Improvement and application of a non-viral episome for mammalian cells

该项目将利用最近在Witten/Herdecke大学细胞生物学研究所构建的非病毒表观染色体载体系统，分析选择哺乳动物复制起点所需的表观遗传学特征，稳定地建立Episome，并在没有着丝粒序列的情况下提供有丝分裂稳定性。该载体在目前测试的所有哺乳动物细胞系中复制，包括原代细胞，并用于产生转基因动物。它的最小功能元素已被描述，因此可以很容易地操纵它。选择一个起源和建立作为一个稳定的Episome可能需要类似或相同的机制。必须采用正确的染色质结构，并且Episome的起源必须分别进入特定的核区域。在这个项目的框架内，将分析染色质结构，即组蛋白编码和DNase I超敏部位与核定位的相关性。通过结合c/.v作用序列，将载体定向到核的特定间隔，结合作为染色质的载体的转染，将增加作为Episome的建立。在没有着丝粒序列的情况下，载体的有丝分裂稳定性似乎也与稳定建立的问题密切相关。因此，原型载体及其衍生物的行为将在整个细胞周期中进行研究。该项目所取得的结果将有助于更深入地理解DNA复制的表观遗传控制，同时也将为合理设计最终可能用于基因治疗的载体提供基础。

This project will exploit a non-viral episomal vector system recently constructed in the Institute of Cell Biology, University Witten/Herdecke to analyze the epigenetic features required for selection of a mammalian origin of replication, to stably establish an episome and to provide mitotic stability in the absence of a centromere sequence. The vector used replicates in all mammalian cell lines tested so far, including primary cells and was used to generate genetically modified animals. Its minimal functional elements have been described and therefore it can be manipulated with ease. Selection of an origin and establishment as a stable episome require probably similar or the same mechanisms. The correct chromatin structure has to be adopted and the episome, respectively origin has to enter a specific nuclear region. In the frame of this project the relevance of chromatin structure, i.e. histone code and DNase I hypersensitive sites, and of nuclear localization will be analyzed. By the incorporation of c/.v-acting sequences targeting the vector to specific compartments of the nucleus combined with transfection of the vector as chromatin will increase establishment as an episome. Mitotic stability of the vectors in absence of a centromere sequence seems also to be closely related to the problem of stable establishment. Therefore, the behaviour of the prototype vector as well as its derivatives will be studied throughout the cell cycle. Results obtained within this project will lead to a deeper understanding of the epigenetic control of DNA replication but will also provide the basis for a rational design of vectors which may eventually be used in gene therapy.