Molecular Biology and Mouse Core

MTCC的分子生物学和小鼠核心组件将通过以下方式为基因转移社区服务： 提供与囊性纤维化（CF）基因转移相关的小鼠模型和获得分子生物学 设备和专业知识。虽然并不总是预测结果在人体试验中，动物模型是 然而对于建立载体的功效和理解基因表达水平是有用的 进行功能矫正。对于设计用于在以下背景下评估新AAV载体的试点研究， 肠道，将提供CFTR缺陷型小鼠模型。这个模型显示了一个非常重要的肠道 表型与人类囊性纤维化（CF）非常相似。此外，对于肺部研究，核心将 提供在近交系背景（C57 BI/6 N）上过表达p-ENaC的小鼠，并且将相同的小鼠繁殖到 CFTR缺陷小鼠。这两种小鼠都提供了一种将基因转移到充满粘液、发炎的 可能是人类CF肺的典型气道。在β-ENaC-CFTR模型中缺乏CFTR提供了一种新的方法。 有机会在一个非常相关的系统中探索CFTR基因转移。为了支持试点项目的工作， 核心还将有助于产生和维持Dnail和选定的粘蛋白缺陷小鼠。作为 第二个主要重点是，核心将通过提供访问来帮助研究人员满足分子生物学需求， 相关设备和开展专业分子生物学服务。为了实现这些目标， 核心将维护和提供专用仪器，特别是评估质量和 定量RNA和DNA，并进行定量实时PCR。核心还将提供qPCR 为需要评估mRNA表达水平的进行基因转移的个人提供服务，例如， 基因转移后CFTR的表达。此外，核心区将是协调 访问和评价机构获得的shRNA慢病毒载体文库。核心将服务于 为了验证可用的shRNA载体的功能性敲低，并随后将质粒提供给 矢量核心的生产。最后，核心将是阿菲耶维奇基因阵列的集中地 分析用于评估p-ENaC过表达小鼠的疾病发展，这一努力将有助于 为了阐明该模型的发病机制，并为生理和细胞的早期变化提供线索， 在CF样疾病的背景下的肺生物学。这些信息很可能会确定新的目标， 疗法分子生物学和小鼠核心已经建立了坚实的设备和专业知识基础。 因此，使用核心资源应能提高整个导弹技术协调委员会工作的效率和质量。

The Molecular Biology and Mouse Core component of the MTCC will serve the gene transfer community by providing mouse models relevant to cystic fibrosis (CF) gene transfer and access to molecular biology equipment and expertise. Although not always predictive of results in human trials, animal models are nonetheless useful for establishing efficacy of vectors and understanding levels of gene expression necessary for functional correction. For pilot studies designed to evaluate new AAV vectors in the context of the gut, the CFTR-deficient mouse model will be provided. This model shows a highly significant gut phenotype that is very similar to human cystic fibrosis (CF). Additionally, for lung studies, the Core will provide p-ENaC over-expressing mice on an inbred background (C57BI/6N) and the same mice bred to the CFTR-deficient mice. Both of these mice provide a model for gene transfer to a mucus-filled, inflamed airway that might be typical of a human CF lung. The lack of CFTR in the (3-ENaC-CFTR model provides an opportunity to explore CFTR gene transfer in a very relevant system. To support pilot project efforts, the Core will also aid in the generation and maintenance of Dnail- and selected mucin-deficient mice. As a second major focus, the Core will assist investigators with molecular biology needs by providing access to relevant equipment and conducting specialized molecular biology services. To reach these objectives, the Core will maintain and provide access to specialized instrumentation, specifically to evaluate the quality and quantity of RNA and DMA and to conduct quantitative real-time PCR. The Core will also provide qPCR services for individuals conducting gene transfer who need to evaluate mRNA expression levels, e.g., expression of CFTR after gene transfer. In addition, the Core will be the centralized location to coordinate access to and evaluation of a library of institutionally obtained shRNA lentiviral vectors. The Core will serve to validate available shRNA vectors for functional knock-down and subsequently provide plasmids to the Vector Core for production. And finally, the Core will be the centralized location for Affymetrix gene array analysis for evaluating the disease development in the p-ENaC over-expressing mice, an effort that will help to unravel the pathogenesis in this model and provide clues to early changes in the physiology and cell biology of the lung in the context of a CF-like disease. Such information may well identify new targets for therapy. The Molecular Biology and Mouse Core has built a solid foundation of equipment and expertise. As such, use of the Core should improve efficiency and quality of the entire MTCC effort.