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

健康的组织正常发育，并通过严格调节的细胞周期（或增殖程序）来维持。细胞周期的延长或缩短是发育、衰老和癌症的基本疾病机制。很可能，许多发育性疾病至少在很大程度上是由细胞增殖的早期缺陷引起的，许多研究现在正在使用这些条件的小鼠模型来探索许多系统中的这一假设。小鼠细胞周期动力学研究通常使用胸苷类似物，如BrdU -在s期合并到DNA中。这些技术依靠多次注射或手术植入微型渗透泵来输送类似物。最近，基因编码的细胞周期探针已经开发出来，这些探针是改进增殖试验的关键，因为它们可以与实时成像相结合，有效地从更少的动物中生成大数据集。其中，现有的最佳模型是R26Fucci2aR（由RLM开发）报告因子模型，因为探针表达的1:1化学计量和cre -重组酶在时空上控制表达的能力。但也存在一些不足：1)不能区分s相和G2/ m相；2) G0中不区分G1和静止细胞。3)探针在技术上很难检测到抗体，限制了它们的活体研究。为了解决这些不足，我们将开发新一代静止与细胞周期指示器（QuCCI）和Rosa26 QuCCI敲入报告小鼠。QuCCi将经过尝试和测试的探针p27K-，hCdt1（Cy-）和hGem（1/110）分别与mCerulean， mCherry和mVenus融合，并具有独特的表位标签。QuCCi可以区分G1、S、G2/M和G0的细胞。该博士生将加入兰开斯特大学（英国）、爱丁堡大学（英国）和麦吉尔大学（加拿大）的现有团队，他们已经为该模型的开发投入了时间和资源。我们组建的支持学生的团队完全可以促进项目，因为我们在开发多顺子结构和细胞系（Mort博士，Ford博士），使用CRISPR/Cas9技术产生转基因小鼠(包括通过注射将大型结构插入双细胞胚胎（Yamanaka博士，Cowan博士）以及执行图像分析和定量成像技术（Mort博士，Ford博士）方面拥有丰富的经验和良好的记录。奥谢教授)。

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).