Combining genotypic and phenotypic analyses on single mutant zebrafish larvae

Abstract

Zebrafish is a powerful animal model used to study vertebrate embryogenesis, organ development and diseases (Gut et al., 2017) [1]. The usefulness of the model was established as a result of various large forward genetic screens identifying mutants in almost every organ or cell type (Driever et al., 1996; Haffter et al., 1996) [2,3]. More recently, the advent of genome editing methodologies, including TALENs (Sander et al., 2011) [4] and the CRISPR/Cas9 technology (Hwang et al., 2013) [5], led to an increase in the production of zebrafish mutants. A number of these mutations are homozygous lethal at the embryonic or larval stages preventing the generation of homozygous mutant zebrafish lines. Here, we present a method allowing both genotyping and phenotype analyses of mutant zebrafish larvae from heterozygous zebrafish incrosses. The procedure is based on the genotyping of the larval tail after transection, whereas phenotypic studies are performed on the anterior part of the zebrafish larvae. • The method includes (i) a protocol for genotyping, (ii) protocols for paraffin embedding and histological analyses, (iii) protocols for protein and histone extraction and characterization by Western blot, (iv) protocols for RNA extraction and characterization by RT-PCR, and (v) protocols to study caudal spinal cord regeneration. • The technique is optimized in order to be applied on single zebrafish embryos and larvae. Keywords: Zebrafish, Genotyping, Protein extraction, Histone extraction, RNA extraction, Histology, Spinal cord regeneration