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

本项目将利用维滕/埃尔德克大学细胞生物学研究所最近构建的非病毒附加体载体系统，分析选择哺乳动物复制起点所需的表观遗传特征，以稳定地建立附加体并在缺少着丝粒序列的情况下提供有丝分裂稳定性。该载体在迄今为止测试的所有哺乳动物细胞系（包括原代细胞）中重复使用，并用于产生转基因动物。它的最小功能元件已被描述，因此它可以很容易地操作。一个起源的选择和作为一个稳定的附加体的建立可能需要相似或相同的机制。必须采用正确的染色质结构，并且附加体，分别起源必须进入特定的核区域。在这个项目的框架内，将分析染色质结构的相关性，即组蛋白编码和DNA酶I超敏感位点，以及核定位。通过掺入将载体靶向至细胞核的特定区室的顺式作用序列，结合载体作为染色质的转染，将增加作为附加体的建立。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，将在整个细胞周期中研究原型载体及其衍生物的行为。在这个项目中获得的结果将导致更深入地了解DNA复制的表观遗传控制，但也将提供一个合理的设计载体，最终可能用于基因治疗的基础。

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.