Development and application of optimised systems for neutral cell marking

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Stable genetic marking has been successfully applied in experimental animal models as well as in clinical studies to follow the fate of cells in vivo over long periods of time. However, we and other groups have shown that the integration of retroviral vectors used for permanent marking may exert a significant influence on the fate of marked cells, as exemplified by the instances of induced clonal dominance or even leukaemia due to insertional mutagenesis. During the last years, particularly in the 1st phase of this project we have developed new gene-transfer vectors, transplantation models, marking strategies and analysis tools. Together these techniques allowed us to examine the regeneration of different organ systems on a single-cell level and to minimise at the same time the influence of the marking on the experimental read-out. Based on this preliminary work the present project aims at the development of optimised systems for an ideally neutral marking of cells. To do so, several innovative tools will be combined, in particular new retroviral vectors with a very low potential for mutagenesis as well as new DNA-barcodes. In preliminary work for this project we have developed retroviral promoter-deficient vectors as well as innovative barcodes with unmatched complexity, the latter in co-operation with bioinformatics scientists. Also, alpharetroviral vectors characterised by a widely neutral insertion pattern and the absence of aberrant splice signals are available for the planned studies. We will test the suitability of our new barcodes as well as the new vectors based on the described technologies for neutral cell marking in our established murine stem cell transplantation model. This will give us the possibility to analyse a nearly "natural" reconstitution of haematopoiesis over time at a clonal level. Comparable in-depth data on the kinetics of haematopoietic regeneration is to our knowledge not available yet.The results of our studies will provide new insights into the biology and homeostasis of haematopoiesis as well as the contribution of individual HSC clones during its regeneration. A better understanding of clonal reconstitution of blood formation is certainly of significant interest from a basis-science point of view, e.g. with regard to the numbers and the regenerative potential of adult, haematopoietic stem cells in the marrow. It is also very important in the context of clinical stem cell transplantation and related gene therapy approaches. Additionally, the present project will establish an innovative technology for the generation barcodes of highest complexity. The new barcode vectors based thereon will be useful for various marking studies and quantitative clonality analyses.