Animal Model Core E

核心E.动物模型核心 项目负责人：克莱尔·史密斯 摘要 了解结核分枝杆菌(Mtb)代谢产物影响人类的机制 结核病(TB)疾病需要实施既可驯服又可控制的动物模型系统 忠实地复制在人类身上观察到的结核病状态。在这里，我们建议利用Collaborative 杂交(CC)，一个遗传多样性的重组近交系小鼠小组，可以无限期地繁殖 重生了。我们之前已经表明，CC小组涵盖了广泛的结核病 特征和感染微环境。CC小鼠为研究特定的结核分枝杆菌提供了一种易于处理的模型 遗传-代谢物对，与表现出有限表型变异的标准近交系小鼠相比。为 例如，在全基因组TnSeq实验中，我们发现在19个高值Mtb代谢基因中，只有 一种在常规C57BL/6J(BL6)小鼠品系中可控生长。然而，超过一半的突变体 研究表明，当在CC小鼠品系中筛选时，体内生长表型。此外，通过对 单个细菌基因在体内控制或不控制结核分枝杆菌存活的宿主遗传背景 史密斯实验室可以开始研究宿主因素对结核分枝杆菌应答的控制。动物核心E将进行 支持项目1和项目3的实验性感染方法，这些项目侧重于毒力相关的血脂和 分别为诊断学。为了支持在项目3中确定结核分枝杆菌代谢物作为诊断测试，我们将 鉴定感染了高和低结核分枝杆菌负荷的小鼠的肺和血清。支持以下努力 了解宿主压力对分枝杆菌被膜内容物的作用在项目1中，我们将产生Mtb 在小鼠体内传代的菌株。扩展我们现有的Tnseq方法以识别分枝杆菌基因 为了控制结核分枝杆菌在体内的生长，我们将筛选并测试50株结核分枝杆菌CRISPR基因敲除株，用于肺结核病 CC菌株的感染情况。其中，5株体内生长最强的Mtb CRISPR基因敲除菌株 表型将作为单基因敲除进一步详细研究，以确定其特定功能。

Core E. Animal Model Core Project Leader: Clare Smith ABSTRACT Understanding the mechanisms through which Mycobacterium tuberculosis (Mtb) metabolites impact human tuberculosis (TB) disease requires implementation of model animal systems that are both tractable and faithfully replicate the TB disease states observed in humans. Here we propose to leverage the Collaborative Cross (CC), a genetically diverse panel of recombinant inbred mice that can be reproducibly and indefinitely regenerated. We have previously shown that the CC panel encompasses a broad spectrum of TB disease traits and infection microenvironments. CC mice provide a tractable model in which to study specific Mtb genetic-metabolite pairs, compared to standard inbred mice that show limited phenotypic variability. For example, in a genome-wide TnSeq experiment, we found that among 19 high value Mtb metabolic genes, only one controlled growth in the conventional C57BL/6J (BL6) mouse strain. However, more than half of mutants studied showed in vivo growth phenotypes when screened across CC mouse strains. Further, through study of the host genetic backgrounds in which individual bacterial genes do or do not control in vivo Mtb survival, the Smith laboratory can begin to study host factors in control of Mtb response. The Animal Core E will conduct experimental infection approaches to support Projects 1 and 3 that focus on virulence associated lipids and diagnostics, respectively. To support identification of Mtb metabolites as diagnostic tests in Project 3, we will characterize lungs and serum from mice infected with a high and low burden of Mtb. Supporting efforts to understand the role of host pressure on the mycobacterial envelope content in Project 1, we will produce Mtb strains passaged in vivo in mice. Extending our existing Tnseq approach to identify mycobacterial genes that control Mtb growth in vivo, we will select and test up 50 pooled Mtb CRISPR knockdown strains for pulmonary infection in CC strains. From these, five Mtb CRISPR knockdown strains with the strongest in vivo growth phenotypes will be further studied in detail as single gene knockdowns to determine their specific functions.