Name: in ovo electroporation in the chicken auditory brainstem
Uploaded: 2017-06-09T13:00:00
Description: Watch this Scientific Journal Video about In Ovo Electroporation in the Chicken Auditory Brainstem at JoVE.com

Vi vil gerne takke Drs. Leslayann Schecterson, Yuan Wang, Andres Barria og Fru Ximena Optiz-Araya til førstehjælp med protokolopsætning og til tilvejebringelse af plasmider. Dette arbejde blev støttet af NIH / NIDCD grant DC013841 (JTS).

Alle procedurer blev godkendt af institutter for institutionelle dyrepleje og brugskomiteer fra Northwestern University og udført i overensstemmelse med de nationale retningslinjer for sundhedspleje for pleje og brug af laboratoriedyr. 1. Æg håndtering <ol><li> Køb befrugtet æg fra en lokal leverandør. Opbevar æg ved 13 ° C i køleskab i højst 5 dage før inkubation. Embryo-levedygtighed falder signifikant efter 1 uge. </li><li> Vask hvert æg med 70% ethanol, før det sættes i i...

<ol>
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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+and+development+of+time+coding+systems.">Evolution and development of time coding systems.</a> Curr Opin Neurobiol. 11 (6), 727-733 (2001).</li><li>Carr, C. E., Soares, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+convergence+and+shared+computational+principles+in+the+auditory+system.">Evolutionary convergence and shared computational principles in the auditory system.</a> Brain Behav Evol. 59 (5-6), 294-311 (2002).</li><li>Rubel, E. W., Parks, T. 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S., Fraser, S. E., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+origins+of+auditory+brain-stem+nuclei+in+the+chick+hindbrain.">Embryonic origins of auditory brain-stem nuclei in the chick hindbrain.</a> Dev Biol. 224 (2), 138-151 (2000).</li><li>Cramer, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EphA4+signaling+promotes+axon+segregation+in+the+developing+auditory+system.">EphA4 signaling promotes axon segregation in the developing auditory system.</a> Dev Biol. 269 (1), 26-35 (2004).</li><li>Korn, M. J., Cramer, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Windowing+chicken+eggs+for+developmental+studies.">Windowing chicken eggs for developmental studies.</a> J Vis Exp. (8), e306 (2007).</li><li>Sanchez, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+culture+of+chicken+auditory+brainstem+slices.">Preparation and culture of chicken auditory brainstem slices.</a> J Vis Exp. (49), (2011).</li><li>Jhaveri, S., Morest, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+architecture+in+nucleus+magnocellularis+of+the+chicken+auditory+system+with+observations+on+nucleus+laminaris:+a+light+and+electron+microscope+study.">Neuronal architecture in nucleus magnocellularis of the chicken auditory system with observations on nucleus laminaris: a light and electron microscope study.</a> Neuroscience. 7 (4), 809-836 (1982).</li><li>Matsui, R., Tanabe, Y., Watanabe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avian+adeno-associated+virus+vector+efficiently+transduces+neurons+in+the+embryonic+and+post-embryonic+chicken+brain.">Avian adeno-associated virus vector efficiently transduces neurons in the embryonic and post-embryonic chicken brain.</a> PLoS One. 7 (11), e48730 (2012).</li><li>Koppl, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auditory+nerve+terminals+in+the+cochlear+nucleus+magnocellularis:+differences+between+low+and+high+frequencies.">Auditory nerve terminals in the cochlear nucleus magnocellularis: differences between low and high frequencies.</a> J Comp Neurol. 339 (3), 438-446 (1994).</li><li>Hyson, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+analysis+of+interaural+time+differences+in+the+chick+brain+stem.">The analysis of interaural time differences in the chick brain stem.</a> Physiol Behav. 86 (3), 297-305 (2005).</li><li>Jones, T. A., Jones, S. M., Paggett, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+of+hearing+in+the+chicken+embryo.">Emergence of hearing in the chicken embryo.</a> J Neurophysiol. 96 (1), 128-141 (2006).</li><li>Saunders, J. C., Coles, R. B., Gates, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+auditory+evoked+responses+in+the+cochlea+and+cochlear+nuclei+of+the+chick.">The development of auditory evoked responses in the cochlea and cochlear nuclei of the chick.</a> Brain Res. 63, 59-74 (1973).</li><li>Woolf, N. K., Ryan, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+auditory+function+in+the+cochlea+of+the+mongolian+gerbil.">The development of auditory function in the cochlea of the mongolian gerbil.</a> Hear Res. 13 (3), 277-283 (1984).</li><li>Walsh, E. J., McGee, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+development+of+auditory+nerve+and+cochlear+nucleus+neuronal+responses+in+kittens.">Postnatal development of auditory nerve and cochlear nucleus neuronal responses in kittens.</a> Hear Res. 28 (1), 97-116 (1987).</li><li>Uziel, A., Romand, R., Marot, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+cochlear+potentials+in+rats.">Development of cochlear potentials in rats.</a> Audiology. 20 (2), 89-100 (1981).</li></ol>

I ovo elektroporation er en metode til at udtrykke eller slå ned gener af interesse for at analysere in vivo genfunktion 8 , 9 . I kyllingembryoen er det en innovativ metode til at udtrykke plasmidkodede gener i forskellige auditive hjernestammeområder 8 . For at sikre optimalt udtryk kræves der flere kritiske trin. Først indsprøjt kun embryoner, hvis otocytter er tydeligt synlige. Hvis otocytter ikke er synlige, er...

Hurtig neural kodning af lyd er afgørende for normale auditive funktioner. Disse omfatter lydplaceringsevner 1 , tale i støjdiskrimination 2 og forståelsen af ​​andre adfærdsrelevante kommunikationssignaler 3 . Analoge neuroner placeret i den hørbare hjernestamme hos både fugle og pattedyr er højt specialiserede til hurtig neural kodning 4 . Disse omfatter kyllingekernens magnocellularis (NM), nucleus laminaris (NL) og deres pattedyranaloger, den anteroventrale cochleære kerne (AVCN) og den mediale overlegne oliven (MSO) henholdsvis 5 . Imidlertid forstås udviklingsmekanismer, der regulerer hurtig neuralkodning, dårligt i den hørbare hjernestamme. Derfor er det fordelagtigt at studere specifikke gener, der er ansvarlige for hurtig neural kodning for bedre at forstå deres udtryk, regulering og funktion i auUdvikling af ditory. Det udviklende kyllingembryo er et effektivt og veletableret forskningsværktøj til at studere grundlæggende biologiske spørgsmål om lydsystemudvikling 6 , 7 . Nylige molekylære fremskridt har behandlet disse biologiske spørgsmål i det udviklende kyllingembryo ved at udtrykke eller slå ned gener af interesse for at analysere in vivo genfunktion 8 , 9 . Undersøgelse af regulatoriske rolle for specifikke gener er en væsentlig fremgang i forståelsen af ​​patologier forbundet med auditory underskud. Her præsenterer vi i ovo elektroporation af plasmidkodede gener i den høreværende hjernestamme, hvor hurtig neural kodning af lyd forekommer 10 . Ved at målrette auditory neurale progenitor regioner associeret med rhombomerer 5 og 6 11 , 12 (R5 /R6) viser vi rumlig kontrol af plasmidtransfektion i NM og NL. Derudover viser vi tidsmæssig regulering af udtryk ved at vedtage et tet-on vektorsystem. Dette er en lægemiddelinducerbar procedure, som udtrykker genene af interesse i nærvær af doxycyclin (Dox) 8 .

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Fertilized white leghorn chicken eggs</td>
			<td>Sunnyside Inc. (Beaver Dam, WI)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Picospritzer</td>
			<td>Parker Hannifin</td>
			<td>052-0500-900</td>
			<td>Picospritzer III, single or dual channel</td>
		</tr>
		<tr>
			<td>Current/voltage stimulator</td>
			<td>Grass Technologies</td>
			<td>SD9</td>
			<td>SD9</td>
		</tr>
		<tr>
			<td>Microfil syringe needles</td>
			<td>World Precision Instruments</td>
			<td>MF28G67-5</td>
			<td>28 Gauge, 67 mm Long, (Pack of 5)</td>
		</tr>
		<tr>
			<td>Electrode holder</td>
			<td>Warner Instruments</td>
			<td>64-1280</td>
			<td>MP Series: Non-Electrical Pressure Applications</td>
		</tr>
		<tr>
			<td>Stimulating microelectrode</td>
			<td>FHC</td>
			<td>PBSA1075</td>
			<td>PBSA1075</td>
		</tr>
		<tr>
			<td>Air tank/regulator</td>
			<td>NU Laboratory Services</td>
			<td></td>
			<td>Air dry 300 CF</td>
		</tr>
		<tr>
			<td>Fast green</td>
			<td>Sigma Aldrich</td>
			<td>F7258-25G</td>
			<td>F7258-25G</td>
		</tr>
		<tr>
			<td>Clear plastic tape</td>
			<td>Scotch</td>
			<td>191</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxycycline hyclate</td>
			<td>Sigma Aldrich</td>
			<td>D9891-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Egg refrigerator</td>
			<td>Vissani Wine Refrigerator</td>
			<td></td>
			<td>13.3-16.1&#176; C (56-61&#176; F)</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Hova-Bator</td>
			<td></td>
			<td>37.8&#176; C (100&#176; F), ~50% humidity</td>
		</tr>
		<tr>
			<td>Dissection scope</td>
			<td>Zeiss</td>
			<td>4.35E+15</td>
			<td>SteREO Discovery, V8 Microscope, 50.4X</td>
		</tr>
		<tr>
			<td>Cold-light source</td>
			<td>Zeiss</td>
			<td>4.36E+15</td>
			<td>CL6000 LED</td>
		</tr>
		<tr>
			<td>Micromanipulators</td>
			<td>Narishige Japan</td>
			<td>Model: MM-3</td>
			<td>2 Micromanipulators</td>
		</tr>
		<tr>
			<td>Capillary tubes</td>
			<td>Sutter Instrument</td>
			<td>BF150-86-10</td>
			<td>Thick-walled borosilicate (dimensions)</td>
		</tr>
		<tr>
			<td>Syringes</td>
			<td></td>
			<td></td>
			<td>1 mL, 3 mL</td>
		</tr>
		<tr>
			<td>Needles</td>
			<td>BD Precision Glide&#160;</td>
			<td></td>
			<td>27 G x 1 1/4, 19 G x 1 1/2</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Stoelting</td>
			<td></td>
			<td>No. 5 Super Fine Dumont</td>
		</tr>
		<tr>
			<td>Egg holder</td>
			<td>Custom Made</td>
			<td></td>
			<td>Clay base works as well</td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instrument</td>
			<td></td>
			<td>Model P-97</td>
		</tr>
		<tr>
			<td>Syringe filter</td>
			<td>Ultra Cruz</td>
			<td>sc-358811</td>
			<td>PVDF 0.22 &#956;m</td>
		</tr>
	</tbody>
</table>

in ovo electroporation in the chicken auditory brainstem

Elektroporation er en metode, der introducerer gener af interesse for biologisk relevante organismer som kyllingembryoen. Det er længe fastslået, at kyllingembryoen er en effektiv forskningsmodel til at studere grundlæggende biologiske funktioner i lydsystemudviklingen. For nylig er kyllingembryoen blevet særlig værdifuld til at studere genekspression, regulering og funktion i forbindelse med hørelse. I ovo kan elektroporation bruges til at målrette auditive hjernestammeområder, der er ansvarlige for højt specialiserede auditive funktioner. Disse regioner omfatter kyllingekernen magnocellularis (NM) og nucleus laminaris (NL). NM og NL neuroner stammer fra særskilte forstadier af rhombomerer 5 og 6 (R5 / R6). Her præsenterer vi i ovo elektroporation af plasmidkodede gener for at studere genrelaterede egenskaber i disse regioner. Vi viser en metode til rumlig og tidsmæssig kontrol af genekspression, der fremmer enten gevinst eller tab af funktionel fænotypees. Ved at målrette auditoriske neurale progenitorregioner associeret med R5 / R6, viser vi plasmidtransfektion i NM og NL. Temporal regulering af genekspression kan opnås ved at vedtage et tet-on-vektorsystem. Dette er en lægemiddelinducerbar procedure, der udtrykker genene af interesse i nærvær af doxycyclin (Dox). I ovo elektroporationsteknikken - sammen med enten biokemiske, farmakologiske og eller in vivo funktionelle analyser - tilvejebringes en innovativ tilgang til undersøgelse af auditiv neuronudvikling og tilhørende patofysiologiske fænomener.

Vi viser her, at i ovo elektroporation tillader genekspression i et normalt udviklende biologisk system. Plasmidkodede gener injiceres fokalt i neuralrøret over R5 / R6. Et skematisk eksempel på elektroden og pipetteplaceringerne i forhold til vigtige anatomiske markører er vist i figur 1A . Den korrekte placering af plasmidinjektion bekræftes 24 timer efter elektroporation og vist i figur 1B . Den målrettede indsprøjtning af plasmider i n...

Watch this Scientific Journal Video about In Ovo Electroporation in the Chicken Auditory Brainstem at JoVE.com

In Ovo Electroporation in the Chicken Auditory Brainstem

Auditoriske hjernestam neuroner af avians og pattedyr er specialiseret i hurtig neurale kodning, en grundlæggende proces for normale hørefunktioner. Disse neuroner stammer fra tydelige forstadier af embryonisk hindbrain. Vi præsenterer teknikker, der anvender elektroporering til at udtrykke gener i baghugns kerneembryoner for at studere genfunktion under auditiv udvikling.

I Ovo Electroporation i Kylling Auditory Brainstem

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that ...

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Developmental Biology

In Ovo Electroporation in the Chicken Auditory Brainstem

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Effektiv Gene Transfer i Chick nethinder for primær cellekultur Studies: An<em&gt; Ex-ovo</em&gt; Elektroporation Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

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Analyse af zebrafisk Larver Skeletmuskel Integritet med Evans blå farvestof

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Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments

Hele Retinale Eksplantater fra kylling Embryoner til Elektroporation og kemisk reagens Behandlinger

The retina is a good model for the developing central nervous system. The large size of the eye and most importantly the accessibility for experimental ...

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Gene Transfer til kylling Auditory Organ med<em&gt; I Ovo</em&gt; Micro-elektroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

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The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and ...

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

Embryo Mikroinjektion og Elektroporering i Chordatzoologi<em&gt; Ciona intestinalis</em

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance ...

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Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Dissektion og immunfluorescent farvning af champignon krop og fotoreceptor neuroner i voksen Drosophila melanogaster hjerner

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

Tre-dimensionelle Organotypic kulturer af vestibulære og auditive sanseorganer

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...

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The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...