Name: a method for microinjection of patiria minata zygotes
Uploaded: 2014-09-01T15:00:00
Description: Watch this Scientific Journal Video about A Method for Microinjection of Patiria minata Zygotes at JoVE.com

Dette arbejde blev støttet af National Science Foundation IOS 0844948 og IOS 1024811

Hold alle havvand eller kunstigt havvand (SW), voksne dyr, og kulturer ved 15 ° C så meget som det er praktisk. Sikre æg og zygoter holdes nedsænket i SW. Kommercielt fremstillede havet salte rekonstitueret med destilleret eller omvendt osmose vand tjener godt som en kilde af SW. Tjek saltholdighed ved hjælp af et hydrometer og justere salte eller vand for at opnå optimale niveau. Hold Vægtfylde niveauer mellem 1,020-1,025. Hold alle glasvarer og plastvarer adskilt fra alt andet labor...

<ol>
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Der er to vigtige skridt, der er svært for nybegyndere i denne teknik, men er afgørende for en vellykket skabe morphant embryoner. Den første er at vælge sunde oocytter, der vil modnes og befrugte ordentligt. Den procentdel af den normale udvikling i en kultur afhænger af sæsonen, sundhed af dyret, og det antal gange, oocytter er blevet høstet fra et enkelt individ. Oocytter tendens til at være af en bedre kvalitet fra april til oktober. Det er vigtigt at se nøje på de oocytter, før du fortsætter til at sikr...

Havet stjerne, Patiria miniata, (almindeligvis kendt som bat-stjerne) fremstår som en interessant og alsidig model for en række cellulære 1- 3, udviklingsmæssige 4,5, evolutionær 6 til 8, og økologiske undersøgelser 9- 11. Voksen P. miniata er fordelt langs Stillehavskysten fra Sitka, Alaska til Baja, Californien 12 og let vedligeholdes i marine akvarier. Oocytter er opnåelige året rundt og hver hun kan kaste titusinder af æg. Oocytter let modnet og befrugtet eksternt 13. De resulterende embryoner er gennemsigtige tillader nem observation; de udvikler synkront, og kun kræver havvand til udvikling. Hele genom samling og flere transcriptomes er også tilgængelige for P. miniata ( <a href="http://www.echinobase.org/Echinobase/">Echinobase.org</a> ). Sådanne fordele gør dem ideelle til en række af forskningog undervisning. I de seneste år, s miniata er blevet en model for udviklingsmæssige genregulatorisk netværk analyser 14.-16. Formålet med sådanne undersøgelser er at identificere hele kompliment af regulerende gener og bestemme netværket af deres samspil. Meget af dette arbejde indebærer forstyrrende genekspression gennem indførelse af antisense-oligonukleotider eller in vitro-syntetiserede mRNA&#39;er. Derudover er cis regulatoriske analyser anvendes til at karakterisere funktionen af regulatorisk DNA 15. Disse analyser kræver indførelse af perturbations- reagenser og / eller DNA-reporter-konstruktioner i embryoner. Endvidere at karakterisere de efterfølgende virkninger af disse forstyrrelser, må man analysere mange embryoner til ændringer i genekspression af potentielle mål. Teknikker til mikroinjektion af mange hundrede zygoter er centrale for dette arbejde. Pighuder, inklusiv P. miniata </em&gt;, kræver mange måneder at nå seksuel modenhed. På grund af dette, er det almindeligvis ikke praktisk at udvikle og vedligeholde transgene linier af disse dyr til forsøg. Derfor opdræt af transgene voksne kan ikke effektivt at skabe modificerede embryoner. I stedet skal forstyrrelse forekommer de novo ved mikroinjektion. Mikroinjektion giver mulighed for at ændre embryoner med reagenser, der ikke er celle-gennemtrængelig. Følgende protokol beskriver en metode til at indføre DNA, mRNA, celle sporstoffer, og morpholino antisenseoligonukleotider ind i hundredvis af befrugtede æg i en 2-3 timers mødet gennem mikroinjektion. Dette giver tilstrækkeligt materiale til en række efterfølgende eksperimenter, herunder, men ikke begrænset til, qPCR, in situ-hybridisering, RNA-Seq og western blotting.

<table border="0" cellpadding="0" cellspacing="0" width="648">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:147px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">1-Methyladenine</td>
			<td style="width:147px;">Acros Organics (Fisher Scientific)</td>
			<td style="width:119px;">AC20131-1000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">190 micron nitex nylon filter</td>
			<td style="width:147px;">Small Parts (originally Sefar)</td>
			<td style="width:119px;">CMN-0185-C/5PK-05</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">100 micron nitex nylon filter</td>
			<td style="width:147px;">Small Parts (originally Sefar)</td>
			<td style="width:119px;">CMN-0105-C/5PK-05</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Polystyrene Petri Dishes, 60mm x 15mm</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:119px;">FB0875713A</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Capillary tubing</td>
			<td style="width:147px;">FHC, Inc</td>
			<td style="width:119px;">30-30-0</td>
			<td style="width:167px;">For pulling microinjection needles</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Model P-97 Needle Puller</td>
			<td style="width:147px;">Sutter Instruments</td>
			<td style="width:119px;">P-97</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="101">
			<td height="101" style="height:101px;width:216px;">Dextran, Rhodamine Green</td>
			<td style="width:147px;">Life Technologies</td>
			<td style="width:119px;">D7163</td>
			<td style="width:167px;">If injecting a GFP expression reporter, it is helpful to substitute Texas Red dextran as an injection tracer</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Instant Ocean Sea Salt</td>
			<td style="width:147px;">Doctors Foster and Smith</td>
			<td style="width:119px;">CD-116528</td>
			<td style="width:167px;">Also available in many pet stores</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microloader Tips</td>
			<td style="width:147px;">Eppendorf</td>
			<td style="width:119px;">5242 956.003</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

a method for microinjection of patiria minata zygotes

Pighuder har længe været en favorit modelsystem til undersøgelser af reproduktion og udvikling, og for nylig til undersøgelse af genregulering og udviklingen af ​​udviklingsprocesser. Havet stjerne, Patiria miniata, er ved at vinde udbredelse som en model for disse typer af undersøgelser, der tidligere blev udført næsten udelukkende i søpindsvin, Strongylocentrotus purpuratus og Lytechinus variegatus. En fordel ved disse modelsystemer er let at fremstille modificerede embryoner, hvor et bestemt gen er op eller nedreguleret, mærkning af en gruppe af celler, eller indføre et reportergen. En enkelt mikroinjektion metode er i stand til at skabe en bred vifte af sådanne modificerede embryoner. Her præsenterer vi en metode til at opnå kønsceller fra P. miniata, der producerer zygoter, og indføre forstyrrende reagenser via mikroinjektion. Sunde morphant embryoner isoleres efterfølgende for kvantitative og kvalitative studier af gene funktion. Tilgængeligheden af ​​genom og transkriptom data for denne organisme har øget de former for undersøgelser, der udføres og den lethed at udføre dem.

Målet med denne protokol er at indføre reagenser i embryoner. Vi demonstrere effektiviteten af ​​protokollen ved at indsprøjte en DNA-reporter-konstrukt, som driver ekspressionen af ​​grønt fluorescerende protein (GFP). Injicerede embryoner udtrykker GFP i klonede patches (figur 4A-B) som DNA inkorporerer under tidlig spaltning. Mange reagenser, der er ønskeligt at indføre i embryoner er giftige i store mængder og til suboptimale partier af embryoner. Toksicitet manifester forsinker udvik...

Watch this Scientific Journal Video about A Method for Microinjection of Patiria minata Zygotes at JoVE.com

A Method for Microinjection of Patiria minata Zygotes

Metoder, der producerer morphant embryoner er afgørende for at studere udviklingsmæssige mekanismer og gen-regulatoriske netværk. Havet stjerne Patiria miniata er en ny model for disse studier. Her præsenterer vi en protokol for opnåelse af kønsceller, der producerer kulturer af embryoner, og hurtig mikroinjektion af zygoter fra denne art.

Metode til Mikroinjektion af<em&gt; Patiria minata</em&gt; Zygoter

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A Method for Microinjection of Patiria minata Zygotes

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