Regulation of protein expression using a cell cyclce-specific degron and its application to functional analysis

fucci1是一种新开发的细胞周期特异性荧光发生器。这是基于多泛素化诱导的Cdt1或Geminin蛋白的细胞周期依赖性降解。我们认为，如果我们将FUCCI中使用的Cdt1或Geminin的一部分与以细胞周期特异性方式控制表达的蛋白质融合，就可以利用这一点。当与鸡DT40细胞系统的强大遗传学结合使用时，这将是最有用的。因此，我们克隆了与人类用于FUCCI的Cdt1或Geminin部分相对应的鸡Cdt1或Geminin。然而，我们无法在野生型DT40细胞中实现高表达水平。我们已经创造了一种条件缺乏Treslin基因的细胞系，该基因是酵母Sld3同源物，对DNA复制的启动至关重要。该细胞系对研究Treslin的功能具有重要意义。

FUCCIis a recently developed cell cycle specific fluorescent maker. This is based on the cell cycle dependent degradation of Cdt1 or Geminin proteins induced by polyubiquitination. We reasoned this can be exploited if we fuse a part of Cdt1 or Geminin used in FUCCI with a protein forcontrolingexpression in a cell cycle specific manner. This would be most useful when used in combination with powerful genetics in chicken DT40 cell system. Thus we cloned chicken Cdt1 or Geminin thatcorresponds to part of human Cdt1 or Geminin used for FUCCI. However, we could not achieve high expression levels in wild type DT40 cells. We havecreated a cell line rendered conditionally deficient in the Treslin gene, which is a yeast Sld3 homologessential for intiation of DNA replication.This cell line would be useful to study Treslin function.