Applied Research

项目摘要/摘要-应用研究 人类生物学和疾病背后的遗传学是复杂的，需要使用“系统遗传学”。 来剖析控制和调节生物过程的无数相互作用。人体研究的工具 虽然已经有了很大的进步，但仍然有必要依赖哺乳动物的模型。为此，出现了新的菌株 已经创造出了更好地模仿人类基因变异程度的老鼠。这些菌株包括 协作杂交(CC)、多样性近交系(DO)和重组近交系小鼠(如BXD)。CC/DO和 BxD平台利用系统遗传学研究所需的自然近交系变异。然而， 使用这些菌株通常需要对小鼠进行额外的基因工程，以创造出“工具菌株”，以便 特定类型的基因操作。这样的品系已经存在于标准近交系(例如C57BL/6J)， 但是，携带普遍需要的基因操作的“工具菌株”目前还不能用于遗传研究。 不同的近交系菌株，如CC/DO、BXD或用于创建这些RI面板的所有创始人近交系菌株。 强大的CC/DO/BXD菌株的使用正在迅速增长，为了促进这些小鼠的有效使用，我们 建议建立一系列工具菌株和小鼠胚胎干细胞(MESC)。要做到这一点，我们将采取 含有新型高效重组酶对接位点的不同自交系的优势 促进使用CRISPR-CAS9编辑来创建新菌株。经过验证的新工具品种将是 通过特殊老鼠品系资源(SMSR)提供。 为达致这个目标，我们会： 从现有的Bxb1整合酶菌株中获得并验证具有胚系能力的mESC系。目标是 这里是创建一个包含Bxb1整合酶位点以及LSL的遗传多样性mESC系的面板。 Cas9-FLAG。这些mESCs将为难以在受精卵中工程的基因座提供工具，或为 可以在体外完成的验证实验。 为CAST/EIJ、PWK/EIJ和DBA/2J近交系生成rosa26-lsl-Cas9-FLAG工具品系。 这些菌株将在没有外源Cas9的情况下实现CRISPR/Cas9的编辑。这便于工程设计 在受精卵中，并允许对体细胞/组织进行简单、精确的工程。目标是创建和验证 这一重要工具品系适用于上面列出的三个高度优先的近交系背景。 为CAST/EIJ和PWK/EIJ近交系背景生成生殖系删除工具品系。 特定DNA序列或工程终止密码子的移除通常使用侧翼来完成 作为cre重组酶催化结构域的loxP位点。经常实施这种方法 使用“胚系cre”菌株，这在遗传多样性的菌株背景上目前是不可用的。因此， 这里的目标是利用我们的Bxb1对接地点菌株来创建rosa26Sox2-cre敲入等位基因 关于CAST/EIJ和PWK/EIJ。

PROJECT SUMMARY/ABSTRACT – Applied Research The genetics underlying human biology and disease is complex and will require the use of “system genetics” to dissect the myriad of interactions that control and regulate biological processes. Tools for human studies have advanced greatly yet there remains a necessary reliance on mammalian models. To this end new strains of mice have been created that better emulate the extent of human genetic variation. These strains include Collaborative Cross (CC), Diversity Outbred (DO) and recombinant inbred mice (e.g. BXD). The CC/DO and BXD platforms harness the natural inbred strain variation needed for systems genetics studies. However, the use of these strains typically requires additional genetic engineering of the mice to create “tool strains” to allow specific types of genetic manipulations. Such strains already exist for standard inbred strains (e.g. C57BL/6J), but “tool strains”, that carry commonly needed genetic manipulations, are not presently available for genetically diverse inbred strains such as CC/DO, BXD, or all of the founder inbred strains used to create these RI panels. The use of the powerful CC/DO/BXD strains is growing rapidly and to facilitate efficient use of these mice we propose to create a series of tool strains and mouse embryonic stem cells (mESC). To do this, we will take advantage of diverse inbred strains that harbor novel, high efficiency recombinase-based docking sites to facilitate the use of CRISPR-cas9 editing for creation of the new strains. Validated new tool strains will be made available through the Special Mouse Strain Resource (SMSR). To achieve this goal we will: Derive and validate germ line competent mESC lines from existing Bxb1 integrase strains. The goal here is to create a panel of genetically diverse mESC lines harboring Bxb1 integrase sites, as well as LSL- Cas9-FLAG. These mESCs will provide tools for engineering loci that are difficult to engineer in zygotes or for validation experiments that can be accomplished in vitro. Generate Rosa26-LSL-Cas9-FLAG tool strains for CAST/EiJ, PWK/EiJ, and DBA/2J inbred strains. These strains will enable CRISPR/cas9 editing in the absence of exogenous Cas9. This facilitates engineering in zygotes and allows for easy, precise engineering of somatic cells/tissues. The goal is to create and validate this important tool strain for the three high priority inbred strain backgrounds listed above. Generate germ line deletion tool strains for CAST/EiJ and PWK/EiJ inbred strain backgrounds. Removal of specific DNA sequences or engineering stop codons is frequently accomplished using flanking loxP sites that serve as catalytic domains for cre recombinase. Implementation of this approach frequently uses “germ line cre” strains, that are not currently available on genetically diverse strain backgrounds. Thus, the goal here is to take advantage of our Bxb1 docking site strains to create Rosa26 Sox2-cre knock-in alleles on CAST/EiJ and PWK/EiJ.