Non-invase high-throughput genotyping of zebrafish larvae (phase I)

斑马鱼幼虫的非侵入高通量基因分型（第一阶段）

• 批准号: 561567-2021
• 负责人: Samarut, Éric
• 金额: $ 8.92万
• 依托单位: Université de Montréal
• 依托单位国家: 加拿大
• 项目类别: Idea to Innovation
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=724264
• 关键词: Non; invase; throughput; genotyping; zebrafish

Zebrafish is a vertebrate model organism that is widely used for genetic studies, functional genomics and biomedical investigations. Particularly, its genetic accessibility and the ease of use of several mutagenesis techniques (including CRISPR/CAS9) made it a model of choice for genetic engineering. As a result, in the last few years, a myriad of genetic models have been developed. Concomitantly, the need for accurate, time- and cost-efficient genotyping techniques has grown, particularly with the aim of genotyping zebrafish larvae as early as possible. Although there are several well established genotyping techniques, those procedures are usually not well-suited for young larvae as they require a significant amount of biological material to be taken from the animal. To circumvent this issue, manual larvae tail-clipping or semi-automated protocols have been established. However, the fact that they are time-consuming, require anesthetizing young larvae and/or necessitate buying costly equipment limit their general use.Here we designed and developed a reusable simple genotyping device that allows for accurate, time-efficient and cost-effective genotyping on non-anesthetized zebrafish larvae from 4 days post-fertilization. Our technique allows the mechanic retrieval of biological material from 96 larvae at a time within 10 minutes that can further be used successfully for genomic DNA extraction. Interestingly, this device does not require specific equipment other than regular laboratory apparatus. Importantly, subsequent genotyping using regular techniques such as PCR/Sanger or HRM analysis was accurate and successful in 98% of the cases. Furthermore, the survival rate of zebrafish larvae undergoing this procedure is more than 97%. We also showed by brain imaging and behavioral recording that the general physiology of the larvae is not affected by the procedure. As a matter of fact, we designed and developed a new reusable device that allows fast and cheap genotyping of zebrafish larvae while keeping them alive. We foresee numerous applications for this simple apparatus and believe it will be of great interest to the entire zebrafish community.

斑马鱼是一种脊椎动物模式生物，广泛用于遗传学研究、功能基因组学和生物医学研究。特别是，它的遗传可及性和几种诱变技术（包括CRISPR/CAS 9）的易用性使其成为基因工程的首选模型。因此，在过去的几年里，已经开发了无数的遗传模型。与此同时，需要准确，时间和成本效益的基因分型技术已经增长，特别是尽早对斑马鱼幼虫进行基因分型的目的。虽然有几种成熟的基因分型技术，但这些程序通常不适合年轻的幼虫，因为它们需要从动物身上采集大量的生物材料。为了避免这个问题，已经建立了手动幼虫剪尾或半自动化方案。然而，事实上，他们是耗时的，需要麻醉年轻的幼虫和/或需要购买昂贵的设备限制了他们的一般use. There我们设计和开发了一个可重复使用的简单的基因分型设备，允许准确，时间效率和成本效益的非麻醉斑马鱼幼虫从受精后4天的基因分型。我们的技术允许在10分钟内从96只幼虫中机械检索生物材料，可以进一步成功地用于基因组DNA提取。有趣的是，除了常规的实验室仪器之外，该设备不需要特定的设备。重要的是，随后使用常规技术如PCR/桑格或HRM分析的基因分型在98%的病例中是准确和成功的。此外，经过该程序的斑马鱼幼体的存活率超过97%。我们还通过脑成像和行为记录表明，幼虫的一般生理学不受程序的影响。事实上，我们设计并开发了一种新的可重复使用的设备，可以快速，廉价地对斑马鱼幼虫进行基因分型，同时保持它们的存活。我们预计这个简单的装置会有许多应用，并相信它会引起整个斑马鱼群落的极大兴趣。