Briggs, Tiernan and Mort, Richard (2024) Development of cell cycle biosensors with improved optical discrimination for use within cell and animal models. PhD thesis, Lancaster University.
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Abstract
Tools to analyse the cell cycle have thus far been limited in various ways. Whilst Fucci represents the gold standard in live cell analysis of proliferation, it provides no insight into mitosis or cells that have exited the cell cycle. Furthermore, whilst the newer Fucci(CA) allows discrimination of G1, S and G2 phases (unlike Fucci which discriminated G1 and S/G2) this construct was comprised of two separate probes and was incapable of G0 or M phase discrimination. Thus, random transgenesis, variegated transgene expression, limited phase discrimination and the lack of a Cre-inducible mouse line results in this construct also being severely limited. Fucci2a addressed some of these issues through self-cleaving peptides but not that of cell cycle phase discrimination. Alternative biosensors such as the p27K quiescent cell biosensor allowed for visualisation of cells that had entered into quiescence and the H1.0 biosensor allowed for visualisation of M phase. Here we report the fusion of Fucci(CA) to the p27K and H1.0 biosensors utilising self-cleaving peptides to produce tricistronic cell cycle biosensors with improved cell cycle phase discrimination and delineation. Improvements to the Fucci probeset allow for accurate identification of S phase as well as the beginning of G1. Whilst multiplexing of these probes allowed for M phase or G0 phase identification depending on the third probeset included. Here we report the generation and validation of both Fucci(CA)-p27k- (Qucci) and H1.0-Fucci(CA) probesets that that can accurately discriminate all cell cycle phases and show improved probe degradation and easier maintenance. We also report the generation of a Creinducible Qucci mouse line and both Qucci and H1.0-Fucci(CA) cell cycle reporter chicken lines.