Abstract
The transition of a proliferating cell into a state of cell quiescence is a key step in organogenesis and tissue homeostasis. It is currently difficult to distinguish between quiescent cells and those that have entered the cell cycle. This study’s objective was to generate a fluorescent‐based genetically encoded reporter capable of differentiating between arrested and actively cycling cells. The Ki67 promoter (Ki67p) was previously shown in our lab to selectively label cells that have entered the cell cycle. However, using Ki67p to drive GFP as a reporter gene results in fluorescent daughter cells due to GFP’s long half‐life (~26h). We hypothesized using the Ki67p to drive expression of the Fluorescent Ubiquitination‐based Cell Cycle Indicator (FUCCI) reporters would enable indirect detection of quiescent (G0) cells and cells in G1/S and S/G2/M. The existing FUCCI reporters employ a constitutively active promoter, thereby preventing discrimination between cells in G0 and cells in G1. To overcome both reporters’ limitations we generated two lentiviral vector constructs with Ki67p upstream of mcherry‐hCdt1(30‐120) and mAG‐hGem(1‐110). We then generated stable double transgenic cell lines (HT1080 and COS cells) that express mCherry‐hCdt1(30‐120) and mAG‐hGem(1‐110) under control of Ki67p. Fluorescence time‐lapse imaging of Ki67p‐FUCCI cells indicates that low‐density plating induces G0 cell cycle withdrawal and concurrent attenuation of Ki67‐FUCCI reporter activity. Low density compared to high density plating increased the number of Ki67p‐FUCCI− HT1080 cells (44% vs. 5%, P<0.05) indicating G0 entry. Moreover, serum starvation induced G1 arrest (91% Ki67p‐mCherry+ without serum vs. 47% with serum, P<0.01). These reporters will enable studies designed to segregate between quiescent and actively proliferating cells in each of the major phases of the cell cycle in living tissue and in real‐time, facilitating studies in stem cell and cancer biology.Grant Funding Source: NIGMS 2‐R25‐GM‐083275, AHA 10SDG2630130
Published Version
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