CORE--TRANSGENIC FACILITY

核心——转基因设施

• 批准号: 3735671
• 负责人: MALCOLM J LOW
• 金额: --
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3735671
• 关键词: biomedical facility; embryonic stem cell; genetically modified animals; laboratory mouse; microinjections; polymerase chain reaction; southern blotting; tissue mosaicism

The mission of the transgenic core is to provide technical expertise, guidance in experimental design, and training opportunities in all aspects of the production of transgenic mice. A core laboratory will prevent the unnecessary and time consuming duplication of effort involved in the development of this technology by multiple investigators. The core will additionally provide better quality control, turn around time, and cost effectiveness than either commercially available services or multiple users within one institution. Projects 1 through 4 will use the core to produce transgenic mice that are essential to addressing a major theme of the Program Project: What are the physiological roles of specific molecular components of the hypothalamic-pituitary-adrenal axis? Project 1 will use transgenic mice that have altered naturietic peptide receptor expression to determine the role of these peptides in the control of ACTH secretion. Project 2 will generate mice with pituitary- specific expression of functional POMC minigenes to genetically "rescue" POMC gene "knock-out" mice produce by homologus recombination. Project 3 will explore the role of pituitary derived beta-endorphin in the stress response by blocking the posttranslational processing of POMC at particular dibasic residues with dominant negative or antisense type transgenes. Project 4 will use the technical expertise and facilities of the core to produce mutant mice carrying either null alleles of the neural-specific MSH receptors produced by homologous recombination in embryonic stem cells or constitutively activated receptors encoded by dominant transgenes. These aims will be accomplished through provision of the following services: 1. Embryo collection, nuclear microinjection, blastocyst injections, and oviduct and uterine transfer of manipulated embryos to generate transgenic mice. 2. Genotyping of transgenic mice by a combination of tail DNA dot-blot, Southern blot, or PCR analysis. 3. Supervision and maintenance of a mouse breeding colony providing inbred FVB mice for embryo donations and including stud and vasectomized males used for the initial production of transgenic embryos, and breeding and pedigree analysis of the transgenic mice. FVB mice from the colony will also be available to all investigators for use as controls in physiology experiments or for tissue harvesting. 4. Assistance in physiology experiments using mice including injections, anesthesia, cannulation, and surgical procedures. 5. Permanent storage of cryopreserved transgenic mouse embryos to protect valuable pedigrees from accidental loss, to minimize the continuous breeding of pedigrees that no longer are immediately needed for experiments, and to provide a potential resource for distribution of selected pedigrees to other qualified investigators outside of the institution.

转基因核心的使命是提供技术专长， 实验设计的指导和所有人的培训机会 转基因小鼠产生的各个方面。 核心实验室将 防止不必要和耗时的努力重复 在多个研究人员开发这项技术的过程中。 这 核心还将提供更好的质量控制，扭转时间， 比市售服务或成本效益 一个机构中的多个用户。 项目1至4将使用 产生转基因小鼠的核心，这对于解决一个专业至关重要 计划项目的主题：生理角色是什么 下丘脑 - 垂体 - 肾上腺轴的特定分子成分？ 项目1将使用改变自然肽的转基因小鼠 受体表达以确定这些肽在 控制ACTH分泌。 项目2将与垂体生成小鼠 - 功能性POMC微型元素的特定表达在遗传上“拯救” POMC基因“敲除”小鼠通过同型重组产生。 项目 3将探索垂体衍生的β-内啡肽在压力中的作用 通过阻止POMC的翻译后处理来响应 具有主要负或反义类型的特定Dibasic残基 转基因。 项目4将使用技术专长和设施 核心产生的突变小鼠，这些突变小鼠带有任何一个无效等位基因 由同源重组产生的神经特异性MSH受体 胚胎干细胞或由组成型活化的受体编码 主要转基因。 这些目标将通过提供实现 以下服务： 1。胚胎收集，核显微注射，胚泡注射和 操纵胚胎的输卵管和子宫转移以产生 转基因小鼠。 2。通过尾巴DNA点印刷的结合转基因小鼠的基因分型， Southern印迹或PCR分析。 3。鼠标繁殖菌落提供的监督和维护 近交FVB小鼠用于胚胎捐赠，包括螺柱和输精管 雄性用于初始产生转基因胚胎和繁殖 和转基因小鼠的血统分析。 来自殖民地的FVB小鼠 所有调查人员也将使用作为控制 生理实验或进行组织收集。 4。使用包括注射在内的小鼠的生理学实验的帮助， 麻醉，插管和手术程序。 5。冷冻保存的转基因小鼠胚胎的永久存储 保护宝贵的血统免受意外损失，以最大程度地减少 不再需要立即需要的血统的连续繁殖 用于实验，并为分发的潜在资源提供 选定的血统会给其他合格的调查员 机构。