Applied Research

应用研究

• 批准号: 10557103
• 负责人: LAURA G REINHOLDT
• 金额: $ 3.61万
• 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/10557103
• 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; 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 已经大大提高了哺乳动物模型，但仍有必要的提醒。为此结束新的菌株 已经创建了更好地模拟人类遗传变异程度的小鼠。这些应变包括 协作交叉（CC），多样性杂种（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性菌株变异。但是， 这些菌株的使用通常需要小鼠的其他基因工程来创建“工具菌株”以允许 特定类型的遗传操作。对于标准的近交菌株（例如C57BL/6J），已经存在此类菌株， 但是，“工具菌株”（通常需要遗传操作）目前不可用于基因上 CC/DO，BXD等各种近交菌株或用于创建这些RI面板的所有创始人近交菌株。 强大的CC/DO/BXD菌株的使用正在迅速增长，以促进这些小鼠的有效使用 提出创建一系列工具株和小鼠胚胎干细胞（MESC）的建议。为此，我们将接受 具有新颖的高效重组酶对接场所的各种近交菌株的优势 促进使用CRISPR-CAS9编辑来创建新菌株。经过验证的新工具应变将是 通过特殊的鼠标应变资源（SMSR）提供。 为了实现这一目标，我们将： 从现有的BXB1集成菌株中得出并验证生殖线胜任的MESC线。目标 这是为了创建一个具有BXB1集成站点以及LSL-的一般多元化的MESC系列。 cas9-fag。这些MESC将为工程基因座提供工具，这些工具很难在Zygotes或 可以在体外完成的验证实验。 生成cast/eij，pwk/eij和dba/2J近交菌株的ROSA26-LSL-CAS9-FLAG工具应变。 这些菌株将在没有外源性CAS9的情况下实现CRISPR/CAS9编辑。这有助于工程 在Zygotes中，可以轻松，精确的体细胞/组织工程。目标是创建和验证 该重要的工具应变，用于上面列出的三个高优先级近交性应变背景。 生成用于铸造/eij和PWK/eij inbred菌株背景的生殖系缺失工具应变。 去除特定的DNA序列或工程终止密码子，经常使用侧面完成 作为CRE重组酶的催化域的LOXP位点。经常实施这种方法 使用当前在一般不同的应变背景上可用的“种系Cre”菌株。那， 这里的目标是利用我们的BXB1对接站点应变来创建ROSA26 SOX2-CRE敲入等位基因 在cast/eij和pwk/eij上。