High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping

Abstract

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short generation time, are easy to handle, display a high reproductive rate, low cost, and are easily amenable to genetic manipulations by microinjection of DNA in embryos. Recent advances in gene editing tools are enabling precise introduction of mutations and transgenes in zebrafish. Disease modeling in zebrafish often leads to larval phenotypes and early death which can be challenging to interpret if genotypes are unknown. This early identification of genotypes is also needed in experiments requiring sample pooling, such as in gene expression or mass spectrometry studies. However, extensive genotypic screening is limited by traditional methods, which in most labs are performed only on adult zebrafish or in postmortem larvae. We addressed this problem by adapting a method for the isolation of PCR-ready genomic DNA from live zebrafish larvae that can be achieved as early as 72 h post-fertilization (hpf). This time and cost-effective technique, improved from a previously published genotyping protocol, allows the identification of genotypes from microscopic fin biopsies. The fins quickly regenerate as the larvae develop. Researchers are then able to select and raise the desired genotypes to adulthood by utilizing this high-throughput PCR-based genotyping procedure.

Genomic evidence supports the introgression between two sympatric stickleback species inhabiting the White Sea basin

“…After phenotypical validation of each stickleback specimen, conducted by in situ dorsal fin spine counting, the anal fin was clipped and preserved in 70% ethanol for genomic DNA isolation, as described previously [ 50 ]. All specimens were caught and immediately released without any further morphological analysis to avoid stressing much the fish.…”

Zebrafish, Medaka and Turquoise Killifish for Understanding Human Neurodegenerative/Neurodevelopmental Disorders

“…It is estimated that approximately 71% of human genes have at least one zebrafish gene orthologue [31]. In addition, approximately 84% of the genes known to be associated with human diseases have orthologues in the zebrafish genome [32]. Small fishes have the advantage of simple procedures for gene editing.…”

Adar-mediated A-to-I editing is required for establishment of embryonic body axes in zebrafish

“…Oligonucleotides used for genotyping are as follows: adar_genom_F 5'-CTAACGCTACACCCTCCTCAGC-3' and adar_genom_R 5'-CGTCTGGTACTGGATAGGCTC -3'. Similarly, 3 dpf larvae from F2 generation were genotyped according to a published protocol [68] and sequenced using the abovementioned pair of oligonucleotides. Expression of zebrafish adar family based on transcriptome profiling of developing embryos (RNA-seq) [54] .…”