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

Summary

Zebrafish have been used as reliable genetic model organisms in biomedical research, especially with the advent of gene-editing technologies. When larval phenotypes are expected, DNA extraction and genotype identification can be challenging. Here, we describe an efficient genotyping procedure for zebrafish larvae, by tail clipping, as early as 72-h post-fertilization.

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.

Introduction

The zebrafish (Danio rerio) is a vertebrate organism widely used as a model for disease investigation as well as for preclinical testing of therapeutic hypotheses^1,2,3. 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 embryos⁴. Through the increasing development of novel transgenic and gene editing technologies such as Zinc-Finger nucleases (ZFN)⁵, TAL-like Effector Nucleases (TALENs)

Protocol

Procedures involving animal subjects have been approved and are in accordance with animal care guidelines provided by the Canadian Council on Animal Care and the University of Ottawa animal care committees.

1. Preparation

1. Prepare the dissection surface by taping a 9 cm Petri dish lid with autoclave tape across its interior surface. Position it under a stereo-microscope.
2. Place 3 - 5 days post-fertilization (dpf) zebrafish larvae in a Petri dish and anesthetize in ˜1.5 mM Tricaine in 1x E3 embryo media (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂, 0.33 mM MgSO₄, pH 7.2).
3. Cut off the end of a P1000 pipette tip w....

Results

The efficiency of the protocoll was demonstrated by genotyping the offspring of an heterozygous cross (aldh7a1^+/ot100, here shown as aldh7a1^+/-) (Figure 3A-C)⁹. The aldh7a1^ot100 mutant allele has a 5-base pair (bp) insertion in the first coding exon of the zebrafish aldh7a1 gene that leads to frameshift and loss-of-function due to an .......

Discussion

Gene editing tools have been employed to precisely introduce mutations and transgenes in zebrafish over the past years^5,6,7,8. When performing disease modeling in zebrafish, early larval or juvenile phenotypes are often observed^9,12,13,14. The protocol presented here .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Petri dish	Corning	430167
Microscalpel	World Precision Instruments	500249
Stereomicroscope	Nikon	SMZ1500
Tricaine/Ethyl 3-aminobenzoate methanesulfonate	Sigma-Aldrich	E10521
96-well Tissue-culture plate	Costar	3595
96-well PCR plate	Fisher	14230232
NaOH	Fisher	S25548A
Tris base	Sigma-Aldrich	T1503
NaCl	Fisher	BP358-10
KCl	Sigma-Aldrich	P3911
MgSO4	Sigma-Aldrich	230391
5% Chelex 100 sodium form	Sigma-Aldrich	C7901
GelRed 1x	Biotium	41003
Boric Acid	Sigma-Aldrich	B6768
Sub-Cell Model 192 system	Bio-Rad	1704508
30 % Acrylamide/Bis Solution, 37.5: 1	Bio-Rad	1610158
GoTaq Green Master Mix, 2X	Promega	M7123
paper wipes (Kimwipes® disposable wipers)	Sigma-Aldrich	Z188956
1kb-plus molecylar weight marker	Thermo Scientific	SM1343
Nuclease free water	Sigma-Aldrich	W4502
Sub-Cell Model 192 system (high-throughput agarose gel system)	Bio-Rad	1704508
vertical gel electrophoresis system Mini-PROTEAN Tetra Cell	Bio-Rad	1658004