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 实验室动物模型在人类疾病的研究中起着重要作用。使用合适的动物是 不仅对基础研究至关重要，而且对于开发治疗和诊断工具的开发至关重要。兔子 是一种经典的实验动物。 通过将可编程核酸酶（例如CRISPR/CAS9）调整为基因靶向平台，我们最近 在兔子中实现了有效的基因敲除和敲除。已经意识到诱导和条件基因 诸如安全港基因座，Cre-loxp和Tet之类的定位工具仍然缺乏成为限制因素 对于这种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。 我们以前表征了Rabbit Rosa26基因座，证明它可以用作安全的港口基因座， 并产生rosa26-egfp loxed兔子。我们还确定了另一个假定的安全港基因座，直系同源 Hipp11（H11）的，在兔基因组中的21染色体上。 在本项目中，我们建议：（i）发展条件敲除（CKO）兔子； （ii）发展诱导 淘汰赛（iko）兔子。为了实现CKO和IKO目标，我们将利用兔子H11（RBH11）基因座作为 第二个安全港基因座进行敲门工作，并开发Cre-loxp，Creer-loxp和Tet On/Off工具。 此外，我们建议开发基于CAS9的CKO/IKO工具。 通过建议的工作，我们将生成多功能工具以实现有条件和诱导的基因靶向 兔子中的工具。这些工具将实现该模型物种中基因表达的空间和时间控制。