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>
	<li>Nichols, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligament+versus+bone+cell+identity+in+the+zebrafish+hyoid+skeleton+is+regulated+by+mef2ca.">Ligament versus bone cell identity in the zebrafish hyoid skeleton is regulated by mef2ca.</a> Development. 143 (23), 4430-4440 (2016).</li><li>Sheehan-Rooney, K., Swartz, M. E., Zhao, F., Liu, D., Eberhart, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ahsa1+and+Hsp90+activity+confers+more+severe+craniofacial+phenotypes+in+a+zebrafish+model+of+hypoparathyroidism,+sensorineural+deafness+and+renal+dysplasia+(HDR).">Ahsa1 and Hsp90 activity confers more severe craniofacial phenotypes in a zebrafish model of hypoparathyroidism, sensorineural deafness and renal dysplasia (HDR).</a> Dis Model Mech. 6 (5), 1285-1291 (2013).</li><li>Cox, S. 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The authors have nothing to disclose.

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 ...

The overall goal of this breeding strategy is to manipulate mutant penetrance upward or downward. This selective breeding method enables a more complete understanding of mutant phenotypes. By generating high and low penetrance mutant strains, the entire phenotypic series of a mutation can be understood.Most zebrafish scale where mutants are recessive lethal, so homozygous mutants die before they can be directly bred. Therefore, we employ the classical selective breeding strategy of progeny testing, which we demonstrate here. We first had the item for this method when attempting to time lapse record ectopic bone cell development.Frustratingly, extra bone cells appeared in it frequently, so we set out to alter mutant penetrants by selective breeding. Although we perform this method on a zebrafish scale with a mutant line, it can also be applied to other zebrafish mutant phenotypes and likely, other organisms. After preparing unselected starting zebrafish stock according to the text protocol, carry out KASP genotyping as follows.Place the zebrafish tail tissue in 50 microliters of lysis buffer in the wells of a 96 well plate. Add 10 microliters of 10 milligrams per milliliter proteinase k per well and digest the tissue samples in a thermocycler at 55 degree celsius for two to five hours. Followed by a 20 minute 94 degree celsius inactivation step.Refrigerate the lysis product after digestion. Use a spectrophotometer to quantify the genomic DNA. Then, using molecular biology grade water, dilute the genomic template so that each reaction contains approximately 20 to 30 nanograms.Add 0.14 microliters of the KASP primer mix and five microliters of the KASP master mix per sample on ice, into a 1.5 milliliter microcentrifuge tube and mix well. Then, briefly centrifuge to collect the contents at the bottom of the tube. Pipette five microliters of primer master mix solution into the wells of a 96 well optical PCR plate suitable for the real time PCR machine being used.Then, pipette five microliters of diluted DNA template samples into each well and aspirate with a pipette to mix. Place an optically clear film seal over the plate, ensuring that the film is fully sealed on each well. Briefly centrifuge the plate at 600 x G to collect the contents at the bottom and to remove bubbles from the mix.After performing the main KASP reaction, view the resulting scatter plot produced by the analysis computer. House the identified heterozygous animals together on the main water system. After the animal recover from the fin clip, set up pairwise intercrosses, use dividers to ensure embryos are synchronized.Collect the embryos, and keep the parental pairs isolated in breeding cages. Monitor isolated adults, so that aggressive fish can be separated or provided with cover. To rear the embryos on day five or six, when the swim bladders is inflated in 75%of a clutch, use a glass pipette to collect phenotypically wild type animals.Place phenotypic wild types in the nursery, recording which parents yielded them. After anesthetizing larvae that do not inflate their swim bladders according to the text protocol, use a wide bore glass pasteur pipette to collect the animals into 1.5 milliliters microcentrifuge tubes. Remove the embryo water from each tube and replace it with one milliliter of 2%paraformaldehyde in 1x PBS.Rock the tubes for one hour to fix the animals. Track the tubes regularly during this and all subsequent rocking steps to ensure no larvae have become stuck to the side or clumped in the bottom of the tube. Use a glass pipette to remove the fixative from the tubes.Then add one milliliter of 50%ethanol. Rock the samples for an additional 10 minutes. Prepare fresh staining solution by adding 20 microliters of 0.5%alizarin red per one milliliter of Alcian premix.Remove the 50%ethanol, and add one milliliter of staining solution to each tube. Then, rock the tubes overnight. Within a few days after bleaching the embryos according to the text protocol, score stained mutant offspring for penetrants.In this example, we score for any occurrence of a topic bone. For the next generation, choose two high penetrants and two low penetrants families. Give each parental pair a sub stock identifier, for example, family 4.01, 02, et cetera.It's also helpful to indicate the or inbred generation number on all labels. House parental pairs on the main system with several mixed sex companion fish. Companion fish can be any zebrafish line with permanent obvious distinguishing features, such as altered pigmentation or fin structure.Label the tanks containing the wild type sibling larvae from selected families with the penetrants from the mutant siblings and raise the fish to adulthood as normal. Selective breeding by progeny testing, should yield a shift in overall penetrants both downward and upward in a few generations. In these examples, apply to the mef2ca b1086 mutant, high or low penetrants of a topic bone between the opercal and the branchiostegal ray in lethal homozygous mutants was selected for.In a successful cast procedure, type groupings of samples corresponding to gene type should be recognized by the computer program and the NTCs should be located near the origin of the plot after sufficient cycles have been performed. As demonstrated here, successful Alcian blue, alizarin red stain will have vibrantly stained bone and cartilage elements that are not transparent or faint. The boundaries of the cartilage elements should appear crisp and individual contracytes will be discernible.Alcian blue that is too concentrated were not clear from other tissues making analysis impossible. Implementing this technique from zebrafish line maintenance will likely alter penetrants in a few generations. Following this procedure, other methods like NC2 hybridization and timelapse imaging studies may yield more consistent results.We use this technique to explore the heritability of phenotype variation. After watching this video, you should have a good understanding of how to selectively breed your mutant strains to alter penetrance, even if the mutation is lethal.

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7.5K visualizações.  University of Colorado Anschutz Medical Campus. The overall goal of this breeding strategy is to manipulate mutant penetrance upward or downward. This selective breeding method enables a more complete understanding of mutant phenotypes. By generating high and low penetrance mutant strains, the entire phenotypic series of a mutation can be understood.Most zebrafish scale where mutants are recessive lethal, so homozygous mutants die before they can be directly bred. Therefore, we employ the classical selective breeding strategy of pro...