Development and validation of a novel strategy to reduce the use of transgenic and knockout mice
01 / 2008 - 01 / 2010
The objective of this project is to create a validated in vitro model system that can replace transgenic or knockout mouse approaches, thereby reducing the number of experimental animals used in the field of skin biology and dermatology. We aim to construct lentiviral vectors with three different promoters to direct gene expression in specific epidermal cell types. Both controlled overexpression and knockdown by siRNA will be pursued. Advanced technology (pDeCap vector) will be used to minimize interferon responses. These new vectors will be used in combination with reconstructed skin models to obtain physiologically relevant phenotypes in vitro. To obtain proof of concept we will apply this system to the recently discovered cystatin M/E pathway, for which we will follow a conventional transgenic and knockout strategy in parallel, to allow comparison and validation. BACKGROUND: The use of knockout or transgenic mouse models is a cornerstone of modern biomedical research and has provided mechanistic insights in biology and pathology. The scientific, medical and commercial importance of these technologies has to be weighed against ethical considerations and societal concern on the use of experimental animals. We are currently using knockout and transgenic approaches to investigate the role of various genes in skin biology and skin diseases, involving hundreds of genetically modified mice per year with minor to severe phenotypes. We reasoned that if a generally applicable strategy could be developed for gene knock-down or overexpression in a model that closely resembles normal human skin, this would lead to a significant reduction of animal use and discomfort. STRATEGY AND VALIDATION: Several state-of-the art technologies will be combined to create a novel in vitro system that is functionally equivalent to transgenic or knockout mouse approaches. This will result in a 3-D tissue culture model of reconstructed human skin in which lentiviral vectors are used for gene knockdown (by delivery of siRNA) or overexpression of certain genes. We will create vectors that use stratum-specific promoters of the involucrin, cytokeratin 10 and cytokeratin 14 genes to direct expression to the correct cell layer (granular, spinous and basal). The pDeCap vector technology will be used to avoid interferon responses. To obtain proof of concept, we will apply this new technology to elucidate a new biochemical pathway that we have recently discovered in human epidermis, involving the cystatin M/E-cathepsin V-cathepsin L-transglutaminase-3 pathway. We have already studied part of this pathway in knockout mouse models, which enables us to compare and validate the proposed technology with previous data obtained from experimental animals.DELIVERABLES: We aim to develop a technology that has the potential to significantly reduce the use of genetically modified animals. The results and research tools (vectors) will be made available to the scientific community without restriction. Dissemination of knowledge will be achieved by scientific publications and by organizing an international meeting on skin models within the European Epidermal Barrier Research Network (E2BRN) and the European Society of Dermatological Research (ESDR). When proven successful for skin research, this approach might initiate applications in other tissues that can be studied in vitro. Areas of application range from basic research to target validation in pharmaceutical industry.