Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK.
Exp Parasitol. 2013 Jun;134(2):249-55. doi: 10.1016/j.exppara.2013.03.017. Epub 2013 Mar 26.
Ectopic expression of either wild type or mutant proteins is a standard method in cell biology, and a vital part of the tool kit of molecular parasitology. During study of protein expression levels mediating intracellular trafficking, we became aware of highly variable expression between experiments. When investigated systematically it became apparent that ectopic expression of proteins from a ribosomal promoter diminished at high cell culture density in bloodstream form Trypanosoma brucei. This phenomenon was not restricted to expression of a specific protein or cell line or the vector backbone. While procyclic form cells did not exhibit detectable density-related expression changes, bloodstream form cells manifest significant reduction in expression at high density, confirmed by qRT PCR, Western blotting and fluorescence microscopy. Culturing in conditioned media unveiled a similar reduction in expression at lower cell densities. Taken together we concluded that this effect is likely related to the influence of a diffusible factor present in conditioned media and has implications for accurate quantification of ectopic expression using transgenic expression systems.
异位表达野生型或突变型蛋白是细胞生物学中的标准方法，也是分子寄生虫学工具包的重要组成部分。在研究介导细胞内运输的蛋白质表达水平时，我们注意到实验之间的表达变化非常大。当系统地研究时，我们发现来自核糖体启动子的蛋白质在布鲁氏锥虫血腔形式的高细胞培养密度下表达减少。这种现象不仅限于特定蛋白质或细胞系或载体骨架的表达。虽然前鞭毛体细胞没有表现出可检测到的密度相关表达变化，但在高细胞密度下，血腔形式的细胞表现出明显的表达减少，通过 qRT-PCR、Western blot 和荧光显微镜得到证实。在条件培养基中培养也揭示了在较低细胞密度下表达的类似减少。综上所述，我们得出结论，这种效应可能与条件培养基中存在的可扩散因子的影响有关，这对使用转基因表达系统准确定量异位表达具有重要意义。
