Development of conditional and inducible gene targeting tools in rabbits

兔子条件性和诱导性基因打靶工具的开发

• 批准号: 9317588
• 负责人: JIE XU
• 金额: $ 18.41万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9317588
• 关键词: Animal Model; Animals; Basic Science; Biocompatible Materials; Biological Models; Biomedical Research; CRISPR/Cas technology; Chromosomes, Human, Pair 21; Cytomegalovirus; DNA cassette; Development; Diagnostic; Event; Family suidae; Gene Expression; Gene Targeting; Genome; Goals; Human; Knock-in; Knock-out; Laboratory Animal Models; LoxP-flanked allele; Modeling; Mus; Orthologous Gene; Oryctolagus cuniculus; Play; Research; Role; Sequence Analysis; Sum; System; Testing; Tetanus Helper Peptide; Tissues; Transgenic Organisms; Variant; Work; animal model development; base; human disease; knockout gene; nuclease; programs; promoter; response; success; therapeutic development; tool

Project Summary Laboratory animal models play an important role in the study of human diseases. Using appropriate animals is critical not only for basic research but also for the development of therapeutics and diagnostic tools. The rabbit is a classical experimental animal species. By adapting the programmable nucleases, such as CRISPR/Cas9, to the gene targeting platform, we recently achieved efficient gene knockout and knock-in in rabbits. It is realized that inducible and conditional gene targeting tools, such as safe harbor locus, Cre-loxP, and Tet, are still lacking which becomes a limiting factor for the broad applicability of this important large animal model system in biomedical research. We previously characterized the rabbit Rosa26 locus, demonstrated that it can be used as a safe harbor locus, and produce Rosa26-EGFP loxed rabbits. We have also identified another putative safe harbor locus, ortholog of Hipp11 (H11), on Chromosome 21 in the rabbit genome. In the present project we propose to: (i) develop conditional knockout (cKO) rabbits; and (ii) develop inducible knockout (iKO) rabbits. To achieve the cKO and iKO goals, we will utilize the rabbit H11 (rbH11) locus as the second safe harbor locus for knock-in work, and to develop Cre-loxP, CreER-loxP and Tet on/off tools. Furthermore, we propose to develop Cas9 based cKO/iKO tools. Through the propose work, we will generate versatile tools to enable conditional and inducible gene targeting tools in rabbits. These tools will realize spatial and temporal control of gene expressions in this model species.

项目摘要 实验动物模型在人类疾病研究中起着重要作用。使用合适的动物是 这不仅对基础研究至关重要，而且对治疗和诊断工具的开发也至关重要。兔子 是一种经典的实验动物。 通过将可编程核酸酶，如CRISPR/Cas9，适应基因靶向平台，我们最近 在兔中实现了高效的基因敲除和敲入。人们认识到，诱导型和条件型基因 安全港基因座、Cre-loxP和泰特等靶向工具仍然缺乏，这成为限制因素 这一重要的大型动物模型系统在生物医学研究中的广泛适用性。 我们先前表征了兔Rosa 26基因座，证明它可以用作安全港基因座， 并产生Rosa 26-EGFP loxed兔。我们还确定了另一个假定的安全港基因座， Hipp 11（H11），位于兔子基因组的21号染色体上。 在本项目中，我们建议：（i）开发条件性敲除（cKO）兔;（ii）开发诱导型 敲除（iKO）兔子。为了实现cKO和iKO目标，我们将利用兔H11（rbH 11）基因座作为靶基因。 第二个安全港位置，用于敲入工作，并开发Cre-loxP、CreER-loxP和泰特开/关工具。 此外，我们建议开发基于Cas9的cKO/iKO工具。 通过提出的工作，我们将产生多功能的工具，使条件和诱导基因打靶 兔子的工具这些工具将实现对该模式物种基因表达的时空调控。