Generation and Preservation of Novel Mouse Models

新型小鼠模型的生成和保存

• 批准号: 8900946
• 负责人: MARK J SHLOMCHIK
• 金额: $ 33.79万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-17 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8900946
• 关键词: Address; Alleles; Animals; Autoimmune Process; Biological Preservation; Breeding; Budgets; Cataloging; Catalogs; Cell Culture Techniques; Cell Line; Chimeric Proteins; Collection; Complex; Consultations; Cryopreservation; DNA; Databases; Disease; Drug Design; Embryo; Engineering; Failure; Fertilization; Fertilization in Vitro; Freezing; Funding; Gene Targeting; Gene-Modified; Generations; Genes; Genetic; Genetically Engineered Mouse; Genetically Modified Animals; Genomics; Genotype; Grant; Immunologics; Infection; Institutes; Institution; Internet; Investments; Knock-out; Knockout Mice; Left; Libraries; Link; Maintenance; Mechanics; Methods; Microinjections; Modification; Mouse Strains; Mus; Mutation; Point Mutation; Procedures; Process; Proteins; Publishing; Reagent; Recovery; Reporter; Research; Research Personnel; Resources; Rheumatism; Risk; Services; Shipping; Ships; Solutions; Speed; Techniques; Technology; Testing; Time; Tissues; Transgenes; Transgenic Organisms; Work; cell type; cost; cost effective; design; design and construction; egg; embryonic stem cell; endonuclease; flexibility; gene function; improved; in vivo; innovation; knockout gene; member; mouse model; mutant; novel; repository; sperm cell; trait; vector; zygote

To define the functions of genes and cell types in vivo, there is no substitute for the power of modifying the mouse germline to generate gene knockouts (KO) and mutants or to express new genes transgenically. For optimal interpretation, genes need to inactivated conditionally or only in certain tissues, or expressed as transgenes in a tissue specific manner in the native genomic context. Using these techniques, investigators worldwide and particularly at Yale, have already been making numerous KO, knockin, transgenic, etc. mouse strains and breeding the modified alleles onto useful genetic backgrounds. These strains, many of which are published, are invaluable resources for research in Rheumatologic diseases, but the maintenance of them is time consuming and expensive, and one cannot envision the endless collection of more and more strains of actively breeding mice. Moreover, infection and breeding problems threaten the existence of many strains, and make the transfer among investigators cumbersome and costly. At the same time, to date the vast majority of engineered mutations have been made by a relatively small number of investigators; these technologies need to be more widely available so that labs that focus on particular genes or processes can have direct access to modifying them, instead of relying on labs that specialize in making KO mice. An important barrier to the technology is that the process is expensive, difficult to master, fraught with potential pitfalls, and time-consuming. In addition, it is limited to targeting ES cells of 129/Sv or (with more difficulty) B6; autoimmune prone strains cannot be directly modified. To address these issues, Core B will undertake 3 Aims. First, to make gene targeting accessible to YRDRCC members, the Core will provide introduce the novel TALEN endonuclease approach to genetic modification of the mouse germline. It allows the targeting of any gene directly in fertilized eggs, without the need for complex constructs (a simple PCR fragment is used), without the need for unwanted selectable markers, and without any ES cell work. TALEN technology can be applied to any strain, allowing us to directly modify autoimmune-prone animals. Most importantly, it is much faster, simpler, and cheaper, which will allow many more investigators to use the approach. Second, the Core will provide cryopreservation capabilities for the cost-effective storage of genetically modified mice. In our first funding cycle we froze sperm from over 380 strains. However, we did not preserve embryos from strains with multiple modified traits, leaving many very valuable lines unpreserved. We now plan to freeze these strains as embryos. Additionally, the core will continue cryopreservation of sperm, which is still required by many investigators. This will produce substantial decreases in costs associated with preservation of mouse strains. Finally, the Core will promulgate a web database of our 380 (and growing strains), facilitate their distribution to others, and in addition begin to freeze sperm of key strains from non- Yale investigators. This Core is thus both innovative and of high impact within and outside of Yale.

要确定基因和细胞类型在体内的功能，没有什么可以替代修改小鼠生殖系以产生基因敲除(KO)和突变或通过转基因表达新基因的能力。为了获得最佳解释，基因需要有条件地或仅在某些组织中失活，或者在天然基因组环境中以组织特有的方式表达为转基因。利用这些技术，世界各地的研究人员，特别是耶鲁大学的研究人员，已经制作了大量的KO、KNOTKIN、转基因等小鼠品系，并将修改后的等位基因培育到有用的遗传背景上。这些品系，其中许多已经发表，是风湿病研究的宝贵资源，但它们的维护既耗时又昂贵，人们无法想象越来越多活跃繁殖的品系的无穷无尽的收集。此外，感染和繁殖问题威胁到许多菌株的存在，并使研究人员之间的转移变得繁琐和昂贵。与此同时，到目前为止，绝大多数工程突变是由相对较少的研究人员制造的；这些技术需要更广泛地获得，以便专注于特定基因或过程的实验室可以直接修改它们，而不是依赖专门制造KO小鼠的实验室。这项技术的一个重要障碍是，这个过程昂贵，难以掌握，充满了潜在的陷阱，而且很耗时。此外，它仅限于靶向129/Sv或(更困难的)B6的ES细胞；自身免疫倾向株不能直接修饰。为了解决这些问题，核心B将实现三个目标。首先，为了使YRDRCC成员能够获得基因打靶，核心将提供引入新的TALEN内切酶方法来对小鼠生殖系进行遗传修饰。它允许直接在受精卵中靶向任何基因，而不需要复杂的结构(使用简单的PCR片段)，不需要不需要的可选择标记，也不需要任何ES细胞工作。Talen技术可以应用于任何菌株，允许我们直接修改易产生自身免疫的动物。最重要的是，它更快、更简单、更便宜，这将允许更多的调查人员使用这种方法。其次，Core将为具有成本效益的转基因小鼠提供冷冻保存能力。在我们的第一个资助周期中，我们冷冻了380多个品系的精子。然而，我们没有保存具有多个修改性状的菌株的胚胎，留下了许多非常有价值的品系没有保存下来。我们现在计划将这些菌株冷冻为胚胎。此外，核心将继续冷冻保存精子，这仍然是许多研究人员的要求。这将大大降低与保存小鼠品系相关的成本。最后，中心将公布我们380个(和正在生长的菌株)的网络数据库，促进它们分发给其他人，此外，还将开始冷冻来自非耶鲁大学研究人员的关键菌株的精子。因此，这一核心既具有创新性，又在耶鲁大学内外具有很高的影响力。