Applied Research

项目概要/摘要-应用研究 人类生物学和疾病背后的遗传学是复杂的，将需要使用“系统遗传学” 剖析控制和调节生物过程的无数相互作用。人类研究工具 已经有了很大的进步，但仍然需要依赖哺乳动物模型。为此，新菌株 已经创造出了更好地模仿人类遗传变异程度的小鼠。这些菌株包括 协作杂交（CC）、多样性远交（DO）和重组近交小鼠（例如BXD）。CC/DO和 BXD平台利用系统遗传学研究所需的天然近交系变异。但 使用这些品系通常需要对小鼠进行额外的基因工程以产生“工具品系”， 特定类型的基因操作。对于标准近交系（例如C57 BL/6 J），已经存在这样的品系， 但是携带通常需要的遗传操作的“工具菌株”目前还不能用于遗传操作， 不同的近交系，例如CC/DO、BXD或用于产生这些RI组的所有创始近交系。 使用强大的CC/DO/BXD品系正在迅速增长，为了促进这些小鼠的有效使用， 提出创建一系列工具品系和小鼠胚胎干细胞（mESC）。为此，我们将 具有新的、高效的基于重组酶的对接位点的多种近交系的优点， 促进使用CRISPR-cas9编辑来创建新菌株。经确认的新工具菌株将 特别小鼠品系资源（Special Mouse Strain Resource，SMSR） 为实现这一目标，我们将： 从现有Bxb 1整合酶菌株中衍生并验证生殖系感受态mESC系。目标 这里是创建一组具有Bxb 1整合酶位点以及LSL-1的遗传多样性mESC系。 Cas9-FLAG。这些mESC将提供用于工程化难以在合子中工程化的基因座的工具，或用于工程化基因座的工具。 可以在体外完成的验证实验。 生成用于CAST/EiJ、PWK/EiJ和DBA/2 J近交系菌株的Rosa 26-LSL-Cas9-FLAG工具菌株。 这些菌株将能够在不存在外源Cas9的情况下进行CRISPR/cas9编辑。这有利于工程 在受精卵中，并且允许体细胞/组织的容易、精确的工程化。目标是创建和验证 该重要工具品系用于上述三种高优先级近交品系背景。 为CAST/EiJ和PWK/EiJ近交系菌株背景生成种系缺失工具菌株。 去除特定的DNA序列或工程化终止密码子通常使用侧翼插入来完成。 作为cre重组酶催化结构域的loxP位点。这种做法经常 使用“生殖系cre”菌株，这是目前在遗传多样性菌株背景上不可用的。因此，在本发明中， 这里的目标是利用我们的Bxb 1对接位点菌株来产生Rosa 26 Sox 2-cre敲入等位基因 在CAST/EiJ和PWK/EiJ上。

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.