Applied Research

应用研究

• 批准号: 10331041
• 负责人: LAURA G REINHOLDT
• 金额: $ 10.54万
• 依托单位: JACKSON LABORATORY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10331041
• 关键词: Address; Alleles; Applied Research; Biological Process; CRISPR/Cas technology; Catalytic Domain; Cells; Communities; Complex; Cryopreservation; DNA; DNA Sequence; DNA cassette; Development; Disease; Docking; ES Cell Line; Electroporation; Embryo; Engineering; Ensure; Enterobacteria phage P1 Cre recombinase; Epitopes; Excision; Gene Activation; Gene Transfer Techniques; Genetic; Genetic Engineering; Genetic Research; Genetic Variation; Genetic study; Genomics; Germ Lines; Goals; Heritability; Human; Human Biology; Human Genetics; In Vitro; Inbred Mouse; Inbred Strain; Inbreeding; Infrastructure; Integrase; Knock-in; Laboratories; Letters; Mediating; Microinjections; Modeling; Molecular; Mouse Strains; Mus; Phenotype; Plasmids; Positioning Attribute; Process; Proteins; Quantitative Trait Loci; RNA; Recombinants; Recommendation; Reporter; Reproducibility; Research; Research Design; Resources; Series; Site; Somatic Cell; Surveys; System; Terminator Codon; Tissues; Transgenes; Validation; Variant; base; embryonic stem cell; experimental study; expression vector; genetic analysis; genetic manipulation; human disease; novel; recombinase; repaired; tool; trait; validation studies; zygote

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.

项目摘要/摘要——应用研究 人类生物学和疾病的遗传学很复杂，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 已经取得了很大进展，但仍然必须依赖哺乳动物模型。为此，新菌株 已经创造出能够更好地模拟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来创建“工具菌株”，以允许 特定类型的基因操作。标准近交系中已经存在此类菌株（例如 C57BL/6J）， 但携带通常需要的基因操作的“工具菌株”目前还无法用于遗传 不同的近交系，例如 CC/DO、BXD 或用于创建这些 RI 组的所有创始人近交系。 强大的 CC/DO/BXD 品系的使用正在迅速增长，为了促进这些小鼠的有效使用，我们 提议创建一系列工具株和小鼠胚胎干细胞（mESC）。为此，我们将采取 具有新颖、高效的基于重组酶的对接位点的多种近交菌株的优势 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 made available through the Special Mouse Strain Resource (SMSR). 为了实现这一目标，我们将： 从现有 Bxb1 整合酶菌株中衍生并验证具有种系能力的 mESC 系。目标 here is to create a panel of genetically diverse mESC lines harboring Bxb1 integrase sites, as well as LSL- Cas9-标志。这些 mESC 将为工程基因座提供工具，这些基因座很难在受精卵中工程化或用于 可以在体外完成的验证实验。 为 CAST/EiJ、PWK/EiJ 和 DBA/2J 近交菌株生成 Rosa26-LSL-Cas9-FLAG 工具菌株。 These strains will enable CRISPR/cas9 editing in the absence of exogenous Cas9.这有利于工程 in zygotes and allows for easy, precise engineering of somatic cells/tissues.目标是创建和验证 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.经常实施这种方法 uses “germ line cre” strains, that are not currently available on genetically diverse strain backgrounds.因此， the goal here is to take advantage of our Bxb1 docking site strains to create Rosa26 Sox2-cre knock-in alleles 关于 CAST/EiJ 和 PWK/EiJ。