Core--Promoter screening

核心--启动子筛选

• 批准号: 6507448
• 负责人: SUSUMU TONEGAWA
• 金额: $ 16.54万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6507448
• 关键词: artificial chromosomes; biomedical facility; gene expression; genetic promoter element; genetic strain; genetic transcription; genetically modified animals; in situ hybridization; laboratory mouse; memory

A major component of this Center is to use genetic manipulation of the mouse brain to unravel the molecular basis of learning and memory. One of the approaches here is to generate strains of mice in which a gene of interest has been eliminated in specific neuronal populations by use of the Cre recombinase. A prerequisite for this strategy is the availability of promoters that target Cre expression to specific populations of neurons in brain regions of interest. The purpose of this core is to identify appropriate genes and generate the targeted Cre mice. The PI proposes to isolate brain region specific transcripts using a combined subtractive hybridization-array screening strategy followed by in situ hybridization. This approach is based on existing commercial methods which require modification in most cases. The applicant is aware of this and has made substantial progress in optimizing the subtraction methodology. An alternative strategy, which the PI may well have considered, is to use in situ hybridization as the secondary screen (rather than using array technology). However, the proximity of the MIT Genome Center, which has extensive expertise in robotic screening methods, also favors the array approach. Following the isolation of transcripts, BACS will be identified for the clones having the requisite neuronal specificity and these used to generate BAC-Cre constructs. The use of BACs to construct transgenic mice has gained popularity and is probably the best approach available (assuming the candidate gene is represented in the commercial BAC libraries). The BAC fusion constructs will be used to make lines of transgenic mice that express Cre recombinase in specific populations of neocortical neurons. Subsequently, these mice will be crossed with lines of mice that harbor a gene of interest flanked by loxP sites.

该中心的一个主要组成部分是使用老鼠大脑的遗传操作来解开学习和记忆的分子基础。这里的方法之一是产生小鼠品系，其中通过使用Cre重组酶在特定神经元群体中消除了感兴趣的基因。该策略的先决条件是启动子的可用性，该启动子将Cre表达靶向到感兴趣的脑区域中的特定神经元群体。该核心的目的是鉴定合适的基因并产生靶向Cre小鼠。PI建议使用组合消减杂交阵列筛选策略，然后进行原位杂交，分离脑区特异性转录本。这种方法是基于现有的商业方法，在大多数情况下需要修改。申请人意识到这一点，并在优化减法方法方面取得了实质性进展。PI可能已经考虑过的另一种策略是使用原位杂交作为二级筛选（而不是使用阵列技术）。然而，在机器人筛选方法方面拥有广泛专业知识的麻省理工学院基因组中心的邻近也有利于阵列方法。 在分离转录物之后，将鉴定具有必需的神经元特异性的克隆的BACS，并且这些克隆用于产生BAC-Cre构建体。使用BAC构建转基因小鼠已经得到普及，并且可能是可用的最佳方法（假设候选基因在商业BAC文库中呈现）。BAC融合构建体将用于制备在新皮层神经元的特定群体中表达Cre重组酶的转基因小鼠品系。随后，这些小鼠将与携带侧接loxP位点的感兴趣基因的小鼠品系杂交。