Laboratoire de Biotechnologie Cellulaire, Institute of Biological and Chemical Processes, Faculty of Basic Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland,
Cytotechnology. 2002 Jan;38(1-3):57-62. doi: 10.1023/A:1021197830091.
Various methods exist to transfect mammalian cells in culture. It is generally accepted that individual methods have to be optimized for each of the cell lines or cell types used. Despitethe use of optimized protocols, significant day-to-day variationsin transfection efficiency regularly occur. We postulate that the;status' of cell populations prior to transfection is involved insuch variability. This study evaluates standardized transfectionsdone at different phases of the cell cycle. Cell synchronizationwas achieved using mimosine. Transfection efficiency was monitored by fluorescence quantification of GFP (Green Fluorescent Protein). We show that transfection using the calcium-phosphate-DNA co-precipitation method, at differentphases of the cell cycle, yields variable expression levels of GFP. Highest GFP expression levels were seen when transfecting cell populations with a dominant representation of S-phase-cells.
有多种方法可用于转染培养中的哺乳动物细胞。通常认为，必须针对所用的细胞系或细胞类型优化各个方法。尽管使用了优化的方案，但转染效率仍会经常出现显著的日常变化。我们假设，转染前细胞群体的“状态”与这种可变性有关。本研究评估了细胞周期不同阶段进行的标准化转染。使用丝裂霉素 C 实现细胞同步化。通过 GFP（绿色荧光蛋白）的荧光定量监测转染效率。我们表明，使用钙-磷酸盐-DNA 共沉淀法在细胞周期的不同阶段进行转染，会导致 GFP 的表达水平发生变化。当用具有 S 期细胞优势的细胞群体进行转染时，观察到最高的 GFP 表达水平。
