Generation and validation of inducible Cre driver lines by enhancer trapping

通过增强子捕获生成和验证诱导型 Cre 驱动线

• 批准号: 7933921
• 负责人: Paul A. Overbeek
• 金额: $ 109.97万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-12 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7933921
• 关键词: Adult; Animal Model; Biomedical Research; Brain; C57BL/6 Mouse; CMV promoter; Chromatin; Complex; Dimensions; Doxycycline; Drosophila genus; Elements; Embryo; Enhancers; Frequencies; Gene Expression; Gene Expression Regulation; Gene Targeting; Generations; Genes; Genetic; Genetic Enhancer Element; Genome; Genomics; In Situ Hybridization; Individual; Infection; Injection of therapeutic agent; Insulator Elements; Introns; Inverse Polymerase Chain Reaction; Knock-in Mouse; Knock-out; Learning; Lentivirus Vector; Link; Medicine; Modeling; Molecular; Mus; Organism; Pattern; Positioning Attribute; Procedures; RNA; Reagent; Regulation; Regulatory Element; Research; Research Personnel; Response Elements; Site; Specificity; Subfamily lentivirinae; TP53 gene; Tamoxifen; Testing; Time; Tissues; Trans-Activators; Transgenes; Transgenic Animals; Transgenic Mice; Transgenic Organisms; Validation; Work; acronyms; base; cell type; college; expression vector; gene function; improved; interest; mouse genome; mutant; overexpression; promoter; recombinase; research study; vector; Adult; Animal Model; Biomedical Research; Brain; C57BL/6 Mouse; CMV promoter; Chromatin; Complex; Dimensions; Doxycycline; Drosophila genus; Elements; Embryo; Enhancers; Frequencies; Gene Expression; Gene Expression Regulation; Gene Targeting; Generations; Genes; Genetic; Genetic Enhancer Element; Genome; Genomics; In Situ Hybridization; Individual; Infection; Injection of therapeutic agent; Insulator Elements; Introns; Inverse Polymerase Chain Reaction; Knock-in Mouse; Knock-out; Learning; Lentivirus Vector; Link; Medicine; Modeling; Molecular; Mus; Organism; Pattern; Positioning Attribute; Procedures; RNA; Reagent; Regulation; Regulatory Element; Research; Research Personnel; Response Elements; Site; Specificity; Subfamily lentivirinae; TP53 gene; Tamoxifen; Testing; Time; Tissues; Trans-Activators; Transgenes; Transgenic Animals; Transgenic Mice; Transgenic Organisms; Validation; Work; acronyms; base; cell type; college; expression vector; gene function; improved; interest; mouse genome; mutant; overexpression; promoter; recombinase; research study; vector

DESCRIPTION (Provided by Applicant): The limited availability of Cre driver lines in the mouse for manipulating the expression of genes in the dimensions of both time and space has severely hampered biomedical research progress. These lines are required for investigators to be able to perform gene overexpression, gene misexpression, and genesilencing experiments in the normal mouse as well as gene-rescue experiments in mutant organisms. Although defined regulatory elements of some genes could, in principle, be used to drive Cre expression with control over the spatial dimension, the regulatory elements that are used to express native genes are numerous, complex, and difficult to dissect into individual elements. In addition, there are very few genes that are expressed in the mouse brain in a very limited spatial domain, such that classical knock in approaches are not anticipated to provide precise spatial specificity on expression. An alternative strategy developed by investigators working with other model organisms, particularly Drosophila, is enhancer trapping. In this strategy, a DMA segment carrying a minimal promoter that can promote the expression of a gene of interest is integrated into the genome at random in transgenic animals, so that any random integrant usurps the activity of a nearby enhancer and this causes the gene of interest to be expressed in time and space according to the determinants of the nearby enhancer. Such strategies have populated the Drosophila research field with thousands of enhancer trap lines that express Gal4 or inducible Gal4 derivatives in virtually any tissue and cell type of the organism and these reagents are the basis for uncountable discoveries. We propose here, a similar strategy in the mouse. We will construct and validate over the next 5 years more than 2500 new enhancer trap lines in the mouse that carry inducible forms of Cre. We anticipate that between 1 and 5 percent of these lines will show specific Cre expression in the brain in both time and space. The project will be performed at the Baylor College of Medicine, known for its emphasis on and excellence in mouse genetics. The project will include investigators that have developed new and inducible enhancer trap vectors in Drosophila and used these to perform large screens for brain expression patterns, outstanding molecular biologists to construct new enhancer trap vectors carrying inducible versions of Cre, mouse geneticists that have led in the construction of hundreds of new mouse transgenic/knock-out models, and experts in the histological analysis of gene expression in the mouse brain using high-throughput, robotically-based workstations for RNA in situ hybridization and other types of histological procedures. The efficiency of obtaining new Cre driver lines offering experimenter control over expression and time and space will be much greater using the random enhancer trapping-based approach compared to strategies for first defining appropriate regulatory elements and/or knocking Cre into defined genes. This is the lesson learned from Drosophila genetics and there is every reason to expect it to hold in the mouse.

描述（由申请人提供）：小鼠中CRE驱动线的可用性有限，用于操纵时间和空间的基因表达，这严重阻碍了生物医学研究的进度。这些线是需要在正常小鼠中进行基因过表达，基因表达和基因实验的基因过表达，以及突变生物体中的基因响应实验所必需的。尽管原则上可以使用某些基因的定义调节元件来控制CRE表达，以控制空间维度，但用于表达天然基因的调节元素是众多，复杂的，并且难以剖析成单个元素。此外，在非常有限的空间结构域中，在小鼠大脑中表达的基因很少，因此，预计在方法中的经典敲击不会提供表达的精确空间特异性。与其他模型生物（尤其是果蝇）一起工作的研究人员制定的替代策略是增强子捕获。在此策略中，携带最小启动子的DMA段可以促进感兴趣基因的表达在转基因动物中随机的基因组中，因此任何随机整合物都会篡夺附近增强子的活性，这会导致根据附近增强器的确定性在时间和空间中表达的兴趣基因。这些策略已经填充了果蝇研究领域，其中数千种增强子陷阱线在几乎任何组织和细胞类型中表达GAL4或可诱导的GAL4衍生物，这些试剂是无数发现的基础。我们在这里提出，这是鼠标中的类似策略。我们将在接下来的5年中构建和验证小鼠中携带可诱导形式的CRE的2500多个新增强器陷阱线。我们预计，这些线的1-5％之间会在时间和空间中显示出大脑中特定的CRE表达。该项目将在贝勒医学院（Baylor College of Medainical）进行，以其对老鼠遗传学的卓越和卓越的重视而闻名。 The project will include investigators that have developed new and inducible enhancer trap vectors in Drosophila and used these to perform large screens for brain expression patterns, outstanding molecular biologists to construct new enhancer trap vectors carrying inducible versions of Cre, mouse geneticists that have led in the construction of hundreds of new mouse transgenic/knock-out models, and experts in the histological analysis of gene expression in the mouse brain using high-throughput,基于机器人的RNA原位杂交和其他类型的组织学程序的工作站。与首先定义适当的调节元件和/或将CRE撞向定义的基因相比，使用基于随机增强子的方法，获得实验者对表达和时间和空间的新CRE驱动线的效率将更大。这是从果蝇遗传学中学到的教训，有充分的理由期望它在鼠标中保持。