Much of our understanding of the regulatory mechanisms governing the cell cycle in mammals has relied heavily on methods that measure the aggregate state of a population of cells. While instrumental in shaping our current understanding of cell proliferation, these approaches mask the genetic signatures of rare subpopulations such as quiescent (G₀) and very slow dividing (SD) cells. Results described in this study and those of others using single-cell analysis reveal that even in clonally derived immortalized cancer cells, ~1-5% of cells can exhibit G₀ and SD phenotypes. Therefore to enable the study of these rare cell phenotypes we established an integrated molecular, computational and imaging approach to track, isolate and genetically perturb single cells as they proliferate. A genetically encoded cell cycle reporter (K67_p-FUCCI) was used to track single cells as they traversed the cell cycle. A set of R-scripts were written to quantify K67_p-FUCCI over time. To enable the further study G_o and SD phenotypes, a live cell imaging system was retrofitted with a micromanipulator to enable single-cell targeting for functional validation studies. Single cell analysis revealed HT1080 and MCF7 cells had a doubling time of ~24h and ~48h, respectively, with a high degree of variability in G₁ and G₂ phase duration. Direct single cell microinjection of mRNA encoding (GFP) achieves detectable GFP fluorescence within ~5 h in both cell types. These findings coupled with the possibility of targeting several hundreds of single cells improves throughput and sensitivity over conventional methods to study rare cell subpopulations.

中文翻译：

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用于单细胞中细胞周期转换的功能基因组研究的方法和传感器。

我们对哺乳动物细胞周期调控机制的理解很大程度上依赖于测量细胞群聚集状态的方法。虽然有助于塑造我们目前对细胞增殖的理解，但这些方法掩盖了罕见亚群的遗传特征，如静止 (G ₀ ) 和非常缓慢分裂 (SD) 细胞。本研究中描述的结果和其他使用单细胞分析的结果表明，即使在克隆衍生的永生化癌细胞中，~1-5% 的细胞也可以表现出 G ₀和 SD 表型。因此，为了能够研究这些稀有细胞表型，我们建立了一种综合的分子、计算和成像方法来跟踪、分离和遗传干扰单个细胞的增殖。基因编码的细胞周期报告基因 (K67 _p -FUCCI) 用于跟踪单个细胞，因为它们穿过细胞周期。编写了一组 R 脚本以随时间量化 K67 _p -FUCCI。为了能够进一步研究 G _o和 SD 表型，活细胞成像系统被改装了一个显微操作器，以实现功能验证研究的单细胞靶向。单细胞分析显示 HT1080 和 MCF7 细胞的倍增时间分别约为 24 小时和 48 小时，G _{1具有高度可变性}和 G ₂阶段持续时间。在两种细胞类型中，直接单细胞显微注射 mRNA 编码 (GFP) 可在约 5 小时内实现可检测的 GFP 荧光。这些发现与靶向数百个单细胞的可能性相结合，提高了研究稀有细胞亚群的传统方法的通量和灵敏度。