Methods and Sensors For Functional Genomic Studies of Cell-Cycle Transitions in Single Cells.

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 (K67p-FUCCI) was used to track single cells as they traversed the cell cycle. A set of R-scripts were written to quantify K67p-FUCCI over time. To enable the further study Go 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.