Molecular characterization and functional analysis of novel ARCI genes involved in differentiation of human epidermis
Final Report Abstract
RNA Seq is a promising method to perform expression analysis for functional studies of the cell. However, this method is too expensive if a larger, representative number of samples has to be analyzed. From the results of the RNA Seq as well as the experience with primary cell culture of mutant keratinocytes, it turns out that the viability of the cells are not guaranteed. Alternative methods, such as CRISPR/Cas-System in combination with 3D skin, are important for functional studies and will be further pursued. We were able to show that 2D differentiation of keratinocytes is so far the best method to perform subcellular studies (e.g. IF). However, 2D differentiation of keratinocytes does not correspond to the natural process of skin formation. The 3D skin models are closer to the real skin than 2D differentiated keratinocytes. With 2D differentiation, previous suggestions that SDR9C7 and SULT2B1 are more highly expressed only in the late differentiated keratinocyte were confirmed. Based on the results in 3D experiment, the localization of SDR9C7 and SULT2B1 could also be confirmed in the skin model. A co-localization of SDR9C7 at the Golgi apparatus could be confirmed in primary keratinocytes. However, this phenomenon is only seen in differentiation with low cell density and does not correspond to skin differentiation. Functional analysis of the CRISPR/Cas9 gene edited cells as well as the creation of 2- and 3D skin models is still ongoing.
