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，并通过严格调控的细胞周期(或增殖程序)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早期细胞增殖缺陷引起的，许多研究现在正在使用这些条件的小鼠模型在许多系统中探索这一假说。小鼠细胞周期动力学研究通常使用胸腺嘧啶核苷类似物，如在S期掺入DNA。这些技术依赖于多次注射或外科手术植入微型渗透泵来提供类似物。最近，基因编码的细胞周期探针已经被开发出来，这些探针是完善增殖分析的关键，因为它们可以与实时成像相结合，有效地从较少的动物中生成大数据集。其中，现有最好的模型是R26Fucci2aR(由RLM开发)报告模型，因为探针表达的化学计量比为1：1，并且能够用Cre重组酶在时空上控制表达。但仍存在一些不足之处：1)它没有区分S期和G2/M期；2)它没有区分G0期细胞和G1期细胞。3)这些探针在技术上很难被检测到，因为抗体限制了它们的活体研究。为了解决这些不足，我们将开发下一代静息和细胞周期指示器(QuCCI)和rosa26QuCCI敲入报告鼠。QuCCi结合了分别与mCerulean、mCherry和mVenus融合的经过试验和测试的探针p27K-、hCdt1(Cy-)和hGem(1/110)，包括独特的表位标签。去CCI可以区分G1、S、G2/M和G0期细胞。这位博士生将加入英国兰开斯特大学、英国爱丁堡大学和加拿大麦吉尔大学的现有团队，这些大学已经投入了时间和资源来开发该模型。我们为支持该项目而组建的团队非常适合推动该项目，因为我们在开发多顺反子结构和细胞系(Mort博士，Ford博士)、使用CRISPR/Cas9技术培育转基因小鼠(包括通过注射到两细胞胚胎中的大型结构物)以及执行图像分析和定量成像技术(Mort博士、O‘Shea教授)方面拥有丰富的经验和可靠的跟踪记录。

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).