Intracellular trafficking membrane fusion

This is in contrast to viruses, where the virus particles also show active transport when present in the cytosol after fusion with the plasma membrane or endosomal membrane [60-62], This is due to the ability of specific proteins of the virus particle to bind motor proteins. Single-particle tracking reveals that the quantitative intracellular transport properties of internalized non-viral gene vectors (e.g., polyplexes) are similar to that of viral vectors (e.g., adenovirus) [63]. Suk et al. showed that over 80% of polyplexes and adenoviruses in neurons are subdiffusive and 11-13% are actively transported. However, their trafficking pathways are substantially different. Polyplexes colocalized with endosomal compartments whereas adenovirus particles quickly escaped endosomes after endocytosis. Nevertheless, both exploit the intracellular transport machinery to be actively transported. 

As human skin fibroblasts are recalcitrant to transfection, we wished to include an easily traceable marker, which can either be used as an in-frame fusion protein, or as a separately expressed protein. The widely applicable green fluorescent protein (GFP) reporter system, originally isolated from the jellyfish Aequorea victoria, and adapted for efficient expression in human cells and for, elevated levels of fluorescence, provides these properties. The GFP system has been used for example, to study protein trafficking to the mitochondrial matrix and the mitochondrial outer membrane. Therefore, by using confocal laser scanning imaging we may get an impression of the intracellular locahzation of a CPTl-GFP fusion protein after transient or stable transfection. 

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