A next generation Quiescence and Cell Cycle Indicator (QuCCi) for the refinement and reduction of animal usage in proliferation assays

Healthy tissues develop normally and are maintained through a tightly regulated cell cycle (or proliferation program). Lengthening or shortening of the cell cycle is a fundamental disease mechanism in development, ageing and cancer. It is likely that many developmental diseases are caused at least in large part by early defects of cell proliferation and many studies are now using mouse models of these conditions to explore this hypothesis in numerous systems. Mouse cell cycle kinetics are often investigated using thymidine analogues such as BrdU - incorporated into DNA during S-phase. These techniques rely on multiple injections or the surgical implantation of mini osmotic pumps to deliver the analogue. Recently, genetically encoded cell cycle probes have been developed and these are key to refining proliferation assays because they can be combined with live-imaging efficiently generating large datasets from fewer animals. Of these, the best existing model is the R26Fucci2aR (developed by RLM) reporter model because of the 1:1 stoichiometry of probe expression and the ability to control expression spatio-temporally with Cre-recombinase. However a number of shortcomings still exist: 1) It does not discriminate S-phase from G2/M-phases; 2) It does not discriminate G1 from quiescent cells in G0. 3) The probes are technically difficult to detect with antibodies limiting them to live studies. In order to address these shortcomings we will develop a next generation Quiescence and Cell Cycle Indicator (QuCCI) and Rosa26 QuCCi knock-in reporter mouse. QuCCi incorporates the the tried and tested probes p27K-,hCdt1(Cy-) and hGem(1/110) fused to mCerulean, mCherry and mVenus respectively including unique epitope tags. QuCCi can discriminate between cells in G1, S, G2/M and G0. The PhD student will join an existing team across Lancaster University (UK), Edinburgh University (UK) and McGill University (Canada) who have already committed time and resources to the development of the model. The team that we have assembled to support the studentship is perfectly placed to facilitate the project because we have extensive experience and proven track records in developing multicistronic constructs and cell lines (Dr. Mort, Dr. Ford), generating transgenic mice using CRISPR/Cas9 technology including the insertion of large constructs using injection into two-cell embryos (Dr. Yamanaka, Dr. Cowan) and in performing image analysis and quantitative imaging techniques (Dr. Mort, Prof. O'Shea).