Shifting Zebrafish Lethal Skeletal Mutant Penetrance by Progeny Testing

Summary

The goal of this protocol is to alter the penetrance of lethal skeletal mutant phenotypes in zebrafish by selective breeding. Lethal mutants cannot be grown to adulthood and bred themselves, therefore this protocol describes a method for tracking and selecting penetrance through multiple generations by progeny testing.

Abstract

Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Interesting phenotypes that inconsistently appear can be difficult to study, and can lead to confounding results. The protocol described here is a straightforward breeding paradigm to increase and decrease penetrance in lethal zebrafish skeletal mutants. Because lethal mutants cannot be selectively bred directly, the classic selective breeding strategy of progeny testing is employed. This method also includes protocols for Kompetitive Allele Specific PCR (KASP) genotyping zebrafish and staining larval zebrafish cartilage and bone. Applying the husbandry strategy described here can increase the penetrance of an interesting skeletal phenotype enabling more reproducible results in downstream applications. In addition, decreasing the mutant penetrance through this selective breeding strategy can reveal the developmental processes that most crucially require the function of the mutated gene. While the skeleton is specifically considered here, we propose that this methodology will be useful for all zebrafish mutant lines.

Introduction

The zebrafish is a powerful model system for understanding skeletal development. With mutant zebrafish strains, biologists can decipher gene function during skeletogenesis. However, zebrafish skeletal mutant phenotypes can present with variable penetrance^1,2,3,4 which can hinder developmental and genetic analyses. The purpose of this method is threefold. First, generating zebrafish mutant lines which consistently produce severe phenotypes enables downstream developmental studies like time-lapse recording⁵ and transpla....

Protocol

All experiments described in this protocol were completed in accordance and compliance with the University of Colorado and the University of Oregon Institutional Animal Care and Use Committees (IACUC).

1. Preparing the Unselected Starting Stock

1. Identify heterozygous carriers of the mutant allele of interest by fin clip¹¹ and genotyping a stock of full-sibling animals by a method of choice, such as KASP^12,13,14. This protocol was performed with the zebrafish mef2ca^b1086 strain.

Results

This protocol is a long-term husbandry technique useful for understanding zebrafish skeletal mutants (Figure 1). Selective breeding by progeny testing should yield a shift in overall penetrance both downward and upward in a few generations (Figure 2). In our previous work, two rounds of selective breeding drove the average penetrance downward from 17% to 3%¹. Similarly, in our upward line, we shifted the a.......

Discussion

Selective Breeding Unveils Subtleties of Gene Function

Shifting mutant phenotypes to be either more or less severe by selective breeding is a straightforward way to gain new insights into gene function. When compared with standard methods of unselected breeding, the protocol presented here can yield a much more complete understanding of mutant phenotypes. Specifically, by generating strains that are severe, the full breadth of mutant phenotypes may be revealed, including some .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Paraformaldehyde, pelleted, solid	Ted Pella Co.	18501	Pelleted PFA is a safer alternative to powdered PFA
Magnesium Chloride, solid	Acros Organics	223210010
10x PBS, Aqueous	Fisher	BP3994
190 proof Ethanol
Alcian Blue, solid	Anatech Ltd.	867	Must be from Anatech
Alizarin Red, solid	Sigma	A5533-25G
Glycerol, liquid	Fisher	BP229 1
Hydrogen peroxide, liquid	Fisher	BP263500
Potassium hydroxide,  solid	Fisher	P250 500
StepOnePlus Real-time PCR Machine	Applied Biosystems
MicroAmp Fast Optical 96-well Reaction Plate with Barcode (0.1 mL)	Applied Biosystems	4346906
Microseal 'B' seal	BioRad	MSB1001
KASP Master Mix, High ROX	LGC	KBS-1016-022	https://www.lgcgroup.com/products/kasp-genotyping-chemistry/#.WOPX41UrKUk
KASP By Design Primer Mix	LGC	KBS-2100-100
Tris HCl, solid	Fisher	BP153 500
potassium chloride, solid	Fisher	BP366 500
Tween-20, liquid	Fisher	BP337 100
Nonidet P40	ThermoFisher	28324
Tricaine-S	Western Chemicals
Proteinase K	Fisher	BP1700 100
T100 Thermal Cycler	BioRad	1861096
Controlled Drop Pasteur Pipets	Fisher	13-678-30
Nanodrop	ThermoFisher		for DNA quantitation