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

下一代静止和细胞周期指示器 (QuCCi)，用于细化和减少增殖测定中动物的使用

• 批准号: 2325673
• 金额: --
• 依托单位: Lancaster University
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2325673
• 关键词: next; generation; Quiescence; Cell; Cycle

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).

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）来维持。细胞周期的延长或缩短是发育、衰老和癌症中的基本疾病机制。许多发育性疾病可能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经常使用胸苷类似物，如BrdU -掺入DNA在S期进行研究。这些技术依赖于多次注射或微型渗透泵的手术植入来递送类似物。最近，已经开发了基因编码的细胞周期探针，这些是改进增殖测定的关键，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，最好的现有模型是R26 Fucci 2aR（由RLM开发）报告模型，因为探针表达的1：1化学计量和用Cre重组酶时空控制表达的能力。然而，仍然存在许多缺点：1）它不能区分S期和G2/M期; 2）它不能区分G1期和G 0期的静止细胞。3)这些探针在技术上很难用抗体检测，限制了它们在活体研究中的应用。为了解决这些缺点，我们将开发下一代静止和细胞周期指示剂（QuCCI）和Rosa 26 QuCCi基因敲入报告小鼠。QuCCi结合了分别与mCerulean、mCherry和mVenus融合的久经考验的探针p27 K-、hCdt 1（Cy-）和hGem（1/110），包括独特的表位标签。QuCCi可以区分G1、S、G2/M和G 0中的细胞。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，他们已经投入了时间和资源来开发模型。我们为支持该项目而组建的团队非常适合促进该项目，因为我们在开发多顺反子构建体和细胞系方面拥有丰富的经验和良好的记录（Dr. Mort，Dr.福特），使用CRISPR/Cas9技术产生转基因小鼠，包括使用注射到两细胞胚胎中插入大构建体（Dr. Yamanaka，Dr.科万）和进行图像分析和定量成像技术（Dr. Mort，Prof. O 'Shea）。