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

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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.