Name: gene transfer into older chicken embryos by ex ovo electroporation
Uploaded: 2012-07-27T12:00:00
Description: Watch this Scientific Journal Video about Gene Transfer into Older Chicken Embryos by ex ovo Electroporation at JoVE.com

Dette arbeidet ble støttet av et stipend fra det tyske Research Foundation (DFG, LU1455/1-1).

1. Ex ovo Kultur <ol><li> Ferske befruktede egg på den lange siden i en tvungen-utkast inkubator (BSS160, Ehret, Tyskland) ved 37,5 ° C med 60% fuktighet for inkubasjon. Egg oppbevares ved 12 ° C i mer enn én uke må ikke brukes. </li><li> Etter inkubering for 2,5 dager (ca stadium HH17), ta ut eggene og merke toppen med en blyant for å indikere retning for sprekkdannelser. </li><li> Før sprengning, hell ca 20 ml sterilt destillert vann i en ren stor petriskål (diameter 145 mm; Greiner Bio-One GmbH, Ty...

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	<li>Momose, T., Tonegawa, A., Takeuchi, J., Ogawa, H., Umesono, K., Yasuda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+targeting+of+gene+expression+in+chick+embryos+by+microelectroporation.">Efficient targeting of gene expression in chick embryos by microelectroporation.</a> Dev. Growth Differ. 41, 335-344 (1999).</li><li>Luo, J., Ju, M. J., Lin, J., Yan, X., Markus, A., Mix, E., Rolfs, A., Redies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cadherin-20+expression+by+motor+neurons+is+regulated+by+Sonic+hedgehog+during+spinal+cord+development.">Cadherin-20 expression by motor neurons is regulated by Sonic hedgehog during spinal cord development.</a> NeuroReport. 20, 365-370 (2009).</li><li>Luo, J., Ju, M. J., Redies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regionalized+cadherin-7+expression+by+radial+glia+is+regulated+by+Shh+and+Pax7+during+chicken+spinal+cord+development.">Regionalized cadherin-7 expression by radial glia is regulated by Shh and Pax7 during chicken spinal cord development.</a> Neuroscience. 142, 1133-1143 (2006).</li><li>Nakamura, H., Katahira, T., Sato, T., Watanabe, Y., 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=Gain-+and+loss-of-function+in+chick+embryos+by+electroporation.">Gain- and loss-of-function in chick embryos by electroporation.</a> Mech. Dev. 121, 1137-1143 (2004).</li><li>Dai, F., Yusuf, F., Farjah, G. H., Brand-Saberi, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi-induced+targeted+silencing+of+developmental+control+genes+during+chicken+embryogenesis.">RNAi-induced targeted silencing of developmental control genes during chicken embryogenesis.</a> Dev. Biol. 258, 80-90 (2005).</li><li>Rao, M., Baraban, J. H., Rajaii, F., Sockanathan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+comparative+study+of+RNAi+methodologies+by+in+ovo+electroporation+in+the+chick+embryo.">In vivo comparative study of RNAi methodologies by in ovo electroporation in the chick embryo.</a> Dev. Dyn. 231, 592-600 (2004).</li><li>Kos, R., Tucker, R. P., Hall, R., Duong, T. D., Erickson, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+introducing+morpholinos+into+the+chicken+embryo.">Methods for introducing morpholinos into the chicken embryo.</a> Dev. Dyn. 226, 470-477 (2003).</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.">A series of normal stages in the development of the chick embryo.</a> J. Morphol. 88, 49-92 (1951).</li><li>Auerbach, R., Kubai, L., Knighton, D., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+procedure+for+the+long-term+cultivation+of+chicken+embryos.">A simple procedure for the long-term cultivation of chicken embryos.</a> Dev. Biol. 10, 391-394 (1974).</li><li>Luo, J., Treubert-Zimmermann, U., Redies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cadherins+guide+migrating+Purkinje+cells+to+specific+parasagittal+domains+during+cerebellar+development.">Cadherins guide migrating Purkinje cells to specific parasagittal domains during cerebellar development.</a> Mol. Cell. Neurosci. 25, 138-152 (2004).</li><li>Luo, J., Redies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+ovo+electroporation+for+gene+transfer+into+older+chicken+embryos.">Ex ovo electroporation for gene transfer into older chicken embryos.</a> Dev. Dyn. 233, 1470-1477 (2005).</li><li>Treubert-Zimmermann, U., Heyers, D., Redies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+axons+to+specific+fiber+tracts+in+vivo+by+altering+cadherin+expression.">Targeting axons to specific fiber tracts in vivo by altering cadherin expression.</a> J. Neurosci. 22, 7617-7626 (2002).</li></ol>

Denne protokollen gir en veiledning for genoverføring til eldre kylling embryoer (f.eks inn i optikken tectum ved E6) ved ex ovo electroporation. Denne metoden utvider bruken av electroporation til eldre kylling embryoer og kan enkelt brukes på mange laboratorier for å studere gen-funksjon in vivo på senere stadier. Embryoer fra E4 til minst E7 kan være vellykket electroporated av denne metoden og de ​​electroporated embryoer kan være overlevelse frem til E15 11. Dessuten hjernen, ...

<table border="1"><tbody><tr><td> Navn </td><td> Firma </td><td> Catalog Number </td></tr><tr><td> Electroporator </td><td> NEPA Gene, Japan </td><td> CUY21-Edit </td></tr><tr><td> Elektroder </td><td> NEPA Gene, Japan </td><td> CUY661-3x7 </td></tr><tr><td> Egg inkubator </td><td> Ehret, Tyskland </td><td> BSS160 </td></tr><tr><td> Embryo inkubator </td><td> BINDER GmbH, Tyskland </td><td></td></tr><tr><td> Fluoriserende mikroskop </td><td> KEYENCE, Deutschland GmbH </td><td> BZ-8000 </td></tr><tr><td> Petriskål </td><td> Greiner Bio-One GmbH, Tyskland </td><td></td></tr><tr><td> Microelectrode avtrekker </td><td> Verden presisjonsutstyr, Tyskland </td><td> PUL-100 </td></tr></tbody></table>

gene transfer into older chicken embryos by ex ovo electroporation

Kyllingen embryo gir en utmerket modellsystem for å studere gen-funksjon og regulering under embryonal utvikling. I ovo electroporation er en kraftig metode for å over-uttrykke eksogene gener eller ned-regulere endogene gener in vivo i kylling embryoer 1. Ulike strukturer som for eksempel DNA plasmider koding gener 2-4, små interfererende RNA (siRNA) 5 plasmider, små syntetiske RNA oligos 6, og Morpholino antisens oligonucleotides 7 kan lett tilført til kylling embryoer ved electroporation. Men anvendelsen av i ovo electroporation begrenset til embryoer ved tidlige stadier inkubering (yngre enn stadium HH20 - ifølge Hamburg og Hamilton) 8 og det er noen ulemper for sin søknad i embryoer på senere stadier (eldre enn stadium HH22 - om lag 3,5 dager med utvikling). For eksempel er vitelline membranen på senere stadier vanligvisalliert stakk til Skal membranen og åpne et vindu i skallet forårsaker brudd på fartøyene, noe som resulterer i død av embryoene; eldre embryoer er dekket av vitelline og allantoic fartøy, der det er vanskelig å få tilgang til og manipulere embryoene; eldre embryoer flytte kraftig og er vanskelig å kontrollere retningen gjennom et relativt lite vindu i skallet. I denne protokollen viser vi en ex ovo electroporation metode for genoverføring til kylling embryoer ved sene stadier (eldre enn scenen HH22). For ex ovo electroporation, er embryoer dyrket i petriskåler 9 og vitelline og allantoic fartøyene utbredt. Under disse forholdene, er de eldre kylling embryoer lett tilgjengelig og manipulert. Derfor overvinner denne metoden ulempene med i ovo electroporation anvendt til de eldre kylling embryoer. Ved hjelp av denne metoden, kan plasmider lett tilført i ulike delerav de eldre kylling embryoer 10-12.

Watch this Scientific Journal Video about Gene Transfer into Older Chicken Embryos by ex ovo Electroporation at JoVE.com

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

En metode for genoverføring til kylling embryoer ved senere inkubering stadier (eldre enn hamburger og Hamilton scenen (HH) 22) beskrives. Denne metoden overvinner ulempene ved<em&gt; I ovo</em&gt; Electroporation gjaldt eldre kylling embryoer og er en nyttig teknikk for å studere gen-funksjon og regulering på eldre utviklingsstadier.

Genoverføring til Eldre Chicken Embryoer av<em&gt; Ex ovo</em&gt; Electroporation

The chicken embryo provides an  excellent model system for studying gene function and regulation during  embryonic development. In ovo electroporation is ...

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Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

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