法，但需要在体内进行准确的温度控制。具有实时反馈控制的磁共振引导聚焦超声(MRI-FUS)允许自动执行预定义的温度-时间轨迹。本研究的目的是基于MRI-FUS产生的明确的局部热疗来论证转基因表达的时空控制。方法利用绿色荧光蛋白(GFP)标记基因的表达。两株细胞系均来源于C6胶质瘤细胞。前者的GFP表达受CMV启动子的控制，而后者受HSP70启动子的控制，因此受热诱导。皮下肿瘤是通过注射免疫缺陷小鼠和大鼠而产生的。在MRI-FUS控制下，肿瘤在42-50℃的温度下持续3-25分钟，并在热休克后24小时进行分析。结果50℃短时间(3min)作用C6细胞后，HSP70表达增强，细胞存活率基本不受影响。在44-48摄氏度的温度范围内也可以清楚地显示诱导表达，但在42摄氏度时不是。结论用MRI-FUS加热可以严格和非侵入性地控制道口中的转基因表达。版权所有(C)2002 John Wiley Sons，Ltd.
Background Among the techniques used to induce and control gene expression, a non-invasive, physical approach based on local heat in combination with a heat-sensitive promoter represents a promising alternative but requires accurate temperature control in vivo. MRI-guided focused ultrasound (MRI-FUS) with real-time feedback control allows automatic execution of a predefined temperature-time trajectory. The purpose of this study was to demonstrate temporal and spatial control of transgene expression based on a well-defined local hyperthermia generated by MRI-FUS.Methods Expression of the green fluorescent protein (GFP) marker gene was used. Two cell lines were derived from C6 glioma cells. The GFP expression of the first one is under the control of the CMV promoter, whereas it is under the control of the HSP70 promoter in the second one and thus inducible by heat. Subcutaneous tumours were generated by injection in immuno-deficient mice and rats. Tumours were subjected to temperatures varying from 42 to 50 degreesC for 3 to 25 min controlled by MRI-FUS and analyzed 24 h after the heat-shock. Endogenous HSP70 expression and C6 cell distribution were also analyzed.Results The results demonstrate strong expression at 50 degreesC applied during a short time period (3 min) without affecting cell viability. Induced expression was also clearly shown for temperature in the range 44-48 degreesC but not at 42 degreesC.Conclusions Heating with MRI-FUS allows a tight and non-invasive control of transgene expression in a turnout. Copyright (C) 2002 John Wiley Sons, Ltd.