A fluorescent reporter detecting precise homologous recombination transgenesis activity

检测精确同源重组转基因活性的荧光报告基因

基本信息

批准号：
9520355
负责人：
Trisha Brock
金额：
$ 75.3万
依托单位：
KDT, INC
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-07-01 至 2019-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9520355
关键词：
Alleles Animal Husbandry Animal Model Animals Biological Models Biomedical Research CRISPR/Cas technology Caenorhabditis elegans Clustered Regularly Interspaced Short Palindromic Repeats Communities Complementary DNA Data Detection Disease Disease model Drug Screening Educational workshop Elements Embryo Emerging Technologies Engineering Event Fishes Fluorescence Funding Gene Transfer Techniques Generations Genes Genetic Genetic Materials Genetic Recombination Genomics Goals Injections International Knock-in Knock-out Location Maintenance Mediating Medical Methods Microinjections Modeling Mutation NIH Program Announcements Phase Point Mutation Population Production Public Health Reagent Reporter Research Research Personnel Savings Services Signal Transduction Small Business Innovation Research Grant Societies Speed System Technology Testing Transgenes Transgenic Model Transgenic Organisms Vertebrates Zebrafish cost drug development drug discovery genetic variant genome editing homologous recombination human disease human model innovation knockout gene new technology recombinational repair response screening tool

项目摘要

The broad, long term objective and specific aims of the fasttrack SBIR research are to create a reagent, RecombiRED, for use in microinjection of embryos for identifying a subset of embryos as recombination competent injections. The ability to quickly identify genome editing events in an embryo allows researcher to quickly make precision animal models of human disease states. Precise animal models allow for greater scientific understanding of a human disease and provides a robust platform for drug discovery screening. The RecombiRED reagent relies on induced fluorescence in response to activation of homologous recombination repair within the embryo. Detection of homologous recombination repair in injected embryos with RecombiRED allows animal husbandry and further screening efforts to be restricted to a subset of embryos by up to 100x for having a desired precise knockin of new genetic material at a specific genetic locus. The phase I research adapts a the RecombiRED reagent developed in C . elegans model organism to have the appropriate expression control elements enabling sufficient activity in zebrafish embryo. In Phase II, the additional research further optimizes parameters of the RecombiRED reagent to minimize false positive and false negative readouts, which then allows maximal forecasting of targeted genome edit events. Finally, a set of 15 realworld transgenesis projects are used as premarket betatest to demonstrate the ubiquity and robustness of the RecombiRED for simplifying discovery of genome editing in the creation of animals models for use in drug discovery.

快速 SBIR 研究的广泛、长期目标和具体目标是创建一个试剂 RecombiRED，用于胚胎显微注射以识别胚胎子集作为重组能力注射的能力，能够快速识别基因组编辑事件。胚胎使研究人员能够快速制作人类疾病状态的精确动物模型。精确的动物模型可以更科学地了解人类疾病和 RecombiRED 试剂为药物发现筛选提供了一个强大的平台。响应于同源重组修复的激活而诱导荧光使用 RecombiRED 检测注射胚胎中的同源重组修复允许畜牧业和进一步的筛选工作仅限于胚胎的一个子集高达 100 倍，可在特定基因上对新遗传物质进行所需的精确敲入第一阶段的研究采用了线虫模型中开发的 RecombiRED 试剂。生物体具有适当的表达控制元件，能够实现足够的活性斑马鱼胚胎。在第二阶段，额外的研究进一步优化了 RecombiRED 试剂可最大限度地减少假阳性和假阴性读数，从而允许最后，一组 15 个现实世界转基因事件。项目被用作上市前测试，以证明该技术的普遍性和稳健性 RecombiRED 可简化动物模型创建过程中基因组编辑的发现在药物发现中的应用。