Raup Alexander, Jérôme Valérie, Freitag Ruth, Synatschke Christopher V, Müller Axel H E
Process Biotechnology, University of Bayreuth, 95440 Bayreuth, Germany.
Macromolecular Chemistry II, University of Bayreuth, 95440 Bayreuth, Germany.
Biotechnol Rep (Amst). 2016 Jun 16;11:53-61. doi: 10.1016/j.btre.2016.05.003. eCollection 2016 Sep.
Non-viral transfection protocols are typically optimized using standard cells and reporter proteins, potentially underestimating cellular or transgene effects. Here such effects were studied for two human (Jurkat, HEK-293) and two rodent (CHO-K1, L929) cell lines and three fluorescent reporter proteins. Expression of the enhanced green fluorescent protein (EGFP) was studied under the control of the human elongation factor 1 alpha promoter and three viral promoters (SV40, SV40/enhancer, CMV), that of ZsYellow1 (yellow fluorescence) and mCherry (red fluorescence) for the CMV promoter. Results varied with the cell line, in particular for the Jurkat cells. Pair-wise co-transfection of the CMV controlled transgenes resulted in a significant fraction of monochromatic cells (EGFP for EGFP/YFP and EGFP/RFP co-transfections, YFP in case of YFP/RFP co-transfections). Only Jurkat cells were almost incapable of expressing YFP. Dilution of the plasmid DNA with a non-expressed plasmid showed cell line dependent effects on transfection efficiency and/or expression levels.
非病毒转染方案通常使用标准细胞和报告蛋白进行优化，这可能会低估细胞或转基因的效应。在此，针对两种人类细胞系（Jurkat细胞、HEK - 293细胞）、两种啮齿动物细胞系（CHO - K1细胞、L929细胞）以及三种荧光报告蛋白研究了此类效应。在人类延伸因子1α启动子以及三种病毒启动子（SV40、SV40/增强子、CMV）的控制下研究了增强型绿色荧光蛋白（EGFP）的表达，针对CMV启动子研究了ZsYellow1（黄色荧光）和mCherry（红色荧光）的表达。结果因细胞系而异，尤其是Jurkat细胞。CMV控制的转基因的成对共转染导致相当一部分细胞呈现单色（EGFP/ YFP和EGFP/RFP共转染时为EGFP，YFP/RFP共转染时为YFP）。只有Jurkat细胞几乎无法表达YFP。用非表达质粒稀释质粒DNA显示出对转染效率和/或表达水平的细胞系依赖性效应。
