Rapid and Efficient Live Zebrafish Embryo Genotyping

Abstract

Most current methods for genotyping zebrafish embryos require sacrifice or raising the animal to at least 1 month of age for fin amputation. These limitations restrict the use of zebrafish and increase time and costs for experiments. This article introduces an innovative method for genotyping live zebrafish embryos. The method utilizes enzyme to extract a small amount of genetic material from the skin tissue of the embryo. Then, using conventional polymerase chain reaction (PCR) strategy, the embryo is genotyped. This approach was successful >95% of the time while maintaining high viability (>90%) of the embryo. This effective method can facilitate high-throughput screening and other applications in zebrafish.

Modeling Lysosomal Storage Diseases in the Zebrafish

“…(asah1a +68/+68 ; asah1b −20/−20 , Table 2, and Supplementary Table S1) zebrafish has also been generated using CRISPR-Cas9, and exhibited reduced size, ceramide accumulation, and early mortality [4 months-post-fertilization (mpf); Zhang X. et al, 2019]. No changes in size, ceramide content, or lifespan were observed in Asah1 single mutants, suggesting that Asah1a and Asah1b possess some functional redundancy (Zhang T. et al, 2019).…”

“…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.…”

“…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. Taken together, these methods provide different options toward rapidly obtaining genotype information at the embryo or larval stage.…”

The FusX TALE Base Editor (FusXTBE) for rapid mitochondrial DNA programming of human cells in vitro and zebrafish disease models in vivo

“…The poor correlation of phenotype to genotype and stochastic mode of transmission makes it challenging to study the biology of the mtDNA-encoded disorders. To overcome this technical hurdle, we adopted a non-invasive enzymatic genotyping approach 22 to screen for embryos harboring positive edits. This approach allowed us to subject the animals to functional assessment or monitoring the germline transmission of the edits in the F1 generation.…”

“…To assess the in vivo editing efficiency of the mitoTALE BE, a method of genotyping was needed that enabled the rapid investigation of the functional readout of mtDNA edits. A non-invasive enzymatic method to extract nuclear DNA from zebrafish embryos was employed with modifications 22 . Larvae aged 3 days post fertilization (dpf) were rinsed thrice in fresh embryo media, followed by three washes in collection buffer (30 mM Tris-HCl, 1X tricaine solution).…”