Name: ploidy manipulation of zebrafish embryos with heat shock 2 treatment
Uploaded: 2016-12-16T13:00:00
Description: Watch this Scientific Journal Video about Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment at JoVE.com

This work was supported by NIH grants R21 HD068949-01 and RO1 GM065303.

Alle eksperimenter dyr blev udført i henhold til University of Wisconsin - Madison og Institutional Animal Care og brug Udvalg (IACUC) retningslinjer (University of Wisconsin - Madison Assurance nummer A3368-01). 1. Valg af Hunner for Egg Collection via afbrudt Parring BEMÆRK: IVF-baserede protokoller stole på ekstrudering af modne æg fra kvinder via manuel tryk 9. Tidligere protokoller har benyttes hundyr direkte fra tanke eller i par parringer uden hunnerne undergår æg frigivelse adfærd...

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Kritiske trin Det er afgørende at arbejde under forhold med effektiv in vitro-befrugtning. For at sikre et godt udbud af modne æg (trin 1), hunner oprettet til parring ikke burde have været oprettet i parring kors i mindst 5 dage og skal vises gravid. Under afbrydelse af avl, kan en observatør overvåge 15-30 tanke tilstrækkeligt for den første forekomst af naturlige æg ekstrudering. Afbrydelse af parring bør ske så hurtigt som muligt, når de første æg er frigivet gennem naturlige p...

Denne protokol tillader manipulation af ploidi i zebrafisk embryoer, såsom i frembringelsen af homozygote gynogenetic diploider fra gynogenetic haploider (figur 1) eller produktionen af tetraploids. Dette opnås ved at inducere en forsinkelse i cytokinese svarende til netop en cellecyklus (figur 2A, 2B). Nøglen én-cyklus forsinkelse i cytokinese opnås ved behandling med varmeshock. Standardprotokollen af Heat Shock (HS) som oprindeligt beskrevet af Streisinger og kolleger involverede en temperatur puls i perioden 13-15 mpf, hvilket resulterer i en én-cyklus celledeling stall under den første cellecyklus 1. Effektiviteten af ​​denne protokol er for nylig blevet forbedret ved at scanne de tidlige cellecyklus med en glidende tidsvindue af varme chokbehandling. Denne scanning identificeret et senere tidspunkt for et varmechok, stadig inden for den første celle cyklus (22-24 MPF), som resulterer i en højere brodeyos med en én-cyklus celledeling stall, som i dette tilfælde påvirker den anden cellecyklus 2. Den iagttagelse, at eksperimentelle manipulationer i den første cellecyklus forstyrre celledeling under den anden cellecyklus og forårsage DNA-indholdet overlapning er også blevet rapporteret i andre fiskearter 3,4. Vi henviser til denne modificerede protokol som Heat Shock 2 (HS2 - udtrykket &quot;2&quot; afspejler, at varmepulsen sker på et senere tidspunkt end standard HS metode, og at cellecyklus forsinkelse forårsaget af HS2 forekommer under den anden cellecyklus ). Disse undersøgelser viste, at grundlaget for cytokinese arrest efter varmechok er inhiberingen af ​​centriole overlapning under varmen puls, som påvirker spindeldannelse og fure induktion i følgende cellecyklus. HS2 resulterer i udbytter af cellecyklusstop nærmer 100%, og satser ploidi dobbeltarbejde op til 4 gange højere end standard HS 2. Embryoner behandlet witha varmechok under blastomer cellecyklus udviser mange skadelige virkninger, tyder på, at varmechok påvirker flere processer, der kræves for celledeling 2. På den anden side, hvis varmen chok påføres forud for indledningen af cellecyklus (tidsperiode 0-30 MPF), det ser ud til at have virkninger i overensstemmelse med specifikke indgreb centriole dobbeltarbejde og synes ikke at påvirke andre væsentlige celle processer 2 . Disse undersøgelser viste, at tiden forud for indledningen af ​​blastomer division synes at være en udviklingsperiode modtagelig for hjælp Heat Shock som et redskab til specifikt at manipulere ploidi gennem centriole hæmning. Den underliggende årsag til den tilsyneladende selektivitet for varmechok på centriole overlapning er ukendt, men kan være relateret til en selektiv nedbrydning af centrosom substrukturer observeret under varmestress i visse celletyper, såsom leukocytter 5. Temporal synkronisering af embryonale deling opnås ved in vitro fertilisering (IVF). Anvendelse af ubehandlet sperm under befrugtning resulterer i diploide embryoner at ved HS2-induceret én-cyklus cytokinese stall bliver tetraploid. Anvendelse af UV-behandlede sædceller, som bærer tværbindinger som inaktiverer sit DNA, resulterer i gynogenetic haploide embryoner 6, som ved HSII-induceret én-cyklus cytokinese stall bliver gynogenetic diploider 2. På grund af den resulterende hele genomet overlapning, sidstnævnte gynogenetic diploider er homozygote på hvert enkelt locus tværs genomet. For koncis, henviser vi til gynogenetic haploide embryoner som &quot;haploider&quot;, og homozygote gynogenetic diploide embryoner som &quot;homozygote diploider&quot;. Hvis levedygtig og frugtbar, kan homozygote diploider bruges til at indlede sterile og dødelige-fri linjer. Direkte homozygoti induceret af HS2 bør også let inkorporeres i genetisk analyse eller genetiske skærme, da homozygote diploider fra hundyr, som er heterozygote bærere af muttioner udviser satser homozygositet ved høje og faste (50%) nøgletal 2. Følgende protokol beskriver trin for at udføre HS2 og inducerer ploidi overlapning med fuld homozygoti. For tetraploid produktion, bør sperm løsning være ubehandlet. For homozygot diploid produktion, bør sperm inaktiveres ved UV-behandling. Endvidere som beskrevet i diskussionen, synlige pigment markører kan også anvendes til at lette identifikation af homozygote diploider. Zebrafisk mate primært i den første 3 timer om indledningen af deres lys cyklus 7, og både voksne og æg er følsomme over for døgnrytmen 8, så de bedste resultater bør ske IVF procedure inden for denne periode.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<colgroup>
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Zebrafish mating boxes</td>
			<td style="width:71px;">Aqua Schwarz</td>
			<td style="width:119px;">SpawningBox1</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">S5886</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KCl</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">P5405</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Na2HPO4</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">S3264</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KH2PO4</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">P9791</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CaCl2</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">C7902</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MgSO4-7H2O</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">63138</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaHCO3</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">S5761</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tricaine</td>
			<td style="width:71px;">Western Chemical</td>
			<td style="width:119px;">Tricaine-D (MS 222)</td>
			<td>FDA approved (ANADA 200-226)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris base</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">77-86-1</td>
			<td>to prepare 1 M Tris pH 9.0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HCl</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">920-1</td>
			<td>to prepare 1 M Tris pH 9.0</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fish net (fine mesh) (4-5 in)</td>
			<td style="width:71px;">PennPlax</td>
			<td style="width:119px;">(ThatFishThatPlace # 212370)</td>
			<td>available in ThatFishThatPlace</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plastic spoon</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>available in most standard stores</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Dissecting scissors</td>
			<td style="width:71px;">Fine Science Tools</td>
			<td style="width:119px;">14091-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dissecting forceps</td>
			<td style="width:71px;">Dumont</td>
			<td style="width:119px;">SS</td>
			<td>available from Fine Science Tools</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dissecting stereoscope (with transmitted light source)</td>
			<td style="width:71px;">Nikon</td>
			<td style="width:119px;">SMZ645</td>
			<td>or equivalent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Reflective light source (LED arms)</td>
			<td style="width:71px;">Fostec</td>
			<td style="width:119px;">KL1600 LED</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petri plates 10 cm diameter</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>any maker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Eppendorf tubes 1.5 ml</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>any maker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ice bucket</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>any maker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipetteman P-1000</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>any maker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipette tips 1000 &#181;l</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>any maker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Narrow spatula</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">14-374</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Depression glass plate</td>
			<td style="width:71px;">Corning Inc</td>
			<td style="width:119px;">722085 (Fisher cat. No 13-748B)</td>
			<td>available from Fisher Scientific</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">UV lamp</td>
			<td style="width:71px;">UVP</td>
			<td style="width:119px;">Model XX-15 (cat No. UVP18006201)</td>
			<td>available from Fisher Scientific. Although not observed by us with this model, some UV sources have been observed to experience a decrease of intensity over time (if this is the case, see Modifications and Troubleshooting)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UV glasses</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>any maker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paper towels</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>any maker</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Kimwipes</td>
			<td style="width:71px;">Kimberly-Clark</td>
			<td style="width:119px;">06-666-11</td>
			<td>available from Fisher Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Timer stop watch</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>any maker</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Wash bottle</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;">24020500</td>
			<td>available from Fisher Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tea strainer</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>available in kitchen stores</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">beakers, 250 ml (2)</td>
			<td style="width:71px;">Corning Inc.</td>
			<td style="width:119px;">1000250</td>
			<td>available from Fisher Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">water bath (2)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>any maker, with accurary to 0.1 C (e.g. Shel Lab H2O Bath Series)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;">Hanks&#8217; Solution 1</td>
			<td style="width:71px;">see above</td>
			<td style="width:119px;">see above</td>
			<td>8.0g NaCl, 0.4g KCl in 100ml ddH2O. Store at 4&#176;C.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hanks&#8217; Solution 2</td>
			<td style="width:71px;">see above</td>
			<td style="width:119px;">see above</td>
			<td>0.358g Anhydrous Na2HPO4, 0.6g KH2PO4 in 100ml ddH2O. Store at 4&#176;C.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hanks&#8217; Solution 4</td>
			<td style="width:71px;">see above</td>
			<td style="width:119px;">see above</td>
			<td>0.72g CaCl2 in 50ml ddH2O. Store at 4&#176;C.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hanks&#8217; Solution 5</td>
			<td style="width:71px;">see above</td>
			<td style="width:119px;">see above</td>
			<td>1.23g MgSO4 &#8729; 7H2O in 50ml ddH2O. Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hank&#39;s Premix</td>
			<td style="width:71px;">see above</td>
			<td style="width:119px;">see above</td>
			<td>add, in the following order: 10.0 ml Solution 1;&#160; 1.0 ml Solution 2;&#160; 1.0 ml Solution 4;&#160; 86.0 ml ddH2O;&#160; 1.0 ml Solution 5. Store at 4&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hanks&#8217; Solution 6</td>
			<td style="width:71px;">see above</td>
			<td style="width:119px;">see above</td>
			<td>0.33g NaHCO3 in 10ml ddH2O. Prepare fresh the morning of the IVF procedure.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hank&#39;s Solution (final solution)</td>
			<td style="width:71px;">see above</td>
			<td style="width:119px;">see above</td>
			<td>Combine 990ul of Hank&#8217;s Premix and 10ul of freshly made Solution 6 (NaHCO3 solution)</td>
		</tr>
	</tbody>
</table>

ploidy manipulation of zebrafish embryos with heat shock 2 treatment

Manipulation af ploidi muliggør nyttige transformationer, såsom diploider til tetraploids eller haploider til diploider. I zebrafisk Danio rerio, specielt dannelsen af homozygote gynogenetic diploider er nyttig til genetisk analyse, fordi det tillader den direkte produktion af homozygoter fra en enkelt heterozygot mor. Denne artikel beskriver en modificeret protokol for ploidi dobbeltarbejde baseret på en varme puls under den første cellecyklus, Heat Shock 2 (HS2). Gennem inhibering af centriole overlapning, denne metode resulterer i et præcist celledeling stall under den anden cellecyklus. Den præcise én-cyklus division stall, koblet til upåvirket DNA dobbeltarbejde, resulterer i hele genomet dobbeltarbejde. Protokoller er forbundet med denne fremgangsmåde, indbefatter æg og sperm indsamling, UV-behandling af sædceller, in vitro-befrugtning og varme impuls til at forårsage en én-cellecyklus division forsinkelse og ploidi overlapning. En modificeret version af denne protokol kan anvendesat fremkalde ploidi ændringer i andre dyrearter.

På trods af en-cellecyklus cytokinese stall, DNA-replikation forekommer normalt i sådanne embryoner, hvilket resulterer i overlapning af DNA-indholdet af embryo (figur 1). Den Streisinger Heat Shock protokol (standard HS) involverer en varme puls i perioden 13-15 minutter efter befrugtning (MPF) og inducerer primært cytokinese anholdelse under den første embryonale celledeling ved 35 MPF 1,2, mens den afledte her beskrevne fremgangsmåde, benævnt Heat sho...

Watch this Scientific Journal Video about Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment at JoVE.com

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

En modificeret protokol for ploidi manipulation anvender en varmechok for at inducere en et-cyklus stall i cytokinese i det tidlige embryon. Denne protokol er påvist i zebrafisk, men kan gælde for andre arter.

Ploidiniveauet Manipulation af zebrafisk embryoner med Heat Shock 2 Behandling

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, ...

Research

JoVE Journal

Developmental Biology

Mechanical Vessel Injury in Zebrafish Embryos

Mekanisk karskade i zebrafisk embryoner

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

Isolering og karakterisering af enkeltceller fra zebrafisk embryoer

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

Generation af Parabiotic zebrafisk embryoner ved Kirurgisk Fusion af Udvikling Blastulae

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for ...

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

Affinity Labeling Påvisning af endogene receptorer fra zebrafisk embryoer

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish ...

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Funktionel Manipulation af Maternal Gene produkter ved hjælp af<em&gt; In vitro</em&gt; Oocytmodning i Zebrafisk

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

Biosensing Motor Neuron Membran Potential i Live Zebrafish Embryoer

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Induktion af hypoxi i levende frø og zebrafiskembryoner

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Anvendelse af RNAi og Heat-shock-induceret transskription faktor udtryk til at omprogrammere kønsceller til neuroner i C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Visualisering af cellulære elektriske aktivitet i zebrafisk tidlige embryoner og tumorer

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Hele Mount Immunhistokemi i Zebrafish Embryoner og Larver

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...