As an in vitro model system, patient-derived epidermolysis bullosa simplex keratinocytes have had an immense impact on what we know today about keratin filament function and their role in disease development. In the absence of gene therapy, screening com pound libraries for new or better drug s is another approach to improve existing treatments for genodermatoses. However inthis study, we report of the potential pitfalls when using this type of cell lines as a "reporter" system. When cell lines with different genetic backgrounds are being used in cellbased assays, the greatest obstacle is to determine the most appropriate culture conditions (i.e., the composition of medium, number of cells plated and number of day s in culture). We demonstrate how culture conditions can greatly interfere with the cellular response in cell-based assays (cell proliferation, metabolic activity and migration), potentially al so giving rise to misleading data.
COBISS.SI-ID: 30115545
We present the potential of inclusion bodies (IBs) as a protein delivery method for polymeric filamentous proteins. We used as cell factory a strain of E. coli, a conventional host organism, and keratin 14 (K14) as an example ofa complex protein. Keratins build the intermediate filament cytoskeleton of all epithelial cells. In order to build filaments, monomeric K14 needs first to dimerize with its binding partner (keratin 5, K5), which is then followed by heterodimer assembly into filaments. We show that K14IBs and K5 plasmid DNA can be imported into cells by means of electroporation. Ones in the cytosol they start forming short filamentous structures reminding of filament precursors.
COBISS.SI-ID: 5014298