Development and application of optimised systems for neutral cell marking

稳定的遗传标记已成功应用于实验动物模型以及临床研究中，以长期跟踪体内细胞的命运。然而，我们和其他研究小组已经表明，用于永久标记的逆转录病毒载体的整合可能会对标记细胞的命运产生重大影响，例如由于插入诱变引起的诱导克隆优势甚至白血病。在过去的几年里，特别是在该项目的第一阶段，我们开发了新的基因转移载体，移植模型，标记策略和分析工具。这些技术使我们能够在单细胞水平上检查不同器官系统的再生，同时最大限度地减少标记对实验读数的影响。基于这一初步工作，本项目旨在开发用于理想中性细胞标记的优化系统。为此，将结合几种创新工具，特别是具有非常低的诱变潜力的新逆转录病毒载体以及新的DNA条形码。在该项目的前期工作中，我们开发了逆转录病毒启动子缺陷载体以及具有无与伦比复杂性的创新条形码，后者与生物信息学科学家合作。此外，以广泛中性插入模式和不存在异常剪接信号为特征的α逆转录病毒载体可用于计划的研究。我们将测试我们的新条形码以及基于所述技术的新载体在我们建立的鼠干细胞移植模型中用于中性细胞标记的适用性。这将使我们有可能在克隆水平上分析造血随时间推移的近乎“自然”的重建。造血再生动力学的比较深入的数据是我们的知识unavailable.The结果，我们的研究将提供新的见解造血的生物学和稳态，以及在其再生过程中的单个HSC克隆的贡献。从基础科学的角度来看，更好地理解血液形成的克隆重建当然具有重大意义，例如关于骨髓中成人造血干细胞的数量和再生潜力。它在临床干细胞移植和相关基因治疗方法的背景下也非常重要。此外，本项目将建立一种创新技术，用于生成最复杂的条形码。基于此的新的条形码载体将用于各种标记研究和定量克隆分析。

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.