由重组酶creflp介导的单细胞标记系统的构建.pdf

摘 要 细胞标记和追踪是细胞生物学和发育生物学研究领域中的一个重要方法和手段。一 种新颖、有效且便于操作的细胞标记和追踪方法有可能对相关领域的研究产生意义深远的 影响。但是，目前还没有一种细胞标记方法可用于准确地追踪单个细胞及其后代。为弥补 这一技术空白，我们设计了一个基于 Cre-loxP 和 Flp-Frt 位点特异性重组系统的多个细胞 标记技术。这一技术的核心包括两个部分：由四个 Frt 片段组成的细胞基因组标签，以及 催化该标签发生随机重组因而可产生八种多样性的 Cre/Flp 双重组酶瞬时表达载体。我们 成功地将单个 Frt 标签插入 293FT ，并验证了 Cre/Flp 双重组酶瞬时表达的特性。利用这 一技术，我们有望从单克隆的 293FT 细胞培养出八种携带永久性不同标签的 293FT 克隆。 通过增加 Frt 片段的数目，理论上，我们将可能对单个组织干细胞进行标记并追踪其后代， 从而直接验证单个组织干细胞是否具有多潜能性。因此，这一新颖、独特的细胞标记技术 将可能对细胞生物学和发育生物学研究产生重要影响。 关键词：单个细胞标记；Cre-loxP；Flp-Frt；瞬时表达；多潜能性 I Abstract Cell labeling and tracing provides a promising approach for studying cell biology and developmental biology. A creative, effective and convenient cell tracing technology is likely to bring about a profound influence to related research areas. Unfortunately, single cell labeling, in contrast to the well-established group marking system, which has the potential to follow the developmental fate(s) of an individual progenitor, has not been reported. Toward this end, we designed an individual cell labeling technique mediated by both Cre-loxP and Flp-Frt site specific recombination systems. The major theme of this technique comprised two parts: a genomic tag containing four Frt sites which was separated by stuffer sequences of different length; and a transiently expressing dual recombinase system (Cre/Flp) which could carry out a random recombination between Frt sites and thus generated eight permutations. In this study, we have successfully inserted one copy of the Frt tag into the genome of 293FT cells, and also verified the transient expression characteristic of the dual recombinase system. By using this technique, we would be able to generate eight 293FT clones carrying distinct Frt tags permanently from the original 293FT clone. T