Abstract
<jats:p> 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 (G<jats:sub>0</jats:sub>) and very slowly 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 G<jats:sub>0</jats:sub> 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<jats:sub>p</jats:sub>-FUCCI) was used to track single cells as they traversed the cell cycle. A set of R-scripts were written to quantify K67<jats:sub>p</jats:sub>-FUCCI over time. To enable the further study G<jats:sub>0</jats:sub> and SD phenotypes, we retrofitted a live cell imaging system 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 ∼24 and ∼48 h, respectively, with high duration variability in G<jats:sub>1</jats:sub> and G<jats:sub>2</jats:sub> phases. 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. </jats:p>
Original language | English |
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Pages (from-to) | 468-477 |
Number of pages | 0 |
Journal | Physiological Genomics |
Volume | 52 |
Issue number | 10 |
DOIs | |
Publication status | Published - 1 Oct 2020 |