Uyttersprot N, Costagliola S, Miot F
Interdisciplinary Research Institute, Free University of Brussels, Belgium.
Mol Cell Endocrinol. 1998 Jul 25;142(1-2):35-9. doi: 10.1016/s0303-7207(98)00122-1.
The introduction of exogenous DNA into mammalian cells is commonly used to study the functions of gene products. However cells in primary culture are usually refractory to most transfection systems. Here we investigated the ability of a new lipid formulation, FuGENE 6 transfection reagent, to promote DNA uptake into dog and human thyroid cells in primary culture. Gene transfer was monitored by the expression of a Green Fluorescent Protein (GFP) reporter gene. We report that FuGENE 6 is particularly suited for the transfection of thyroid cells and does not interfere with their normal growth. Optimization of the experimental conditions, such as DNA amount, DNA/lipid ratio, cell density and incubation with the transfection mixture, was achieved by evaluating the percentage of GFP-expressing cells by FACS analysis. FuGENE 6 allowed us to obtain 8-15% thyrocytes expressing the reporter gene which represents an efficiency 100-fold superior to other transfection methods.
将外源DNA导入哺乳动物细胞常用于研究基因产物的功能。然而，原代培养的细胞通常对大多数转染系统具有抗性。在此，我们研究了一种新型脂质制剂FuGENE 6转染试剂促进DNA导入原代培养的犬和人甲状腺细胞的能力。通过绿色荧光蛋白（GFP）报告基因的表达监测基因转移。我们报告称，FuGENE 6特别适合甲状腺细胞的转染，且不干扰其正常生长。通过流式细胞术分析评估GFP表达细胞的百分比，实现了对实验条件的优化，如DNA量、DNA/脂质比、细胞密度以及与转染混合物的孵育时间。FuGENE 6使我们能够获得8%-15%表达报告基因的甲状腺细胞，其效率比其他转染方法高100倍。
