Systems for rapid generation of zebrafish mutants and zebrafish embryo handling

快速生成斑马鱼突变体和斑马鱼胚胎处理的系统

基本信息

批准号：
9909292
负责人：
Josh Leitch Bonkowsky
金额：
$ 21.99万
依托单位：
NANONC, INC.
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-01 至 2022-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9909292
关键词：
Academia Adult Age Alleles Animal Model Animals Automation Biomedical Research Blood Clustered Regularly Interspaced Short Palindromic Repeats Communities Development Devices Disease Drug Screening Drug usage Electroporation Embryo Failure Gene Transfer Techniques Generations Genetic Genotype Human Human Development Industry Injections Larva Manuals Medical Medical Research Methods Modeling Molecular Mutagenesis Mutation Neurologic Organ Pathway interactions Pharmaceutical Preparations Phenotype Problem Solving Process Research Research Methodology Research Project Grants Robotics System Techniques Technology Testing Therapeutic Trials Time Training Transgenic Organisms Vertebrates Work Zebrafish base cost drug discovery experience experimental study genome editing high throughput technology human disease instrument knock-down mechanical device mouse development mutant new technology novel novel therapeutics screening tool

项目摘要

Project Summary Zebrafish is an important vertebrate model organism for biomedical research. However, the full potential of zebrafish research has not been realized, in particular for drug discovery and for large-scale model generation, because of insufficient technologies to handle and genotype animals. Genotyping currently is a time, labor, and training intensive process. Embryos must either be raised to adulthood or embryos must be sacrificed to determine genotypes; mutants are difficult to genotype; and screens or drug/therapeutics trials cannot be performed on animals of known genotype until an older age. Finally, high-throughput technologies based on advances in genomic editing technology such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are limited by a requirement for manual screening. Nanonc, which in the past year has brought the first-of-its-kind live embryo genotyping device ZEG to market (https://www.wfluidx.com), proposes implementation of two novel technologies to empower zebrafish model generation and use: the transformation of genome-editing via electroporation; and the automation of embryo handling. This is because transgenesis/mutagenesis and zebrafish embryo handling are essentially identical in approach to methods used in the 1980s. Mutagenesis, or transgenesis, are performed by manual injection into embryos. While CRISPR mutagenesis in zebrafish is highly efficient and typically achieves bi-allelic knock-down in the injected (G0) animal, the manual requirement limits the total number of animals that can be generated, which limits downstream applications such as new transgenic line generation or use of mutants for screening. Drug screens could be performed on F0 larvae, but the requirement to have humans do the injection limits the number of animals that can be used. The other major problem is that handling of embryos is performed by manual pipette transfer, for example, into 96-well plates, that can require a single user to dedicate up to 30’ per plate by moving embryos one at a time. To solve these problems, we propose the development of two products that will integrate with the commercially available ZEG product: First, developing an electroporation system for high- throughput CRISPR mutagenesis and transgenesis in zebrafish (‘Zapper’). Electroporation techniques have been shown capable of delivering molecular constructs to zebrafish embryos in proof-of-concept experiments, but have not been tested for CRISPR mutagenesis/transgenesis or for scalability. We will test, develop, and implement an electroporation-based system for delivery of constructs to zebrafish embryos. Second, we will develop a zebrafish embryo handling system for rapid loading of embryos (‘Zipper’). Drug or mutant screening in 96- or 324-well plates, or the ZEG (Zebrafish Embryo Genotyping) device, require laborious manual loading/unloading of zebrafish embryos. Robotic options cost in excess of $100,000 and are difficult to trouble-shoot or to interchange between uses. Our lower-cost mechanical device for the rapid dispensing of zebrafish embryos is novel, patentable, and would find immediate use in labs working with zebrafish in both academia and industry sectors.

项目摘要斑马鱼是生物医学研究的进口繁殖模型。研究尚未实现处理和基因型动物的技术不足。必须将胚胎侵犯，或者必须牺牲胚胎以确定基因型；已知的基因型的动物，直到年龄较大。编辑技术（例如定期间隔短的短质体重复序列（CRISPR））的编辑技术受到限制对手动筛查的要求。基因分型设备ZEG到市场（https://www.wfluidx.com），建议实施技术赋予斑马鱼模型生成和使用：通过电穿孔的基因组编辑的转化；胚胎处理的自动化是因为转基因/mutajenesis和斑马鱼胚胎处理在1980年代使用的方法中基本相同。手动注射到胚胎中，而斑马鱼中的crisspr mutajenesis是高效的在注射的（G0）动物中敲门的双重击倒，手动要求限制了可以的动物总数生成，它限制了下游应用，例如新的转基因线基因或使用突变体的应用筛查可以在F0幼虫上进行，但对人类的要求限制可以使用的动物数量。通过手动移液器转移，例如，将单个用户投入30' 通过一次移动胚胎来解决胚胎。将与Commermercial可用的ZEG产品集成：首先，开发用于高的电穿孔系统斑马鱼（Zapper'）中的吞吐量CRISPR Mutajenesis和Transgensis 显示能够在证明概念实验中向斑马鱼胚胎传递分子构建体，但尚未测试CRISPR MUTAJENESIS/转基因或可伸缩性。基于电穿孔的系统，用于将构建体传递到斑马鱼胚胎。斑马鱼的胚胎处理系统快速加载胚胎（“拉链”）。或324孔板或ZEG（斑马鱼胚胎基因分型）设备，需要费力的手动加载/卸载斑马鱼胚胎的成本超过$ 100,000 两次用途。并会发现在Accadia和行业领域与斑马鱼合作的实验室中的使用。