Name: shifting zebrafish lethal skeletal mutant penetrance by progeny testing
Uploaded: 2017-09-01T15:00:00
Description: Watch this Scientific Journal Video about Shifting Zebrafish Lethal Skeletal Mutant Penetrance by Progeny Testing at JoVE.com

We would like to thank Chuck Kimmel for guidance, John Dowd for help in developing this breeding strategy, Macie Walker for her work in perfecting the skeletal stain, and Charline Walker and Bonnie Ullmann for helpful zebrafish advice. This work was supported by K99/R00 DE024190 to JTN.

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
<ol>
	<li>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 KASP12,1...

<ol>
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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 ...

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 penetrance1,2,3,4 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 transplantation6. These sorts of studies can be crippled by attempting to study phenotypes that only manifest inconsistently. Second, inbreeding zebrafish strains can decrease genetic background variation, thus promoting experimental consistency and reproducibility. For example, performing all in situ hybridization analyses on one selectively inbred strain can reduce confounding variability and strengthen conclusions. Third, generating severe and mild strains will reveal the entire phenotypic series that can result from a particular mutation.
At first glance, selective breeding of lethal mutants seems impossible. How can one breed for penetrance when the animals that are scored for selection are dead? Fortunately, methods for selective breeding by family selection, specifically progeny testing, have demonstrated effectiveness in livestock breeding programs for many years7,8. These programs are mainly used for selective breeding for traits that are only present in one sex, like milk production in cows or egg production in hens. The males of these species cannot be scored directly, but their progeny are scored and a value is then assigned to the parents. Borrowing from this strategy, the protocol presented here involves scoring the fixed and stained mutant offspring from a pair of zebrafish that are heterozygous for a mutant gene of interest. The penetrance of a phenotype in the homozygous lethal mutant offspring is assigned to the parents when deciding which individuals will produce the next generation in the line. We find that this method is an effective means of shifting penetrance in zebrafish lethal skeletal mutants1.
Similar to other studies, this selective breeding protocol takes under consideration criteria like clutch size, survival of offspring, normal development of embryos, and sex ratio9. However, these factors are all considered in the context of a mutant background with the objective of shifting the mutant penetrance. Therefore, this protocol extends previous selective breeding paradigms by offering a method to strengthen developmental mutant analyses as well as increase background homogeneity.
This protocol requires extensive genotyping, so it is important to develop a reliable, rapid genotyping protocol in advance. There are many genotyping protocols available10,11, however we find the KASP genotyping12,13,14 is faster, more cost efficient, and more reliable than methods based on restriction enzyme cleavage of amplified sequences10. Therefore, we include a KASP protocol in this work. Additionally, we focus on skeletal mutant phenotypes in this protocol and include a procedure for Alcian Blue/Alizarin Red staining modified from previous protocols15.
The method described here is a straightforward strategy for shifting lethal mutant penetrance upward or downward. While this protocol focuses on skeletal mutant phenotypes, we believe it will be a useful strategy for husbandry of all mutant zebrafish lines. In fact, the utility of this breeding strategy likely extends beyond zebrafish. We predict that this protocol can be modified to shift penetrance in a broad range of organisms. Shifting lethal penetrance by progeny testing can help push forward the progress of any developmental geneticist.

<table border="0" cellpadding="0" cellspacing="0" width="627">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Paraformaldehyde, pelleted, solid</td>
			<td style="width:125px;">Ted Pella Co.</td>
			<td style="width:119px;">18501</td>
			<td style="width:167px;">Pelleted PFA is a safer alternative to powdered PFA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium Chloride, solid</td>
			<td style="width:125px;">Acros Organics</td>
			<td>223210010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10x PBS, Aqueous</td>
			<td style="width:125px;">Fisher</td>
			<td style="width:119px;">BP3994</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">190 proof Ethanol</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Alcian Blue, solid</td>
			<td style="width:125px;">Anatech Ltd.</td>
			<td style="width:119px;">867</td>
			<td>Must be from Anatech</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Alizarin Red, solid</td>
			<td style="width:125px;">Sigma</td>
			<td style="width:119px;">A5533-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycerol, liquid</td>
			<td style="width:125px;">Fisher</td>
			<td style="width:119px;">BP229 1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hydrogen peroxide, liquid</td>
			<td style="width:125px;">Fisher</td>
			<td style="width:119px;">BP263500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Potassium hydroxide,&#160; solid</td>
			<td style="width:125px;">Fisher</td>
			<td style="width:119px;">P250 500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">StepOnePlus Real-time PCR Machine</td>
			<td style="width:125px;">Applied Biosystems</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">MicroAmp Fast Optical 96-well Reaction Plate with Barcode (0.1mL)</td>
			<td style="width:125px;">Applied Biosystems</td>
			<td style="width:119px;">4346906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microseal &#39;B&#39; seal</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;">MSB1001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KASP Master Mix, High ROX</td>
			<td style="width:125px;">LGC</td>
			<td>KBS-1016-022</td>
			<td>https://www.lgcgroup.com/products/kasp-genotyping-chemistry/#.WOPX41UrKUk</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KASP By Design Primer Mix</td>
			<td style="width:125px;">LGC</td>
			<td style="width:119px;">KBS-2100-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris HCl, solid</td>
			<td style="width:125px;">Fisher</td>
			<td style="width:119px;">BP153 500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">potassium chloride, solid</td>
			<td style="width:125px;">Fisher</td>
			<td style="width:119px;">BP366 500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tween-20, liquid</td>
			<td style="width:125px;">Fisher</td>
			<td style="width:119px;">BP337 100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nonidet P40</td>
			<td style="width:125px;">ThermoFisher</td>
			<td>28324</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tricaine-S</td>
			<td style="width:125px;">Western Chemicals</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Proteinase K</td>
			<td style="width:125px;">Fisher</td>
			<td style="width:119px;">BP1700 100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T100 Thermal Cycler</td>
			<td style="width:125px;">BioRad</td>
			<td>1861096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Controlled Drop Pasteur Pipets</td>
			<td style="width:125px;">Fisher</td>
			<td>13-678-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nanodrop</td>
			<td style="width:125px;">ThermoFisher</td>
			<td style="width:119px;"></td>
			<td>for DNA quantitation</td>
		</tr>
	</tbody>
</table>

shifting zebrafish lethal skeletal mutant penetrance by progeny testing

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.

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...

Watch this Scientific Journal Video about Shifting Zebrafish Lethal Skeletal Mutant Penetrance by Progeny Testing at JoVE.com

Shifting Zebrafish Lethal Skeletal Mutant Penetrance by Progeny Testing

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.

Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Interesting phenotypes that inconsistently appear can be ...

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