Abstract
ABSTRACTAdvances in genome engineering have resulted in the generation of numerous zebrafish mutant lines. A commonly used method to assess gene expression in the mutants is in situ hybridisation (ISH). Because the embryos can be distinguished by genotype after ISH, comparing gene expression between wild-type and mutant siblings can be done blinded and in parallel. Such experimental design reduces the technical variation between samples and minimises the risk of bias. This approach, however, requires an efficient method of genomic DNA extraction from post-ISH fixed zebrafish samples to ascribe phenotype to genotype. Here we describe a method to obtain PCR-quality DNA from 95-100% of zebrafish embryos, suitable for genotyping after ISH. In addition, we provide an image analysis protocol for quantifying gene expression of ISH-probed embryos, adaptable for the analysis of different expression patterns. Finally, we show that intensity-based image analysis enables accurate representation of the variability of gene expression detected by ISH and that it can complement quantitative methods like qRT-PCR. By combining genotyping after ISH and computer-based image analysis, we have established a high-confidence, unbiased methodology to assign gene expression levels to specific genotypes, and applied it to the analysis of molecular phenotypes of newly generated lmo4a mutants.
Highlights
The emergence of genome engineering technologies, including zinc-finger nucleases (ZFNs) (Doyon et al, 2008; Meng et al, 2008), transcription activator-like effector nucleases (TALENs) (Huang et al, 2011) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 (Hwang et al, 2013), has enabled zebrafish researchers to generate a wide range of mutant lines by precisely targeting genomic loci with high efficiency (Varshney et al, 2015)
Hot Sodium Hydroxide and Tris (HotSHOT) genomic DNA extraction is suitable for PCR amplification in in situ hybridisation (ISH)-probed embryos Our aim was to establish a fast and cost-effective way to extract DNA from individual embryos after ISH
When comparing two different commercially available PCR master mixes, we found that the JumpStartTM REDTaq® ReadyMixTM performed more consistently on fixed ISH-probed genomic DNA samples than PhireTM Green HotStart II PCR Master Mix (Fig. S1)
Summary
The emergence of genome engineering technologies, including zinc-finger nucleases (ZFNs) (Doyon et al, 2008; Meng et al, 2008), transcription activator-like effector nucleases (TALENs) (Huang et al, 2011) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas (Hwang et al, 2013), has enabled zebrafish researchers to generate a wide range of mutant lines by precisely targeting genomic loci with high efficiency (Varshney et al, 2015) These methods provide alternatives to morpholino oligonucleotides (MOs), which have been used for gene knockdown studies for over 15 years The efficiency of these DNA extraction and genotyping methods has not been adequately demonstrated
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