PLOS ONE Journal Publication 2018

An automated system for rapid cellular extraction from live zebrafish embryos and larvae: Development and application to genotyping

Abstract

Zebrafish are a valuable model organism in biomedical research. Their rapid development, ability to model human diseases, utility for testing genetic variants identified from next-generation sequencing, amenity to CRISPR mutagenesis, and potential for therapeutic compound screening, has led to their wide-spread adoption in diverse fields of study. However, their power for large-scale screens is limited by the absence of automated genotyping tools for live animals. This constrains potential drug screen options, limits analysis of embryonic and larval phenotypes, and requires raising additional animals to adulthood to ensure obtaining an animal of the desired genotype. Our objective was to develop an automated system that would rapidly obtain cells and DNA from zebrafish embryos and larvae for genotyping, and that would keep the animals alive. We describe the development, testing, and validation of a zebrafish embryonic genotyping device, termed “ZEG” (Zebrafish Embryo Genotyper). Using microfluidic harmonic oscillation of the animal on a roughened glass surface, the ZEG is able to obtain genetic material (cells and DNA) for use in genotyping, from 24 embryos or larvae simultaneously in less than 10 minutes. Loading and unloading of the ZEG is performed manually with a standard pipette tip or transfer pipette. The obtained genetic material is amplified by PCR and can be used for subsequent analysis including sequencing, gel electrophoresis, or high-resolution melt-analysis. Sensitivity of genotyping and survival of animals are both greater than 90%. There are no apparent effects on body morphology, development, or motor behavior tests. In summary, the ZEG device enables rapid genotyping of live zebrafish embryos and larvae, and animals are available for downstream applications, testing, or raising.

Introduction

Zebrafish (Danio rerio) is a small vertebrate model system widely used by the biomedical research community. Zebrafish have rapid development, have transparent embryos, are inexpensive, can generate large numbers of offspring, and have a large variety of molecular and imaging tools available. The zebrafish body plan, organs, and genome are conserved with other vertebrates including in particular humans [1]. Recent work demonstrates that zebrafish can be used for drug discovery in human diseases [2], and for understanding the pathogenicity of mutations discovered by next-generating sequencing approaches in patients [3]. Despite the widespread use of zebrafish, automated research tools for working with zebrafish embryos have not developed at the same pace as the research methodologies. There is a bottleneck requiring skilled labor, which in turn has led to limitations on drug and mutant screens, and an inability to capitalize on the potential for identifying new therapies or to interrogate chemical-genetic pathways. A zebrafish screen for mutagenesis or for identifying transgenic offspring can involve timeand labor-intensive genotyping of hundreds to thousands of zebrafish. Further, the rapidly expanded use of CRISPR technologies for mutagenesis and knock-in also could be facilitated by rapid genotyping of live embryos. Currently for genotyping, embryos are grown to adult age (two to three months) before manual fin clipping. Fin clipping requires a trained technician four to six hours to prepare cells and genotype 96 fish; as well as the effort and expenses of raising more adult fish than may be ultimately needed. Alternatively, zebrafish embryos or larvae can be sacrificed and genotyped. If individual animals need to be distinctly genotyped this is even more laborious, and obviously additional testing or use of the animals is not possible. There are a few options that are laborious and slow for manual genotyping of live embryos and larvae [4, 5], but these are impractical on a larger scale. Ideally, technology to rapidly genotype zebrafish embryos (24–72 hours post-fertilization (hpf)) without harming the fish would facilitate current screens, and could lead to future applications that are not feasible considerations currently. While a variety of microfluidic-based approaches have been reported for sorting, visualizing, or monitoring zebrafish [6, 7], they have not been used for genotyping. We describe our development and optimization of an automated high-throughput device that can genotype live zebrafish embryos and larvae. Based on previous promising proof-ofconcept techniques using microfluidic-based fin-clipping or chorionic fluid genetic analyses for genotyping that we developed [8], we now tested and refined a device to generate cells and usable DNA for genotyping, including for analyses by PCR, agarose gel electrophoresis, HRMA (high resolution melt analysis), and sequencing.

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