Department of Molecular Pathology and Innovative Therapies-Histology, Marche Polytechnic University, Ancona, Italy.
Microsc Res Tech. 2010 Mar;73(3):229-33. doi: 10.1002/jemt.20779.
In the last years, physical and chemical methods of plasmid delivery have revolutionized the efficiency of nonviral gene transfer, and the success of gene therapy is largely dependent upon the development of gene-delivery methods. The nonviral techniques that lead to a direct transfer of DNA into tissue fragments, like electroporation (EP) and lipofection delivery systems are still insufficiently investigated. Our aim was to test the efficiency of EP and lipofection protocols in endometrial and testicular tissue fragments, using a naked plasmid DNA encoding green fluorescent protein (GFP). Because the transfection efficiency depends upon several factors, we tried to optimize the transfection conditions by testing different lipofectamine 2000 and plasmid ratios, electrical parameters, and culture after transfection. Our results show that these two nonviral methods of gene delivery are feasible and efficient in gene transfection of endometrial and testicular tissue biopsies. We found that the most performing ratio of plasmid:lipofectamine was 10:50 for transient lipofection, whereas two pulses for 10 s at 960 microF of capacitance, 200 V of voltage were the most favorable electrical parameters for EP efficiency in the presence of 5 microL of phMGFP plasmid. After lipofection and EP, the highest GFP intensity was observed respectively after 48 and 72 h of tissue fragment culturing. In conclusion, nonviral methods are attractive for an improvement of the gene therapy and our protocol could provide useful indications for in vivo gene therapy applications.
在过去的几年中，物理和化学方法的质粒传递使非病毒基因转移的效率发生了革命性变化，基因治疗的成功在很大程度上取决于基因传递方法的发展。导致 DNA 直接转移到组织片段的非病毒技术，如电穿孔（EP）和脂质体转染系统，仍未得到充分研究。我们的目的是使用编码绿色荧光蛋白（GFP）的裸质粒 DNA 测试 EP 和脂质体转染方案在子宫内膜和睾丸组织片段中的效率。由于转染效率取决于多个因素，我们通过测试不同的脂质体 2000 和质粒比例、电参数和转染后的培养来尝试优化转染条件。我们的结果表明，这两种非病毒基因传递方法可有效且高效地转染子宫内膜和睾丸组织活检。我们发现，瞬时脂质体转染的最佳质粒：脂质体比例为 10:50，而在存在 5 μL phMGFP 质粒的情况下，电穿孔效率的最有利电参数为 960 μF 电容的两个 10 s 脉冲，200 V 电压。转染和 EP 后，分别在组织片段培养 48 和 72 小时后观察到最高的 GFP 强度。总之，非病毒方法对于改善基因治疗具有吸引力，我们的方案可为体内基因治疗应用提供有用的指示。
