Highly efficient cell-mediated gene transfer using non-viral vectors and FuGene™6:: in vitro and in vivo studies
Highly efficient cell-mediated gene transfer using non-viral vectors and FuGene™6:: in vitro and in vivo studies
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DOI：
10.1007/pl00000769
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发表时间：
2000-08-01
影响因子：
8
通讯作者：
Sylvén, C
中科院分区：
文献类型：
--
作者：
Hellgren, I;Drvota, V;Sylvén, C
本研究旨在开发一种高效的心血管基因治疗的非病毒基因传递系统。我们研究了新的转染剂Fugene(TM)6的转染率和毒性，并与另外两种转染剂TFX(TM)-50和LipoTaxi(TM)进行了比较。在体内实验中，通过将携带有Fugene(TM)6基因的成纤维细胞移植到肌肉内，初步研究了高效基因传递的条件。从皮肤中分离人和兔成纤维细胞，分别用编码血管内皮细胞生长因子(VEGF)和β-半乳糖苷酶的phVEGF165或pCMVβ-Gal载体进行培养和转染。研究了DNA用量和DNA与转染剂的比例对质粒摄取的影响。在被测试的转染剂中，只有Fugene(TM)6为细胞定位(β-半乳糖苷酶)和分泌型(血管内皮生长因子)基因产物提供了高效和剂量依赖的质粒转移。当用四甲基偶氮唑盐分析时，Fugene(TM)6在低剂量下没有毒性。将优化的条件应用于体内报告基因的传递。兔肌肉注射体外转基因的成纤维细胞。与体外研究一样，体外转基因的成纤维细胞在体内表现出高效的基因表达。组织切片用巨噬细胞特异性免疫染色进行分析。成纤维细胞注射区未见炎症迹象。本研究表明FuGenc(TM)6是一种高效的转染剂，可用于原代人和兔成纤维细胞的体外子病毒转染法和体内治疗性非病毒基因转染法。
The present study was undertaken to develop an efficient non-viral gene delivery system for cardiovascular gene therapy. We investigated transfection efficiency and toxic properties of the new transfection reagent, FuGene(TM)6, and compared it with two other transfection reagents, Tfx(TM)-50 and LipoTaxi(TM). For in vivo experiments, the plasmid was delivered intramuscularly via transplantation of fibroblasts transfected with plasmid and FuGene(TM)6. Conditions for efficient gene delivery were initially studied in vitro. Human and rabbit fibroblasts were isolated from skin, cultured and transfected with phVEGF165 or pCMV beta gal plasmids, coding for vascular endothelial growth factor (VEGF) or beta-galactosidase, respectively. The effect of the DNA amount and the DNA:transfection reagent ratio on plasmid uptake were studied. Of the transfection reagents tested, only FuGene(TM)6 provided high-efficiency and dose-dependent plasmid transfer both for cell-localised (beta-galactosidase) and secreted (VEGF) gene products. When analysed with an MTT assay, FuGene(TM)6 showed no toxicity at low doses. Optimised conditions were applied for in vivo reporter gene delivery. Rabbits were injected intramuscularly with ex vivo-transfected fibroblasts. As in in vitro studies, ex vivo-transfected fibroblasts showed highly efficient gene expression in vivo. Tissue sections were analysed with macrophage-specific immunostaining. No signs of inflammation were seen in the region of fibroblast injection. This study demonstrates that FuGenc(TM)6 is a highly efficient transfection reagent that may be useful for in vitro son-viral transfection of primary human and rabbit fibroblasts and for in vivo therapeutic non-viral gene delivery.