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.

Modeling Lysosomal Storage Diseases in the Zebrafish

“…To address the aforementioned issues, several approaches have been developed that rely on genetic material from fin (Samuel et al, 2015), chorionic fluid (Samuel et al, 2015), or skin (Lambert et al, 2018;Zhang X. et al, 2019). Using replica molding, Samuel et al (2015) designed two microfluidic devices that allow isolation of either chorionic fluid from individual embryo via microchannel-facilitated chorion rupture, or fin tissue from individual larvae via a multichannel system for larva positioning and fin removal, at a success rate of 78% for the former and 100% for the latter.…”

“…Using replica molding, Samuel et al (2015) designed two microfluidic devices that allow isolation of either chorionic fluid from individual embryo via microchannel-facilitated chorion rupture, or fin tissue from individual larvae via a multichannel system for larva positioning and fin removal, at a success rate of 78% for the former and 100% for the latter. More recently, the ZEG (Zebrafish Embryo Genotyper) system was developed that removes skin tissue from embryos or larvae via vibration over a rough glass surface at the rate of 24 fish per 10 min, with >90% success rate and no subsequent changes in larva behavior (Lambert et al, 2018). Another method toward skin cell isolation involves controlled enzyme digestion; by carefully optimizing proteinase K treatment parameters, Zhang X. et al (2019) have demonstrated isolation of genetic material from larval skin tissue in a 96-well plate with >95% success rate and >90% viability.…”

Modeling Neuronal Diseases in Zebrafish in the Era of CRISPR

“…The mutation is subsequently confirmed by PCR and sequencing. A ZEG device enables genotyping while keeping the embryos alive [ 67 ]. To avoid mosaicism and phenotype variability, a homozygotic mutant line can be produced by outcrossing mosaic crispants to wild- type and then inbred F1 until homozygous are found.…”

“…Genotype screening can be performed in a portion of the mosaic F0 by a quick procedure such as TIDE (Tracking of Indels by Decomposition), HMA (heteroduplex mobility assay), or T7 endonuclease digestion, and then confirmed by PCR and sequencing. To keep the genotyped embryos alive, a ZEG device can be used instead of using the whole embryo for the analysis [ 67 ]. For a clear example of this procedure, see [ 101 ].…”

Rapid and Efficient Live Zebrafish Embryo Genotyping

“…However, using current methods for genotyping zebrafish, embryos must be sacrificed; or fish must be raised to at least 1 month old to have a fin clip collected. 1 Methods have been described for genotyping of live embryos; however, these require special instruments 2,3 or tedious surgical procedures. 4,5 These limitations increase time and cost for experiments and prohibit larger-scale phenotypic screens.…”