Disease Models and Mechanisms Core

核心B项目概要/摘要过去十年， 技术到生物医学科学。其中最显著的成就是整个 基因组测序允许对无数生物体的基因组DNA结构的完全了解， CRISPR/Cas9基因组编辑允许随时操纵这些基因组， 支持复杂生物系统（尤其是人类）的蛋白质组学分析的进展。为了使 研究人员要在人类肾脏疾病的研究中取得重大进展，我们现在必须 将这些进展应用于体内哺乳动物模型、体外细胞系统和定向分子评估 人类样本。疾病模型和机制核心的总体目标是促进 这些方法的应用有利于和支持基于人类肾脏疾病的机制研究。在 为了实现这一目标，核心小组已经在专门针对减少 阻碍使用者进行床边到实验台研究（目标1、2和3）的障碍，以及那些寻求 在床旁验证实验室检查结果（目标4和5）。 支持那些追求对人类基因组最近财富的机械理解的用户 序列信息，前两个目标提供互补的专业知识，在操纵小鼠基因组， 目的是开发能够忠实复制人类疾病发病机理的矫形动物模型和细胞系。 核心的第一个目标是支持使用细菌人工染色体作为执行 对特定基因的复杂修饰，包括插入多个表位标签、荧光标签、点标记、 突变和缺失，然后在小鼠品系中表达。第二个目标是提供一个免费的 使用CRISPR/Cas9进行基因组编辑以将遗传变体引入细胞系和小鼠的方法 模型核心的第三个组成部分用于分配小鼠品系和细胞系， 以最低的成本回收价格向用户授予许可。这包括Rosa-DTRfl的分发 “终结者”小鼠，用于分离大量高度纯化的非永生化肾细胞类型。的 核心的最后两个组成部分已经开发出来，以支持那些希望将实验室研究结果转化为 更好地了解人类肾脏疾病。在第四个目标中，为用户提供以下方面的资源和专门知识： 对人类尿液进行靶向蛋白质组学研究，以定量评估>250种尿蛋白。在最后的目标中， 核心已经开发了一种多路复用的方法，可以同时分析多达42个 使用成像质谱细胞术测定人肾活检标本中的蛋白质。这些综合服务 推进肾脏研究领域，增强NIDDK的使命，对更广泛的肾脏研究具有价值。 研究社区。

Core B Project Summary/Abstract The past decade has seen quantum advances in the applications of technology to biomedical sciences. Among the more remarkable achievements has been the advent of whole genome sequencing allowing complete knowledge of the genomic DNA structure of innumerable organisms, CRISPR/Cas9 genome editing allowing ready manipulation of these genomes, and the advent of technological advances supporting proteomic analyses of complex biological systems, most notably humans. In order for researchers to achieve major advances in the study of human kidney diseases, it is imperative that we now apply these advances to in vivo mammalian models, in vitro cell systems and directed molecular assessments of human samples. The overarching objective of the Disease Models and Mechanisms Core is to facilitate application of these approaches to benefit and support human kidney disease-based mechanistic studies. In order to achieve this, the Core has developed research expertise in areas specifically targeted to reduce the barriers that hinder users pursuing bedside to bench research (Aims 1, 2 and 3) as well as those who seek to validate bench findings at the bedside (Aims 4 and 5). To support users who are pursuing mechanistic understanding of the recent wealth of human genome sequence information, the first 2 aims provide complementary expertise in manipulating the mouse genome in order to develop orthologous animal models and cell lines that faithfully replicate human disease pathogenesis. The first aim of the Core provides support for using bacterial artificial chromosomes as a means to perform complex modifications of specific genes including insertion of multiple epitope tags, fluorescent tags, point mutations, and deletions, followed by expression in mouse strains. The second aim provides a complimentary approach to genome editing using CRISPR/Cas9 for introducing genetic variants into cell lines and mouse models. The third component of the Core serves to distribute mouse strains and cell lines, for which distribution permission is granted, to users at a minimal cost-recovery price. This includes distribution of the Rosa-DTRfl “Terminator” mouse for isolation of large quantities of highly purified, non-immortalized renal cell types. The last two components of the Core have been developed to support users who wish to translate bench findings to better understand human kidney disease. In the fourth Aim, users are provided with resources and expertise in performing targeted proteomics of human urine to quantitatively assess >250 urinary proteins. In the final aim, the Core has developed a multiplexed approach to simultaneously analyze the amount and location of up to 42 proteins in human kidney biopsy specimens using Imaging Mass Cytometry. These combined services advance the fields of renal research, enhance the mission of NIDDK and are of value to the broader renal research community.