Name: in ovo electroporation in embryonic chick retina
Uploaded: 2012-02-05T13:00:00
Description: Watch this Scientific Journal Video about In Ovo Electroporation in Embryonic Chick Retina at JoVE.com

Vi vil gerne takke hr. Maaz Enver for brug af hans HD-kamera (Cannon VIXIA HFS100). Dette arbejde blev støttet delvist af tilskud fra NIH (EY018738), New Jersey Kommissionen om Spinal Cord Forskning (08 til 3074-SCR-E-0 og 10 til 3091-SCR-E-0), og Busch Biomedical Research Award.

Da nogle dele af protokollen udføres som tidligere beskrevet i andre Jové videoer 2 og papirer 3 med mindre ændringer, vil vi ikke diskutere dem i detaljer her. 1. Æg Håndtering og Needle Forberedelse <ol><li> Æg kan lagres i en vin køligere ved ca 13 ° C i op til 1 uge. Hvis temperaturen er for høj, vil embryoner begynde at udvikle unormalt, medens lavere temperatur forårsager høj dødelighed. Når du er klar til at inkubere tage æggene fra vinkøler og s...

<ol>
	<li>Doh, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+retinal+cell+development+in+chick+embryo+by+immunohistochemistry+and+in+ovo+electroporation+techniques.">Analysis of retinal cell development in chick embryo by immunohistochemistry and in ovo electroporation techniques.</a> BMC. Dev. Biol. 10, (2010).</li><li>Blank, M. C., Chizhikov, V., Millen, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Ovo+Electroporations+of+HH+Stage+10+Chicken+Embryos.">In Ovo Electroporations of HH Stage 10 Chicken Embryos.</a> J. Vis. Exp. (9), e408-e408 (2007).</li><li>Nakamura, H., Funahashi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+DNA+into+chick+embryos+by+in+ovo+electroporation.">Introduction of DNA into chick embryos by in ovo electroporation.</a> Methods. 24, 43-48 (2001).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.+1951.">A series of normal stages in the development of the chick embryo. 1951.</a> Dev. Dyn.. 195, 231-272 (1992).</li><li>Hamburger, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stage+series+of+the+chick+embryo.">The stage series of the chick embryo.</a> Dev. Dyn. 195, 273-275 (1992).</li></ol>

Målrettet retinal injektion og i ovo elektroporation i embryonale nethinden E4 tidspunkt kan målrette retinale stamceller, hvilket resulterer i evnen til at visualisere alle de seks store nethinden celletyper på enkelt celle niveau. I ovo elektroporation i HH10 (~ E1.5) rettet mod optic vesikel er i stand til transficere celler der udvikler til dannelse øjet. Imidlertid, disse celler have en meget høj omsætning på dette tidspunkt og denne metode er ikke specifik for retinale celler. Det kan vær...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer </td></tr><tr><td> Befrugtede patogenfrie (SPF) hvide italiener hønseæg </td><td> Sunrise Farms (Catskill, NY) </td><td></td><td></td></tr><tr><td> Kylling ægget inkubator </td><td> GQF fremstilling, Savannah, GA </td><td> Model-1550 </td><td> Indstil til 60% fugtighed og 37,5 ° C. </td></tr><tr><td> Glas kapillarrørene </td><td> Verdens Precision Instrument Inc. </td><td> TW150F-4 </td><td></td></tr><tr><td> 0,1 ml sprøjte </td><td> Hamilton Co Reno, Nevada </td><td> Gastæt 1710 </td><td> Alternativt 1ml sprøjte kan anvendes så godt </td></tr><tr><td> MASTERFLEX Silicone (peroxid) Tubing </td><td> Cole-Parmer </td><td> HV-96.400-13 </td&gt; <td> Skære et lille stykke (1 cm) for fastgørelse glasset nål til sprøjten </td></tr><tr><td> Pincet </td><td> Dumont </td><td> AA </td><td></td></tr><tr><td> Mikromanipulator </td><td> Verdens Precision Instrument Inc. </td><td> M3301-M3 </td><td></td></tr><tr><td> Plasmid-DNA </td><td></td><td></td><td> pCAG-GFP blev lånt fra Dr. Connie Cepko </td></tr><tr><td> Fast Green FCF </td><td> Sigma-Aldrich </td><td> F7252 </td><td> Fortynde det til at 0,025% med PBS </td></tr><tr><td> Impulsgeneratoren </td><td> Harvard Apparatus, MA </td><td> BTX ECM 830 </td><td> Firkantsvingning generator </td></tr><tr><td> Elektroder </td><td> Harvard Apparatus, MA </td><td> BTX model 514 </td><td> Vore elektroder blev anbragt 3-5 mm fra hinanden </td></tr><tr><td> Monokrom Digital Camera </td><td> Zeiss, Tyskland </td><td> Enxiocam MRM </td><td></td></tr><tr><td> Fluorescerende Dissection Microscope </td><td> Leica Microsystems, Tyskland </td><td> Leica MZ16FA </td><td></td></tr><tr><td> Opretstående Fluorescens Microscope </td><td> Zeiss, Tyskland </td><td> Zeiss Axio Imager A1 </td><td></td></tr></tbody></table>

in ovo electroporation in embryonic chick retina

Kylling embryonale nethinden er et fremragende værktøj til at studere retinal udvikling i højere hvirveldyr. Grund af store størrelse og ekstern udvikling, er det forholdsvis meget nemt at manipulere den chick embryoniske nethinden anvendelse af rekombinant DNA / RNA-teknologi. Elektroporering af DNA / RNA konstrukter ind i den embryoniske nethinden have en stor fordel at studere genregulering i retinale stamceller / progenitor celler under retinal udviklingen. Forskellig type af assays såsom reportergen assay, gen over-ekspression, knock genet ned (shRNA) etc. kan udføres ved anvendelse af elektroporation teknikken. Denne video demonstrerer målrettede retinal indsprøjtning og in ovo elektroporering ind i den embryoniske kylling retina i det Hamburger og Hamilton stadium 22-23, hvilket er omkring embryonisk dag 4 (E4). Her viser vi en hurtig og bekvem in ovo elektroporering teknik, hvorved en plasmid-DNA der udtrykker grønt fluorescerende protein (GFP) som en markør bliver direkte leveres ind ichick embryonisk subretinale rum og efterfulgt af elektriske impulser at lette DNA-optagelse ved retinale stam / progenitor celler. Den nye metode til retinal injektion og elektroporation ved E4 tillader visualisering af alle retinale celletyper, herunder den sene-fødte neuroner 1, som har været vanskeligt med den konventionelle metode af injektion og elektroporation ved E1.5 2.

Watch this Scientific Journal Video about In Ovo Electroporation in Embryonic Chick Retina at JoVE.com

In Ovo Electroporation in Embryonic Chick Retina

Det overordnede mål for denne video er at vise, hvordan du udfører målrettet retinal injektion og<em&gt; In ovo</em&gt; Elektroporering af DNA / RNA konstrukter ind i kyllingens embryoniske retina i det Hamburger og Hamilton stadium 22-23, hvilket er omkring embryonisk dag 4 (E4). Denne teknik er meget nyttigt at undersøge genekspression, genregulering, og morfologiske forandringer i udviklingen af ​​chick nethinden.

I Ovo Elektroporation i embryonisk Chick Retina

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is ...

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Biology

In Ovo Electroporation in Embryonic Chick Retina

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