Cell density-dependent ectopic expression in bloodstream form Trypanosoma brucei

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.