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: 7676892
• 负责人: Paul A. Overbeek
• 金额: $ 104.21万
• 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/7676892
• 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

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）：小鼠中用于在时间和空间维度上操纵基因表达的Cre驱动系的可用性有限，这严重阻碍了生物医学研究进展。研究人员需要这些细胞系才能在正常小鼠中进行基因过度表达、基因错误表达和基因沉默实验，以及在突变生物体中进行基因拯救实验。虽然原则上某些基因的已定义调控元件可用于通过控制空间维度来驱动 Cre 表达，但用于表达天然基因的调控元件数量众多、复杂且难以分解为单个元件。此外，在小鼠大脑中在非常有限的空间域中表达的基因非常少，因此传统的敲入方法预计无法提供精确的表达空间特异性。研究人员与其他模式生物（特别是果蝇）合作开发的另一种策略是增强子捕获。在该策略中，携带可促进目的基因表达的最小启动子的DMA片段被随机整合到转基因动物的基因组中，因此任何随机整合体都会篡夺附近增强子的活性，从而导致目的基因根据附近增强子的决定因素在时间和空间上表达。这些策略使果蝇研究领域充满了数千种增强子捕获系，这些增强子捕获系几乎在生物体的任何组织和细胞类型中表达 Gal4 或诱导型 Gal4 衍生物，而这些试剂是无数发现的基础。我们在这里建议在鼠标中采用类似的策略。我们将在未来 5 年内在小鼠体内构建并验证 2500 多个携带诱导型 Cre 的新增强子捕获系。我们预计这些细胞系中的 1% 到 5% 将在时间和空间上在大脑中显示出特定的 Cre 表达。该项目将在贝勒医学院进行，该学院以其对小鼠遗传学的重视和卓越而闻名。该项目将包括在果蝇中开发新的诱导型增强子陷阱载体并使用这些载体对大脑表达模式进行大屏幕筛选的研究人员、构建携带诱导型 Cre 的新增强子陷阱载体的杰出分子生物学家、领导构建数百个新的小鼠转基因/敲除模型的小鼠遗传学家以及使用高通量、 用于 RNA 原位杂交和其他类型组织学程序的机器人工作站。与首先定义适当的调控元件和/或将 Cre 敲入定义的基因的策略相比，使用基于随机增强子捕获的方法获得新的 Cre 驱动系，为实验者提供对表达、时间和空间的控制的效率将要高得多。这是从果蝇遗传学中学到的教训，并且有充分的理由期望它在小鼠身上也能成立。