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

Vi vil gjerne takke Chuck Kimmel for veiledning, John Dowd for hjelp i å utvikle denne avl strategi, Macie Walker for sitt arbeid i å perfeksjonere skjelettlidelser flekken, og Charline Walker og Bonnie Ullmann for nyttig sebrafisk råd. Dette arbeidet ble støttet av K99/R00 DE024190 til JTN.

alle eksperimenter beskrevet i denne protokollen ble avsluttet i samsvar og overholdelse av University of Colorado og University of Oregon institusjonelle Animal Care og bruk komiteer (IACUC).
 1. forbereder umerkede starter aksjen <ol><li>identifiserer heterozygote bærere av det muterte allelet rundt av fin utklipp 11 og genotyperingteknologi et lager av full-sibling dyr av en metode for valg, som KASP 12 , 13...

<ol>
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Selektiv formering avsløre nyanser av gen funksjon
Skiftende mutant fenotyper å være mer eller mindre alvorlige av selektiv formering er en grei måte å få ny innsikt i gen funksjon. Sammenlignet med standard metoder for umerkede avl, kan protokollen presenteres her gi en mer komplett forståelse av mutant fenotyper. Spesielt ved å generere stammer som er alvorlige, kan den fulle bredden av mutant fenotyper bli avslørt, inkludert noen som var unobservable med umerkede a...

Sebrafisk er en effektiv modell for å forstå utvikling av skjelettet. Med mutant sebrafisk stammer, kan biologer dechiffrere gen funksjon under skeletogenesis. Imidlertid kan sebrafisk skjelettlidelser mutant fenotyper presentere med variabel penetrance1,2,3,4 som kan hindre utviklingsmessige og genetiske analyser. Formålet med denne metoden er tredelt. Først kan generere sebrafisk mutant linjer som konsekvent produserer alvorlig fenotyper nedstrøms utviklingsmessige studier som tid-lapse innspillingen5 og transplantasjon6. Disse slags studier kan bli ødelagt av prøver å studere fenotyper som bare inkonsekvent. Andre kan inbreeding sebrafisk stammer redusere genetisk bakgrunn variasjon, dermed fremme eksperimentelle konsistens og reproduserbarhet. For eksempel kan alle i situ hybridisering analyser på en selektivt innavlet belastning redusere forvirrende variasjon og styrke konklusjoner. Tredje vil genererer alvorlig og milde stammer avdekke hele fenotypiske serien som kan følge av en bestemt mutasjon.
Ved første øyekast, virker selektiv formering dødelige mutanter umulig. Hvordan kan en rase for penetrance når dyrene som scoret for valg er døde? Heldigvis har metoder for selektiv formering av familien utvalg, spesielt avkom testing vist effektivitet i husdyr programmer for mange år7,8. Disse programmene brukes hovedsakelig for selektiv formering for egenskaper som finnes bare i ett kjønn, som melk produksjon i kyr eller eggproduksjon i hens. Menn av disse artene kan være scoret direkte, men deres avkom scoret og verdien tilordnes deretter til foreldrene. Låne fra denne strategien, omfatter protokollen presenteres her scoring faste og farget mutant avkom fra et par sebrafisk som er heterozygote for en muterte genet av interesse. Penetrance av en fenotypen i homozygous dødelig mutant avkom er tildelt foreldrene når du bestemmer hvilke personer vil produsere neste generasjon i linjen. Vi finner at denne metoden er et effektivt middel for skiftende penetrance i sebrafisk dødelige skjelettlidelser mutanter1.
I likhet med andre studier, denne selektiv formering protokollen tar under vurdering kriterier som clutch størrelse, overlevelse av avkom, normal utvikling av embryo og sex ratio9. Men er disse faktorene alle vurdert i sammenheng med en mutant bakgrunn med sikte på å skifte den muterte penetrance. Derfor varer denne protokollen tidligere selektiv formering paradigmer tilbyr en metode for å styrke developmental mutant analyser samt øke bakgrunnen homogenitet.
Denne protokollen krever omfattende genotyperingteknologi, så det er viktig å utvikle en pålitelig, rask genotyperingteknologi protokoll på forhånd. Det er mange genotyperingteknologi protokoller tilgjengelig10,11, men vi finner de KASP genotyperingteknologi12,13,14 koster raskere, mer effektiv og mer pålitelig enn metoder basert på begrensning enzym cleavage forsterket sekvenser10. Derfor inkludere vi en KASP protokoll i dette arbeidet. I tillegg vi fokusere på skjelettlidelser mutant fenotyper i denne protokollen og inkludere en prosedyre for Alcian blå/Alizarin rød flekk endret tidligere protokoller15.
Metoden beskrevet her er en enkel strategi for skiftende dødelige mutant penetrance oppover eller nedover. Mens denne protokollen fokuserer på skjelettlidelser mutant fenotyper, tror vi det vil være en nyttig strategi for dyrehold alle mutant sebrafisk linjer. Faktisk nytten av dette avl strategi sannsynlig strekker seg utover sebrafisk. Vi spår at denne protokollen kan endres for å skifte penetrance i et bredt spekter av organismer. Skiftende dødelige penetrance avkom tester kan hjelpe push fremover fremdriften for alle utviklingsmessige genetiker.

<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

Sebrafisk mutant fenotyper er ofte ufullstendig penetrant, bare manifestert i noen mutanter. Interessant fenotyper som inkonsekvent vises kan være vanskelig å studere, og kan føre til confounding resultater. Protokollen beskrevet her er en enkel avl paradigmet for å øke og redusere penetrance i dødelige sebrafisk skjelettlidelser mutanter. Fordi dødelige mutanter ikke kan være selektivt avlet direkte, brukes den klassiske selektive formering strategien avkom testing. Denne metoden inneholder også protokoller for Kompetitive allelet bestemt PCR (KASP) genotyperingteknologi sebrafisk og flekker larver sebrafisk brusk og bein. Bruke dyrehold strategi beskrevet her kan øke penetrance av en interessant skjelettlidelser fenotypen aktivere flere reproduserbar resultater i nedstrøms programmer. I tillegg kan reduserer det muterte penetrance gjennom denne selektiv formering strategi avsløre utviklingsprosesser som krever mest avgjørende funksjonen av muterte genet. Skjelettet er spesielt som her, foreslår vi at denne metodikken vil være nyttig for alle sebrafisk mutant linjer.

Denne protokollen er en langsiktig dyrehold teknikk nyttig for forståelse sebrafisk skjelettlidelser mutanter (figur 1). Selektiv formering avkom tester skal gi et skifte i generell penetrance både nedover og oppover i noen generasjoner (figur 2). I vår tidligere arbeid kjørte to rundene av selektiv formering den gjennomsnittlige penetrance nedover fra 17% til 3%1. Tilsvarende i vår oppadgående linje...

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

Målet med denne protokollen er å endre penetrance av dødelige skjelettlidelser mutant fenotyper i sebrafisk ved selektiv avl. Dødelige mutanter kan dyrkes til voksen og bred seg, derfor denne protokollen beskriver en metode for sporing og velge penetrance gjennom flere generasjoner av avkom testing.

Skiftende sebrafisk dødelige skjelettlidelser Mutant Penetrance av avkom 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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