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 arbejde blev støttet af en bevilling fra den tyske Research Foundation (DFG, LU1455/1-1).

1. Ex ovo Culture <ol><li> Friske befrugtede æg er lagt på den lange side i en tvungen udkast inkubator (BSS160, Ehret, Tyskland) ved 37,5 ° C med 60% fugtighed for inkubering. Æg opbevaret ved 12 ° C i længere end en uge, bør ikke anvendes. </li><li> Efter inkubation i 2,5 dage (ca. etape HH17), tage æggene og mærke toppen med en blyant for at angive retning for revner. </li><li> Før revner, hæld ca 20 ml sterilt destilleret vand i en ren stor petriskål (diameter 145 mm; Greiner Bio-One GmbH, Tys...

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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 protokol giver en guide til genoverførsel i ældre kyllingeembryoer (f.eks i den optiske tectum på E6) ved ex ovo elektroporation. Denne fremgangsmåde omfatter anvendelse af elektroporation til ældre kyllingeembryoner og kan let anvendes til mange laboratorier for at studere genfunktion in vivo på senere tidspunkter. Embryoer fra E4 til mindst E7 held kan elektroporeret ved denne fremgangsmåde, og de ​​elektroporerede embryoner kan overlevelse indtil E15 11. Udover hjernen, k...

<table border="1"><tbody><tr><td> Navn </td><td> Firma </td><td> Katalognummer </td></tr><tr><td> Elektroporator </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> Fluorescensmikroskop </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> Mikroelektrode aftrækker </td><td> Verdens Precision Instruments, Tyskland </td><td> Pul-100 </td></tr></tbody></table>

gene transfer into older chicken embryos by ex ovo electroporation

Den kyllingembryo tilvejebringer en fremragende model-system til undersøgelse genfunktioner og regulering under embryonisk udvikling. In ovo elektroporering er en kraftig metode til over-udtrykke eksogene gener eller nedregulere endogene gener in vivo i kyllingeembryoer 1. Forskellige konstruktioner, såsom DNA-plasmider kodende gener 2-4, små interfererende RNA (siRNA) plasmider 5 lille syntetisk RNA oligoer 6, og morpholino antisense-oligonukleotider 7 let kan transficeres ind i kyllingeembryoer ved elektroporering. Dog er anvendelsen af in ovo elektroporation begrænset til embryoner på tidlige inkubationstider stadier (yngre end etape HH20 - i henhold til Hamburg og Hamilton), 8, og der er nogle ulemper for dens anvendelse i embryoner i senere faser (ældre end etape HH22 - cirka 3,5 dag til udvikling). For eksempel er vitellinmembranen på senere stadier usuallieret holdt sig til De underretter membranen og åbne et vindue i skallen forårsager brud på de fartøjer, hvilket resulterer i død af embryoner; ældre embryoner er omfattet af vitellin og allantois fartøjer, hvor det er vanskeligt at få adgang til og manipulere de embryoner, ældre embryoner flytte kraftigt og er vanskelig at styre orienteringen gennem et relativt lille vindue i skallen. I denne protokol vi vise en ex ovo elektroporation metode til genoverførsel i kyllingeembryoer på sene stadier (ældre end etape HH22). For ex ovo elektroporation, er embryoner dyrkes i petriskåle 9 og vitellin og allantois skibe er meget udbredt. Under disse betingelser, er de ældre kyllingeembryoer let adgang til og manipulere. Derfor er denne metode overvinder ulemperne ved in ovo elektroporering anvendes på gamle kyllingeembryoner. Anvendelse af denne fremgangsmåde, kan plasmider let transficeres i forskellige deleDe ældre kyllingeembryoner 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 fremgangsmåde til genoverførsel til kyllingeembryoner på senere inkubering trin (ældre end Hamburger og Hamilton trin (HH) 22) er beskrevet. Denne fremgangsmåde overvinder ulemperne<em&gt; In ovo</em&gt; Elektroporation anvendes til ældre kyllingeembryoer og er en nyttig teknik til at studere gen-funktion og regulering på ældre udviklingsstadier.

Gene Transfer i ældre kyllingeembryoer af<em&gt; Ex ovo</em&gt; Elektroporation

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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