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.
Highlights
Zebrafish (Danio rerio) is a small vertebrate model system widely used by the biomedical research community
We found that while a chorionic fluid device was able to obtain genetic material, that it had variable and inconsistent sensitivity that ranged as low as 0% but was typically 30%
We examined whether the Zebrafish Embryo Genotyper (ZEG) could obtain sufficient DNA for PCR to be analyzed by agarose gel electrophoresis
Summary
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
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