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 zouden graag bedanken Chuck Kimmel voor begeleiding, John Dowd voor hulp bij de ontwikkeling van deze strategie fokken, Macie Walker voor haar werk in het perfectioneren van de skeletal vlek, en Charline Walker en Bonnie Ullmann voor nuttig zebrafish advies. Dit werk werd gesteund door K99/R00 DE024190 te JTN.

alle experimenten beschreven in dit protocol werden voltooid in overeenstemming en naleving van de Universiteit van Colorado en de Universiteit van Oregon institutionele Animal Care en gebruik commissies (IACUC).
 1. voorbereiding van de niet-geselecteerde beginnen voorraad <ol><li>identificeren heterozygoot dragers van de mutant allel van belang door fin bijknippen 11 en genotypering een voorraad van full-broer/zus dieren door een methode van keuze, zoals Kawasaki ...

<ol>
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Selectieve fokken onthult de subtiliteit van de genfunctie
Verschuiven van de mutant fenotypen om meer of minder ernstig door selectief fokken is een eenvoudige manier om te krijgen van nieuwe inzichten in genfunctie. In vergelijking met standaardmethoden voor niet-geselecteerde fokken, kan het protocol hier gepresenteerd een veel vollediger begrip van mutant fenotypen opleveren. In het bijzonder door het genereren van spanningen die ernstig zijn, kan de volledige breedte van ...

De zebravis is een krachtig modelsysteem voor het begrijpen van de ontwikkeling van het skelet. Met mutant zebrafish stammen, kunnen biologen ontcijferen genfunctie tijdens skeletogenesis. Skelet mutant fenotypes van zebravis kunnen echter presenteren met variabele doordringendheid1,2,3,4 die ontwikkelingsstoornissen en genetische analyses kan belemmeren. Het doel van deze methode is drievoudig. Eerst, krommen zebrafish mutant die consequent ernstige fenotypen produceren kunt downstream developmental studies zoals time-lapse opname5 en -transplantatie6. Dit soort studies kunnen worden verlamd door wilt studeren fenotypen die alleen inconsistent manifesteren. Ten tweede, inteelt zebrafish stammen kan verminderen variatie in genetische achtergrond, dus het bevorderen van experimentele consistentie en reproduceerbaarheid. Bijvoorbeeld, kunnen uitvoeren van alle in situ hybridisatie analyses op een selectief ingeteelde stam verminderen van storende variabiliteit en versterken van conclusies. Ten derde, genereren van ernstige en milde stammen zal onthullen de volledige fenotypische serie die uit een bepaalde mutatie voortvloeien kunnen.
Selectieve fokken van dodelijke mutanten lijkt op het eerste gezicht onmogelijk. Hoe kan één RAS voor doordringendheid, wanneer de dieren die worden gescoord voor selectie dood zijn? Gelukkig, methoden voor selectieve fokken door familie selectie, specifiek nakomelingen testen, gebleken doeltreffendheid in veeteelt programma's voor vele jaren7,8. Deze programma's worden voornamelijk gebruikt voor selectieve fokken voor trekken die slechts in één geslacht, zoals melkproductie in koeien of eiproductie kippen aanwezig zijn. De mannetjes van deze soorten kunnen niet rechtstreeks worden gescoord maar hun nageslacht worden gescoord, en vervolgens een waarde wordt toegewezen aan de ouders. Lenen van deze strategie, gaat het hier gepresenteerde protocol scoren de vaste en gebeitst gemuteerde nakomelingen van een paar van de zebravis die heterozygoot voor een gemuteerde gen van belang zijn. De doordringendheid van een fenotype in de homozygoot dodelijke gemuteerde nakomelingen wordt toegewezen aan de ouders bij de beslissing welke personen zal produceren de volgende generatie in de lijn. Wij vinden dat deze methode een effectief middel is van het verschuiven van doordringendheid in zebrafish dodelijke skelet mutanten1.
Gelijkaardig aan andere studies, neemt dit protocol selectief fokken onder overweging criteria zoals koppeling grootte, overleven van nakomelingen, normale ontwikkeling van embryo's en sex-ratio9. Deze factoren worden echter alle beschouwd als in het kader van een mutant achtergrond met de doelstelling van het verschuiven van de mutant doordringendheid. Daarom breidt dit protocol vorige selectief fokken paradigma's door het aanbieden van een methode om te versterken developmental mutant analyses, evenals het verhogen van de homogeniteit van de achtergrond.
Dit protocol vereist uitgebreide genotypering, dus is het belangrijk om een betrouwbare, snelle genotypering protocol op voorhand. Er zijn vele genotypering protocollen beschikbaar10,11, maar we vinden de Kawasaki Advanced Safety genotypering12,13,14 is sneller, meer kosten efficiënte en betrouwbaarder dan methoden op basis van restrictie-enzym splitsing van versterkte sequenties10. We nemen daarom een Kawasaki Advanced Safety-protocol in dit werk. Bovendien, we focus op skelet mutant fenotypen in dit protocol en omvatten een procedure voor Alcian blauw/Alizarine rood kleuring gemodificeerde van eerdere protocollen15.
De hier beschreven methode is een eenvoudige strategie voor het verschuiven van dodelijke mutant doordringendheid, naar boven of naar beneden. Terwijl dit protocol is gericht op het skelet mutant fenotypen, wij geloven dat het zal een nuttige strategie voor veehouderij van alle mutant zebrafish regels. In feite, overstijgt het nut van deze fokken strategie waarschijnlijk zebrafish. We voorspellen dat dit protocol kan worden gewijzigd om te verschuiven van doordringendheid in een brede waaier van organismen. Dodelijke doordringendheid verschuiven door het testen van de nakomelingen kan helpen duwen voorwaarts de voortgang van elk ontwikkelings geneticus.

<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

Mutant fenotypes van zebravis zijn vaak onvolledig penetrant, alleen in sommige mutanten manifesteren. Interessante fenotypen die inconsistent worden weergegeven kunnen moeilijk zijn om te studeren, en kunnen leiden tot verwarrende resultaten. Het protocol beschreven hier is een eenvoudige fokken paradigma te verhogen of doordringendheid in dodelijke zebrafish skelet mutanten. Omdat dodelijke mutanten kunnen niet selectief rechtstreeks worden gefokt, is de strategie van de klassieke selectief fokken van nakomelingen testen werkzaam. Deze methode omvat ook protocollen voor Kompetitive Allele specifieke PCR (Kawasaki Advanced Safety) genotypering zebravis en kleuring larvale zebrafish kraakbeen en bot. Toepassing van de strategie van de veehouderij die hier worden beschreven kunt verhogen de doordringendheid van een interessante skelet fenotype waardoor meer reproduceerbare resultaten in downstream toepassingen. Bovendien kan verminderen de mutant doordringendheid via deze selectieve fokken strategie onthullen de ontwikkelings processen waarvoor de functie van het gemuteerde gen meest cruciaal. Terwijl het skelet speciaal hier wordt geacht, stellen wij voor dat deze methode nuttig voor alle zebrafish mutant lijnen zijn zal.

Dit protocol is een lange termijn veehouderij techniek handig voor begrip zebrafish skelet mutanten (Figuur 1). Selectieve fokken door het testen van de nakomelingen moet opleveren een verschuiving in de algehele doordringendheid zowel neerwaartse en opwaartse in een paar generaties (Figuur 2). In onze eerdere werk reed twee rondes van het selectieve fokken naar beneden de gemiddelde doordringendheid van 17% tot 3%<sup class="xre...

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

Het doel van dit protocol is te wijzigen van de doordringendheid van dodelijke skelet mutant fenotypen in zebrafish door selectief fokken. Dodelijke mutanten naar volwassenheid kunnen niet worden gekweekt en zelf gefokt, dus dit protocol beschrijft een methode voor het bijhouden en selecteren doordringendheid door meerdere generaties nakomelingen testen.

Zebravis dodelijke skelet Mutant doordringendheid verschuiven door nakomelingen testen

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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