Ringraziamo Humana Press per il permesso di pubblicare la pipetta figura microiniezione di fabbricazione, che è originariamente apparso a pagina 130 di riferimento 11; Larry Dlugas e Steven Wong, OHSU Ministero delle Comunicazioni educativi, per videografia, Larry Dlugas per il design e video editing; Adam M. O &#39;Quinn, Senior Designer, Trion / Envirco per la progettazione di nostra consuetudine orizzontale, cappa a flusso laminare e Les Goldsmith per fornire lo schema tecnico; Victor Monterroso, MV, MS, PhD e Tom Chatkupt, DVM, OHSU Dipartimento di Medicina comparativa, per la guida con il nostro animali protocollo di cura, tecniche chirurgiche, e profilattico analgesia regime; Marcel Perret-Gentil, DVM, MS, per condividere la sua dispensa sulle tecniche di sutura veterinaria, Edward Porsov, MS, per la progettazione la nostra workstation video di Adobe Premiere Pro microscopia computer; e Leah bianco e Jonas Hinckley dei LNS didascalie (Portland, OR). Questo lavoro è stato sostenuto dalle concessioni dal National Institute on Deafness e other disturbi della comunicazione: DC 008595 R01 e R01 DC 008595-04S2 (a JB) e P30 DC005983 (Oregon Hearing Research Core Center Grant, Peter Gillespie, Principal Investigator).

1. Laparotomia ventrale <ol><li> Anestetizzare una diga i cui embrioni sono al giorno embrionale 11,5 (E11.5, a mezzogiorno del giorno viene rilevata una spina vaginale è il giorno 0,5 dello sviluppo embrionale) mediante iniezione intraperitoneale di soluzione di sodio pentobarbital anestesia (7,5 pl per grammo di peso corporeo). Soluzione di lavoro anestetico: 180 pl di 50 mg / mL di sodio pentobarbital, 100 pl di etanolo assoluto, 320 pl di 65 mg / ml di solfato di magnesio acquoso (modula tono uterina), e 400 pl di glicole propilenico (miscibile veicolo acquoso e organico componenti). </li><li> Valutare la completezza di anestesia effettuando prove di stimoli nocivi: squeeze zampa; risposta e lampeggiano al tocco sulla guancia e vibrisse; pizzico coda. Applicare una pomata oftalmica sterile per le cornee. </li><li> Accorciare il pelo addominale dalla zona sovrapubica alla gabbia toracica, con forbici e una lama fine (Oster # 40 della lama). Disinfettare l&#39;addome con il 70% etanolo, 10% di iodio povidone (Betadine), unnd etanolo al 70%, sequenzialmente. Luogo del mouse addome rivolto verso il basso su un telino sterile e quindi impostare su una piastra elettrica o piatto caldo (37 ° C) per 2-5 minuti. </li><li> Incidere la cute addominale sulla linea mediana ventrale con palla punta forbici. Estendere l&#39;incisione di 10-14 mm. Identificare la linea alba, uno avascolare, fascia bianca tessuto connettivo lungo la linea mediana ventrale della parete addominale. Incidere la linea alba con palla punta forbici ed estendere il 10-14 mm incisione. Lavare immediatamente con l&#39;addome preriscaldato (37 ° C), Ringer lattato soluzione. </li><li> Esternalizzare i due corni dell&#39;utero con una pinza ad anelli senza applicare una pressione eccessiva sui siti di impianto. Irrigare le corna uterine esternalizzati con preriscaldata, soluzione di Ringer lattato. </li></ol> 2. Microiniezione Transuterine <ol><li> Realizzare una parete spessa, borosilicato pipetta microiniezione capillare di vetro, con le seguenti caratteristiche: 12-16 μm diametro esterno ed uno smusso 20 gradi. Su un P-97 Sutter estrattore pipetta con filamento piccola scatola, utilizzare il seguente programma: calore = test di rampa più 3 unità; pressione = 200; tirare = 0; velocità = 46; tempo = 100). Con il BV-10 Beyeler Sutter, utilizzare la 104C (oro) disco abrasivo per pipette di grande diametro. Risospendere plasmide di espressione a 3-4 pg / pl in fosfato di calcio libero soluzione salina tamponata (pH 7,2-7,4). Aggiungi cristallino verde veloce al DNA concentrato, vortex delicatamente per 30 secondi, e far girare a 10.000 g per 10 secondi. La quantità minima di verde veloce necessaria per seguire visivamente l&#39;efficacia dell&#39;iniezione viene determinato empiricamente. La pipetta in sostegno degli smussato con il DNA / fast soluzione verde. Collegare la pipetta caricato microiniezione al titolare pipetta della pressione dell&#39;iniettore (Picospritzer con&gt; 99% di azoto puro come fonte di gas). </li><li> Transilluminate l&#39;utero a bassa intensità, luce alogena per visualizzare l&#39;embrione all&#39;interno del suo sito di impianto. Identificare il beating cuore, vescicole cerebrali, gemme degli arti, nascente 4 ° ventricolo del cervello posteriore, e gli occhi. Irrigare l&#39;utero ogni 2 minuti con preriscaldata e soluzione di Ringer lattato per mantenere l&#39;idratazione. Applicare una leggera pressione sull&#39;utero di riorientare l&#39;embrione e identificare i punti di repere anatomici di cui sopra. </li><li> Orientare l&#39;embrione per identificare la vena principale cui testa anteriore e posteriore fianco rami del territorio mesenchimali in cui il otocyst risiede. Il otocyst appropriato non può essere visto da transilluminazione dell&#39;utero. Il otocyst si trova a metà strada tra i rami anteriore e posteriore della vena testa primaria che, insieme al tronco principale della vena, formare la forma dei montanti o pali su un campo di football americano. Il otocyst è a metà strada tra i montanti. </li><li> Inserire la pipetta iniezione attraverso l&#39;utero in una traiettoria in linea con la posizione del presunto otocyst. Pulse il microiniettore volta dopo aver attraversato il uterci visualizzare il colorante tracciante e il approssimare la posizione della punta della pipetta. Far avanzare la pipetta sotto controllo micrometro e pulsare di nuovo per valutare la profondità. Ulteriori avanzare la pipetta nel mesenchima laterale della testa e il polso più volte. Il targeting otocyst successo rivelerà la forma affusolata del condotto endolinfatico dorsalmente e capesante forma a conchiglia del vestibolo. Rilasciare la pressione sul utero, rimuovere la pipetta dalla dell&#39;embrione / utero in un unico movimento, e subito irrigare l&#39;utero con preriscaldata, soluzione di Ringer lattato. </li></ol> 3. Elettroporazione in vivo <ol><li> Irrigare l&#39;utero con la soluzione di Ringer lattato. Soluzione appena applicato Ringer lattato è necessario accoppiare elettricamente la pagaia in stile elettrodi verso l&#39;utero. Inumidire le superfici degli elettrodi di tungsteno con il Ringer lattato. Centro il otocyst iniettato nel percorso degli elettrodi. Delicatamente comprimere l&#39;utero con il pa elettroporazioneddles. Il catodo è in contatto con la parete uterina laterale al otocyst iniettata e l&#39;anodo è in contatto con la parete uterina adiacente alla otocyst non iniettati delle cavie. Attivare un quadrato treno di impulsi d&#39;onda con l&#39;interruttore a pedale sul elettroporatore. Parametri di elettroporazione sono: 5, 50 msec impulsi a 43 volt per impulso e 950 msec ritardo interpulso. Immediatamente irrigare l&#39;utero dopo il treno di impulsi viene consegnato. Registrare la corrente erogata al tessuto: 60-100 mAmps è sufficiente per trasfettare progenitori epiteliali dell&#39;orecchio. Iniettare e elettroporare 4-6, gli embrioni E11.5 per diga. </li><li> Effettuare un secondo, microiniezione indipendente transuterine acquosa di destrano fluorescente (Alexa 488 se il plasmide di espressione codifica una proteina fluorescente rossa o Alex Fluor 594 se il plasmide di espressione codifica una proteina verde fluorescente) nel ventricolo 4 ° di tali embrioni cui interno orecchie accuratamente iniettato e almeno 60 mAmps di corrente per impulso era delivered durante l&#39;elettroporazione. Il destrano fluorescente sarà rilevabile al momento della nascita nel cervello posteriore e consentire la selezione di cuccioli cui interno sono state manipolate le orecchie durante l&#39;embriogenesi (vedi 3.5). </li><li> Irrigare le corna uterine con il Ringer lattato. Reinserire le corna uterine nella cavità addominale. Sciacquare la cavità uterina con 2-4 ml di preriscaldata, soluzione di Ringer lattato e consentire l&#39;overflow di drenare fuori dal sito di incisione sul telino sterile. Sostituire il drappeggio a secco, materiale sterile. Suturare la parete addominale con un non-taglio ago e un 6-0 sutura riassorbibile. Si preferisce un filza, bloccando ogni punto altro, sia per la parete addominale e la pelle. </li><li> Asciugare fur le dighe &#39;e amministrare un non-steroidei anti-infiammatori come il Meloxicam per iniezione sottocutanea. Riportare la diga per la gabbia di recupero preriscaldata in un telino sterile. Monitorare e registrare le respirazioni dighe &quot;, sito pervietà dell&#39;incisione e della vagina per lo scarico di sangue. Il sanguinamento è rari, ma se presente e senza sosta, l&#39;eutanasia la diga, mentre lei è ancora sotto anestesia. Nota: il momento in cui riprende conoscenza e tenta di deambulare. Riportare la diga per la colonia del mouse quando mostra segni di mangiare e bere e ha iniziato a costruire il nido. In genere, questo si verifica entro 12 ore. </li><li> Alla nascita (postnatali giorno 0), il flash della regione rombencefalo di ogni pup nella lettiera per rilevare la fluorescenza destrano utilizzando un microscopio stereofluorescence dissezione con GFP o Texas Red filtro impostato a seconda dei casi. Ritorna alla diga di allattamento solo i cuccioli che visualizzano l&#39;etichettatura rombencefalo. </li></ol> 4. Risultati rappresentativi <img src="/files/ftp_upload/3653/3653fig1.jpg" alt="Figura 1" /> Figura 1. Elettroporazione trasferimento genico mediato alla coclea sviluppo. La otocyst E11.5 stato iniettato un plasmide di espressione codifica maggiore proteina fluorescente verde (EGFP) ed elettroporati (impulsi TRAnei parametri: cinque, 43 impulsi volt a 50 msec / impulsi e 950 msec di ritardo interpulso). A) Un orecchio rappresentante interno da un giorno postnatale 6 (P6) cucciolo otocyst cui è stato iniettato e elettroporate E11.5 a dimostrare l&#39;espressione EGFP dalla base attraverso il turn centro della coclea. La parete laterale della coclea è stato rimosso dal mezzo giro e solo apice. E11.5 progenitori che danno origine al vertice non sono state trasfettate. B) mount intero immunocolorazione della coclea in (A) con il marcatore delle cellule dei capelli, 7a miosina (MYO7A), indica che l&#39;espressione EGFP segue la traiettoria dell&#39;organo del Corti ed è grossolanamente localizzato l&#39;epitelio capelli cellula portante sensoriale. C) Un orecchio rappresentante interno da un embrione E18.5 otocyst cui è stato iniettato e elettroporate a E11.5. La proiezione laser confocale dimostra espressione EGFP in MYO7A-positivi cellule ciliate sensorie. D) la proiezione laser confocale dell&#39;epitelio cocleare sensoriale dell&#39;organo del Corti E18.5 indicando EGFP expression nelle cellule ciliate interne (IHC), le cellule ciliate esterne (OHC), le cellule interne phalangial (IPC), cellule pilastro (pc), e le cellule Deiters &#39;(dc). La barra della scala in (B) si applica a (A).

<ol>
	<li>Gillespie, P. G., Muller, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanotransduction+by+hair+cells:+models,+molecules,+and+mechanisms.">Mechanotransduction by hair cells: models, molecules, and mechanisms.</a> Cell. 139, 33-44 (2009).</li><li>Bok, J., Chang, W., Wu, 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=Patterning+and+morphogenesis+of+the+vertebrate+inner+ear.">Patterning and morphogenesis of the vertebrate inner ear.</a> Int. J. Dev. Biol. 51, 521-533 (2007).</li><li>Kelley, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cell+fate+in+the+sensory+epithelia+of+the+inner+ear.">Regulation of cell fate in the sensory epithelia of the inner ear.</a> Nat. Rev. Neurosci. 7, 837-849 (2006).</li><li>Ohyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP+signaling+is+necessary+for+patterning+the+sensory+and+nonsensory+regions+of+the+developing+mammalian+cochlea.">BMP signaling is necessary for patterning the sensory and nonsensory regions of the developing mammalian cochlea.</a> J. Neurosci. 30, 15044-15051 (2010).</li><li>Pan, W., Jin, Y., Stanger, B., Kiernan, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signaling+is+required+for+the+generation+of+hair+cells+and+supporting+cells+in+the+mammalian+inner+ear.">Notch signaling is required for the generation of hair cells and supporting cells in the mammalian inner ear.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 15798-15803 (2010).</li><li>Gubbels, S. P., Woessner, D. W., Mitchell, J. C., Ricci, A. J., Brigande, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+auditory+hair+cells+produced+in+the+mammalian+cochlea+by+in+utero+gene+transfer.">Functional auditory hair cells produced in the mammalian cochlea by in utero gene transfer.</a> Nature. 455, 537-541 (2008).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide for the Care and Use of Laboratory Animals. , (2010).</li><li>Matsuda, T., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+expression+of+transgenes+introduced+by+in+vivo+electroporation.">Controlled expression of transgenes introduced by in vivo electroporation.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 1027-1032 (2007).</li><li>Chen, C., Smye, S. W., Robinson, M. P., Evans, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+electroporation+theories:+a+review.">Membrane electroporation theories: a review.</a> Med. Biol. Eng. Comput. 44, 5-14 (2006).</li><li>Saito, T. <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+electroporation+in+the+embryonic+mouse+central+nervous+system.">In vivo electroporation in the embryonic mouse central nervous system.</a> Nat. Protoc. 1, 1552-1558 (2006).</li><li>Brigande, J. V., Gubbels, S. P., Woessner, D. W., Jungwirth, J. J., Bresee, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation-mediated+gene+transfer+to+the+developing+mouse+inner+ear.">Electroporation-mediated gene transfer to the developing mouse inner ear.</a> Methods Mol. Biol. 493, 125-139 (2009).</li><li>Morsli, H., Choo, D., Ryan, A., Johnson, R., Wu, 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=Development+of+the+mouse+inner+ear+and+origin+of+its+sensory+organs.">Development of the mouse inner ear and origin of its sensory organs.</a> J. Neurosci. 18, 3327-3335 (1998).</li><li>Sher, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+embryonic+and+postnatal+development+of+the+inner+ear+of+the+mouse.">The embryonic and postnatal development of the inner ear of the mouse.</a> Acta. Otolaryngol. , 1-77 (1971).</li><li>Sheffield, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+vector+tropism+for+supporting+cells+in+the+developing+murine+cochlea.">Viral vector tropism for supporting cells in the developing murine cochlea.</a> Hear Res. 277, 28-36 (2011).</li><li>Bedrosian, J. C. <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+delivery+of+recombinant+viruses+to+the+fetal+murine+cochlea:+transduction+characteristics+and+long-term+effects+on+auditory+function.">In vivo delivery of recombinant viruses to the fetal murine cochlea: transduction characteristics and long-term effects on auditory function.</a> Mol. Ther. 14, 328-335 (2006).</li><li>Reisinger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+functional+equivalence+of+otoferlin+and+synaptotagmin+1+in+exocytosis.">Probing the functional equivalence of otoferlin and synaptotagmin 1 in exocytosis.</a> J. Neurosci. 31, 4886-4895 (2011).</li><li>Magnani, E., Bartling, L., Hake, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+Gateway+to+MultiSite+Gateway+in+one+recombination+event.">From Gateway to MultiSite Gateway in one recombination event.</a> BMC Mol. Biol. 7, 46 (2006).</li><li>Perret-Gentil, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of Veterinary Suturing. , .</li><li>Oesterle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> P-1000 &#38; P-97 Pipette Cookbook. , (2011).</li></ol>

Non ci sono conflitti di interesse dichiarati.

Trasferimento del gene per l&#39;orecchio del mouse in via di sviluppo interno: L&#39;orecchio interno del mouse si sviluppa dalla placode otic durante la prima settimana di sviluppo post-impianto 12,13. Di giorno embrionale 9.5 (E9.5), il placode ha invaginato e trasformato in una vescicola piena di liquido chiamato otocyst 2. Precursori Otic in vescicole dare origine alle cellule sensoriali e nonsensory all&#39;interno dell&#39;orecchio interno matura così come i neuroni che inn...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> Micro Caso sterilizzazione </td><td> ROBOZ </td><td> RS-9900a </td><td> 8X8.5X1.25 pollici </td></tr><tr><td> Ball-punta forbici </td><td> Strumenti di Scienze Belle </td><td> 14109-09 </td><td></td></tr><tr><td> Anello pinza </td><td> Strumenti di Scienze Belle </td><td> 11106-09 </td><td> 4,8 millimetri ID/6mm OD </td></tr><tr><td> Adson tessuto Pinza </td><td> Strumenti di Scienze Belle </td><td> 11027-12 </td><td></td></tr><tr><td> Needle conducente </td><td> Strumenti di Scienze Belle </td><td> 12502-12 </td><td></td></tr><tr><td> Allergy siringa Tray </td><td> Becton Dickison </td><td> 305536 </td><td></td></tr><tr><td> Suture 6-0 </td><td> Syneture </td><td> GL-889 </td><td> 0,7 sutura metrica gastrointestinale </td></tr><tr><td> Ringer lattato iniezione USP </td><td> Baxter </td><td> 2B2323 </td><td></td></tr><tr><td> Veloce verde </td><td> Sigma Aldrich </td><td> F7258 </td><td></td></tr><tr><td> Capillare di vetro borosilicato </td><td> Harvard Apparatus </td><td> 30-0053 </td><td></td></tr><tr><td> Soluzione di sodio Nembutal </td><td> OVATION Pharmaceuticals Inc. </td><td> NDC 67386-501-52 </td><td></td></tr><tr><td> MgSO4.7H2O </td><td> Fisher Scientific </td><td> M63-500 </td><td></td></tr><tr><td> Glicole propilenico </td><td> Fisher Scientific </td><td> P355-1 </td><td></td></tr><tr><td> Etanolo </td><td> Sigma Aldrich </td><td> E7023-500 </td><td></td></tr><tr><td> Meloxicam </td><td> Boehringer Ingeheim </td><td> NADA 141-219 </td><td></td></tr><tr><td> Micropipetta Puller </td><td> Sutter Instruments </td><td> P-97 </td><td> FB255B filamento box; consultare Cookbook pipetta da strumenti Sutter </td></tr><tr><td> Microelettrodo Beyeler </td><td> Sutter Instruments </td><td> BV-10 </td><td> Disco smussatura 104C per pipette di grandi dimensioni, consultare il manuale per smussare la teoria </td></tr><tr><td> Micropipetta titolare </td><td> Warner Instruments </td><td> MP-S15T </td><td> Per pipette 1,5 millimetri di diametro esterno e la porta di pressione femmina per tubi Picospritzer. </td></tr><tr><td> Pinzetta-style elettrodo </td><td> Protech International Inc. </td><td> CUY650P5 </td><td> 5 mm di diametro esterno </td></tr><tr><td> Piazza Onda Elettroporatore </td><td> Protech International Inc. </td><td> CUY21EDIT </td><td> Pedale raccomandata </td></tr><tr><td> PICOSPRITZER III </td><td> Parker Hannifin </td><td> 051-0500-900 </td><td> Pedale raccomandata </td></tr><tr><td> Controllo manuale Micromanipolatore </td><td> Harvard Apparatus </td><td> 640056 </td><td></td></tr><tr><td> Flusso laminare orizzontale banco pulito </td><td> Envirco </td><td></td><td> Modifiche personalizzate LF 630-10554. Vedi informazioni supplementari per disegno dello schema cappuccio. </td></tr><tr><td> Leica stereofluorescence dissezione microcope con motore Lumencor SOLA luce </td><td> Bartels e Stout e Lumencor </td><td> MZ10F con motore Lumencor SOLA luce </td><td> Footpedals di concentrare i MZ10F e per attivare l&#39;illuminatore SOLA sono raccomandati </td></tr><tr><td> Alexa Fluor 594 destrano </td><td> Invitrogen </td><td> D22913 </td><td> 10mg/ml, acquoso </td></tr><tr><td> Alexa Fluor 488 destrano </td><td> Invitrogen </td><td> D22910 </td><td> 10mg/ml, acquoso </td></tr><tr><td> Enviro-dri </td><td> Pastore Altre PapERS </td><td></td><td> <a href="http://www.ssponline.com" target="_blank">www.ssponline.com</a> </td></tr></tbody></table>

gene transfer to the developing mouse inner ear by in vivo electroporation

L&#39;orecchio interno dei mammiferi ha 6 epiteli sensoriali distinti: 3 creste in ampolle dei canali semicircolari, macule nel utricolo e sacculo, e l&#39;organo del Corti nella coclea a spirale. Le creste e macule contengono cellule ciliate vestibolari che trasducono stimoli meccanici per la pongono al servizio della speciale senso di equilibrio, mentre le cellule uditive dei capelli in dell&#39;organo del Corti sono i trasduttori primari per l&#39;audizione 1. Il destino specifica cella in questi epiteli sensoriali e morfogenesi dei canali semicircolari e la coclea hanno luogo durante la seconda settimana di gestazione nel topo e sono in gran parte completati prima della nascita 2,3. Studi sullo sviluppo dell&#39;orecchio interno del mouse sono regolarmente condotti da raccolta embrioni transgenici in diverse fasi embrionali o post-natale al fine di conoscere le basi molecolari di cellulare e / o morfologica fenotipi 4,5. Ipotizziamo che il trasferimento del gene per l&#39;orecchio interno del mouse in via di sviluppo in utero &lt;/ Em&gt; nel contesto di guadagno-e la perdita di funzione studi rappresenta un approccio complementare alla transgenesi mouse tradizionale per l&#39;interrogatorio dei meccanismi genetici alla base dei mammiferi lo sviluppo dell&#39;orecchio interno 6. Il paradigma sperimentale per condurre studi misexpression gene nel topo in via di sviluppo dell&#39;orecchio interno dimostrato qui si risolve in tre fasi generali: 1) laparotomia ventrale; 2) microiniezione transuterine e 3) elettroporazione in vivo. Laparotomia ventrale è una tecnica di sopravvivenza del mouse che permette di esternalizzazione chirurgica dell&#39;utero per accedere sperimentale per gli embrioni impiantati 7. Microiniezione Transuterine è l&#39;uso di smussati, micropipette capillari di vetro per introdurre plasmide di espressione nel lume della vescicola otica o otocyst. In vivo elettroporazione è l&#39;applicazione di onda quadra, diretti impulsi di corrente per guidare l&#39;espressione plasmidico in 8-10 cellule progenitrici. 11. Mouse tecniche sperimentali embriologici può essere difficile da imparare dalla prosa e immagini fisse da solo. Nel presente lavoro, dimostriamo i 3 passaggi della procedura di trasferimento genico. La maggior parte critica, abbiamo distribuire microscopia video digitale per mostrare con precisione come: 1) identificare l&#39;orientamento dell&#39;embrione in utero, 2) riorientare embrioni per il targeting iniezioni al otocyst; 3) microinject DNA miscelata con una soluzione tracciante colorante nella otocyst al giorno e 11,5 embrionali 12,5; 4) elettroporare il otocyst iniettato; e 5) embrioni etichette elettroporate per la selezione post-natale alla nascita. Forniamo esempi rappresentativi di successo trasfettate orecchio interno, di una guida pittorica per le cause più comuni di mistargeting otocyst, discutere su come evitare i più comuni errori metodologici e attuali orientamenti per la scrittura di un utero in gene trasferimento degli animali protocollo di cura.

Watch this Scientific Journal Video about Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation at JoVE.com

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

L&#39;orecchio interno è un topo placode derivato il cui organo sensoriale per lo sviluppo del programma è elaborato durante la gestazione. Definiamo uno<em&gt; In utero</em&gt; Tecnica di trasferimento genico composto da tre fasi: laparotomia ventrale del mouse, microiniezione transuterine e<em&gt; In vivo</em&gt; Elettroporazione. Usiamo la microscopia video digitale per dimostrare le critiche tecniche sperimentali embriologici.

Gene Transfer per l&#39;orecchio del mouse Sviluppo Inner<em&gt; In Vivo</em&gt; Elettroporazione

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

gene-transfer-to-developing-mouse-inner-ear-vivo

Research

JoVE Journal

Neuroscience

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

13.9K Views.  Oregon Health & Science University. L'orecchio interno è un topo placode derivato il cui organo sensoriale per lo sviluppo del programma è elaborato durante la gestazione. Definiamo uno In utero Tecnica di trasferimento genico composto da tre fasi: laparotomia ventrale del mouse, microiniezione transuterine e In vivo Elettroporazione. Usiamo la microscopia video digitale per dimostrare le critiche tecniche sperimentali embriologici.

Video: Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

Gene consegna di cervello di ratto post-natale da parte di non-ventricolare iniezione plasmidi e Elettroporazione

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

Ricerca

Neuroscienze

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

Trasfezione delle cellule gangliari della retina di topo In vivo Elettroporazione

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to cis-regulatory elements (CREs), short (&tilde;300-600 bp) stretches of genomic DNA which can lie upstream, downstream, or within the introns of the genes they control. CREs (i.e., enhancers/promoters) typically consist of multiple clustered binding sites for both transcriptional activators and repressors1-3. They serve as logical integrators of transcriptional input giving a unitary output in the form of spatiotemporally precise and quantitatively exact promoter activity. Most studies of mammalian cis-regulation to date have relied on mouse transgenesis as a means of assaying the enhancer function of CREs4-5. This technique is time-consuming, costly and, on account of insertion site effects, largely non-quantitative. On the other hand, quantitative assays for mammalian CRE function have been developed in tissue culture systems (e.g., dual luciferase assays), but the in vivo relevance of these results is often uncertain. 
Electroporation offers an excellent alternative to traditional mouse transgenesis in that it permits both spatiotemporal and quantitative assessment of cis-regulatory activity in living mammalian tissue. This technique has been particularly useful in the analysis of cis-regulation in the central nervous system, especially in the cerebral cortex and the retina6-8. While mouse retinal electroporation, both in vivo and ex vivo, has been developed and extensively described by Matsuda and Cepko6-7,9, we have recently developed a simple approach to quantify the activity of photoreceptor-specific CREs in electroporated mouse retinas10. Given that the amount of DNA that is introduced into the retina by electroporation can vary from experiment to experiment, it is necessary to include a co-electroporated 'loading control' in all experiments. In this respect, the technique is very similar to the dual luciferase assay used to quantify promoter activity in cultured cells. 
When assaying photoreceptor cis-regulatory activity, electroporation is usually performed in newborn mice (postnatal day 0, P0) which is the time of peak rod production11-12. Once retinal cell types become post-mitotic, electroporation is much less efficient. Given the high rate of rod birth in newborn mice and the fact that rods constitute more than 70% of the cells in the adult mouse retina, the majority of cells that are electroporated at P0 are rods. For this reason, rod photoreceptors are the easiest retinal cell type to study via electroporation. The technique we describe here is primarily useful for quantifying the activity of photoreceptor CREs.

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

This protocol describes a simple and inexpensive way to quantify the activity of cis-regulatory elements (i.e., enhancer/promoters) in living mouse retinas via explant electroporation. DNA preparation, retinal dissection, electroporation, retinal explant culture, and post-fixation analysis and quantification are described.

Quantificare le attività di Cis-Regolamentazione Elementi nella retina di topo Elettroporazione Espianto

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

In elettroporazione in vivo di Retina topo in via di sviluppo

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

Mouse in Utero elettroporazione: controllato Transefection Gene spazio-temporale

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

Isolando LacZ cellule che esprimono da tessuti dell&#39;orecchio interno del mouse in citometria a flusso

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

Imaging ad alta risoluzione dal vivo del comportamento delle cellule in via di sviluppo neuroepitelio

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

Registrazioni patch clamp in cellule interne dell&#39;orecchio Capelli Isolato da Zebrafish

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

La manipolazione genetica del topo sviluppo dell&#39;ipotalamo attraverso<em&gt; In utero</em&gt; Elettroporazione

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and genetic mechanisms that contribute to hearing loss. Introduction of vectors into the inner ear must be done in a way that widely distributes the agent throughout the cochlea while minimizing injury to the existing structures. This manuscript describes a post-auricular surgical approach that can be used for mouse cochlear therapy using molecular, pharmacologic, and viral delivery to mice postnatal day 10 and older via the round window membrane (RWM). This surgical approach enables rapid and direct delivery into the scala tympani while minimizing blood loss and avoiding animal mortality. This technique involves negligible or no damage to essential structures of the inner and middle ear as well as neck muscles while wholly preserving hearing. To demonstrate the efficacy of this surgical technique, the vesicular glutamate transporter 3 knockout (VGLUT3 KO) mice will be used as an example of a mouse model of congenital deafness that recovers hearing after delivery of VGLUT3 to the inner ear using an adeno-associated virus (AAV-1).

The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane.

Metodo chirurgico per Virally Mediated Gene consegna all&#39;orecchio mouse interno attraverso la finestra rotonda a membrana

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and ...