Vorremmo ringraziare il Sig. Maaz Enver per l&#39;utilizzo della sua macchina fotografica HD (Cannon VIXIA HFS100). Questo lavoro è stato sostenuto in parte da sovvenzioni dal NIH (EY018738), New Jersey Commissione per la ricerca del Midollo Spinale (08-3074-SCR-E-0 e 10-3091-SCR-E-0), e Busch Award Ricerca Biomedica.

Poiché alcune parti del protocollo vengono eseguiti come precedentemente descritto in altri video Jové 2 e testi 3 con modifica minore non parleremo quelli in dettaglio qui. 1. Egg manipolazione e preparazione Needle <ol><li> Le uova possono essere memorizzati in un wine cooler a circa 13 ° C per fino a 1 settimana. Se la temperatura è troppo alta, embrioni inizierà a sviluppare anormalmente, mentre più bassa temperatura provoca mortalità elevata. Una volta che siete pronti ad incubare togliere le uova dal raffreddamento di vino e impostare le uova in posizione verticale con l&#39;estremità più grande up. Conservare le uova della temperatura ambiente per almeno 2 ore prima di metterli nel 37.5 ° C incubatore. </li><li> Incubare le uova per 96-100 ore che è di circa 4 giorni embrionali per ottenere gli embrioni che sono alla Hamburger e lo stadio Hamilton 22 4-5. </li><li> Preparare aghi micropipette da tubi di vetro tirato capillari e rompere la punta sotto un micro dissezionecampo di applicazione con una pinzetta per ottenere una apertura punta circa 0,1 micron di diametro e un cono 20 mm. Aghi con le grandi punte hanno difficoltà a perforare la membrana vitellina mentre i più piccoli hanno difficoltà a punte di carico e fornire la soluzione di DNA. </li><li> Applicare l&#39;ago da ml 0,1 siringhe Hamilton a tenuta di gas montato su un micromanipolatore. Usare un piccolo pezzo di tubo in silicone Masterplex per attaccare l&#39;ago alla siringa. Dal momento che i collegamenti siano saldi non c&#39;è bisogno di aggiungere olio minerale per suggellare questo allegato. </li><li> Mix 2 pl di soluzione di DNA plasmide reporter con una concentrazione compresa 3-6 ug / pl e 0,2 pl di verde veloce (0,025%) su un pezzo di parafilm. Veloce colorante verde aiuterà a visualizzare l&#39;iniezione. Lentamente, caricare l&#39;ago con la miscela. </li><li> Per liberare la membrana vitellina dalla membrana interna ruotare delicatamente l&#39;uovo circa 180 ° e attendere per qualche minuto quindi ruotarlo in posizione originale e impostarlo per l&#39;elettroporazione. </li><li> Pulire la forceps e guscio d&#39;uovo con il 70% di etanolo per evitare l&#39;infezione per l&#39;embrione. </li><li> Fare un piccolo foro sul l&#39;uovo immediatamente sopra la cella aria con un paio di dimensioni pinze AA. Fare attenzione a non rompere il guscio dell&#39;uovo. Rimuovere piccoli pezzi uno alla volta per fare una piccola finestra. Rimuovere con cura la membrana interna utilizzando le pinze, senza toccare la membrana vitellina. </li></ol> 2. Iniezione ed elettroporazione <ol><li> Per evitare danni al cervello o, il cuore della posizione di contra &#39;ago lateralmente al fascio principale dei vasi sanguigni che entrano l&#39;occhio e rivolta verso il becco. </li><li> Pierce attraverso la membrana vitellina, sclera, retina e vitreo da una spinta improvvisa lieve dell&#39;ago. Se l&#39;ago perfora attraverso l&#39;altra estremità dell&#39;occhio essa dovrebbe essere alright meno che non danneggia qualsiasi vena principale sangue. </li><li> Tiri indietro lentamente lo dell&#39;ago al bordo dell&#39;apertura e posizionarlo quasi tangente alla parete esterna del bulbo oculare. </li><li> Insert l&#39;ago nello spazio sub retinica tra la sclera e retina. </li><li> Iniettare il DNA finché non sarà possibile visualizzare la soluzione verde riempire il lato del bulbo oculare e spingendo verso l&#39;interno della retina creando un rigonfiamento. </li><li> Se la vostra collocazione dell&#39;ago non è corretta allora vedrete la soluzione di DNA diffusione all&#39;interno del vitreo riempire il centro del bulbo oculare. Inoltre, se si danneggiare la retina troppo allora vedrete la soluzione di DNA è venuta fuori del bulbo oculare. </li><li> Rimuovere lentamente l&#39;ago e porre immediatamente gli elettrodi in parallelo dentro l&#39;uovo dopo l&#39;immersione in PBS. Spingere verso il basso gli elettrodi di immergerci il liquido amniotico in un modo in modo che l&#39;occhio iniettato si trova tra gli elettrodi. Evitare di toccare qualsiasi nave importante il sangue o il cuore con gli elettrodi, mentre per questi ultimi. L&#39;elettrodo negativo dovrebbe essere al lato iniezione in modo che il DNA può essere trasportato da sub spazio retinica nella retina verso elettrodo positivo. Electroincorporare l&#39;retina con 5 impulsi di 15V per 50 ms, con intervalli di 950 ms. </li><li> Rimuovere con cura gli elettrodi e sigillare la finestra delle uova con pezzi di nastro adesivo trasparente. </li><li> Etichetta e data l&#39;uovo iniettato prima di riporlo per l&#39;incubatrice. Tipicamente, ci vogliono circa 3 a 5 minuti per completare l&#39;intero processo elettroporazione. </li><li> Espressione GFP può essere visto come precoce da 8 ore dalla elettroporazione. Tuttavia, è possibile attendere che l&#39;embrione raggiunge lo stadio desiderato prima della raccolta. </li></ol> 3. Risultati rappresentativi Nel nostro studio, usiamo vari costrutti plasmidici per studiare la regolazione dell&#39;espressione genica che ha coinvolto lo sviluppo delle cellule della retina. In questo video pCAG-GFP (controllo trasfezione) è stato usato per seguire una iniezione di successo e elettroporazione. Tuttavia, qualsiasi costrutto plasmidico con gene reporter (GFP, RFP ecc) può essere utilizzato. Anche se l&#39;espressione GFP può essere visto a partire da 8 ore dopo l&#39;electroporation, di solito la raccolta inizia l&#39;uovo il giorno 6 (E6) e poi. Elettroporate retine sono stati sezionati fuori dell&#39;embrione e analizzato al microscopio a fluorescenza dissezione prima di incasso e di sezionamento. Tipicamente, espressione genica reporter può essere visto almeno in un quarto della retina dopo una elettroporazione di successo (Fig. 1). I tessuti della retina transfettate sono stati ulteriormente analizzati attraverso il sezionamento per la visualizzazione chiara delle morfologie cellulari. Immunoistochimica utilizzando marcatori di tipo specifico di cellule (Brn3a, Pax6 ecc) ha permesso la caratterizzazione delle cellule GFP-specifica espressione (Fig. 2). <img src="/files/ftp_upload/3792/3792fig1.jpg" align="Figure 1" /> Figura 1. Elettroporazione successo di plasmide reporter GFP risultati positivi. Pollo embrionale retine sono state iniettate ed elettroporati al giorno embrionale 4 e raccolte al giorno embrionale 6. Almeno il 25% della retina con successo trasfettata (A = Vista dall&#39;alto, B= Vista dal basso). Scale Bar = 1mm. <img src="/files/ftp_upload/3792/3792fig2.jpg" align="Figure 2" /> Figura 2. Caratterizzazione di GFP esprimere cellule della retina mediante immunoistochimica. GFP esprimere tessuti retinici in fase E7 sono stati fissati e sezionati. Queste sezioni sono state poi colorate con vari marcatori cellulari specifici. Brn3a è stato utilizzato per determinare cellule gangliari (A), mentre Pax6 è stato utilizzato per determinare orizzontali, amacrine e le cellule gangliari (B). Scala bar = 50 micron.

<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>

Tutti gli esperimenti sugli animali sono stati approvati dal Animal Care Comitato Istituzionale e Servizi alla Rutgers University.

L&#39;iniezione mirata della retina e in elettroporazione ovo in retina embrionale allo stadio E4 possono specificatamente colpire le cellule progenitrici della retina conseguente la capacità di visualizzare tutti e sei i principali tipi di cellule della retina a livello di singola cellula. In elettroporazione ovo a HH10 (~ E1.5) rivolte alla vescicola ottica è in grado di trasfettare cellule che si sviluppano a formare l&#39;occhio. Tuttavia, queste cellule hanno un fatturato molto...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> Fecondate esenti da organismi patogeni (SPF) Leghorn Bianco di pollo uova </td><td> Farms Sunrise (Catskill, NY) </td><td></td><td></td></tr><tr><td> Incubatrice per uova di pollo </td><td> GQF produzione, Savannah, GA </td><td> Modello-1550 </td><td> Imposta al 60% di umidità e 37,5 ° C. </td></tr><tr><td> Tubi di vetro capillari </td><td> Mondiale Precision Instrument Inc. </td><td> TW150F-4 </td><td></td></tr><tr><td> 0,1 ml siringa </td><td> Hamilton Co. Reno, Nevada </td><td> A tenuta di gas 1710 </td><td> Siringa alternativa 1ml può essere utilizzato come pure </td></tr><tr><td> Masterflex Silicone (perossido) Tubi </td><td> Cole-Parmer </td><td> HV-96400-13 </td&gt; <td> Tagliare un pezzetto (1 cm) per fissare l&#39;ago alla siringa di vetro </td></tr><tr><td> Pinzetta </td><td> Dumont </td><td> AA </td><td></td></tr><tr><td> Micromanipolatore </td><td> Mondiale Precision Instrument Inc. </td><td> M3301-M3 </td><td></td></tr><tr><td> DNA plasmidico </td><td></td><td></td><td> pCAG-GFP è stato preso in prestito dal Dr. Connie Cepko </td></tr><tr><td> Verde fast FCF </td><td> Sigma-Aldrich </td><td> F7252 </td><td> Diluire al 0,025% con PBS </td></tr><tr><td> Generatore di impulsi </td><td> Harvard Apparatus, MA </td><td> BTX ECM 830 </td><td> Generatore di onda quadra </td></tr><tr><td> Elettrodi </td><td> Harvard Apparatus, MA </td><td> BTX modello 514 </td><td> I nostri elettrodi sono stati distanziati 3-5 mm l&#39;uno dall&#39;altro </td></tr><tr><td> Fotocamera digitale monocromatica </td><td> Zeiss, Germania </td><td> Axiocam MRM </td><td></td></tr><tr><td> Dissezione microscopio a fluorescenza </td><td> Leica Microsystems, Germania </td><td> Leica MZ16FA </td><td></td></tr><tr><td> Upright microscopio a fluorescenza </td><td> Zeiss, Germania </td><td> Zeiss Axio Imager A1 </td><td></td></tr></tbody></table>

in ovo electroporation in embryonic chick retina

Retina embrionale di pollo è un ottimo strumento per studiare lo sviluppo della retina nei vertebrati superiori. Causa delle grandi dimensioni e sviluppo esterna, è comparativamente molto facile manipolare la retina embrionale pulcino usando ricombinante DNA / tecnologia RNA. Elettroporazione del DNA / RNA costrutti nella retina embrionale hanno un grande vantaggio per lo studio della retina regolazione genica in cellule staminali / progenitrici durante lo sviluppo della retina. Diverso tipo di saggi quali saggio gene reporter, genica sovra-espressione, gene abbattere (shRNA) ecc può essere eseguita utilizzando la tecnica elettroporazione. Questo video dimostra l&#39;iniezione mirata della retina e in elettroporazione ovo nella retina pulcino embrionale alla Hamburger e Hamilton fase di 22-23, che è di circa 4 giorni embrionale (E4). Qui mostriamo una rapida e conveniente in tecnica elettroporazione ovo quale un DNA plasmide che esprime proteina verde fluorescente (GFP) come marcatore viene consegnato direttamente nelpulcino embrionale spazio sottoretinico e seguiti da impulsi elettrici per facilitare l&#39;assorbimento del DNA da parte della retina cellule staminali / progenitrici. Il nuovo metodo di iniezione retinica ed elettroporazione presso E4 permette la visualizzazione di tutti i tipi di cellule retiniche, compreso il tardo-nato neuroni 1, che è stato difficile con il metodo convenzionale di iniezione ed elettroporazione a 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

L&#39;obiettivo generale di questo video è quello di mostrare come eseguire l&#39;iniezione mirata della retina e<em&gt; In ovo</emElettroporazione&gt; del DNA / RNA costrutti nella retina pulcino embrionale alla Hamburger e lo stadio Hamilton 22-23, che è di circa 4 giorni embrionale (E4). Questa tecnica è molto utile per studio dell&#39;espressione genica, regolazione genica, e cambiamento morfologico in sviluppo retina pulcino.

In elettroporazione Ovo in Retina Chick Embryonic

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

in-ovo-electroporation-in-embryonic-chick-retina

Research

JoVE Journal

Biology

In Ovo Electroporation in Embryonic Chick Retina

14.3K Views.  University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School. L'obiettivo generale di questo video è quello di mostrare come eseguire l'iniezione mirata della retina e In ovo del DNA / RNA costrutti nella retina pulcino embrionale alla Hamburger e lo stadio Hamilton 22-23, che è di circa 4 giorni embrionale (E4). Questa tecnica è molto utile per studio dell'espressione genica, regolazione genica, e cambiamento morfologico in sviluppo retina pulcino.

Video: In Ovo Electroporation in Embryonic Chick Retina

In Ovo Electroporations of HH Stage 10 Chicken Embryos

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of molecular biological techniques, the chick has become a useful system in which to study gene regulation and function.  By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function.  Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements.  Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive.  This video demonstrates first how to make a window in the eggshell to manipulate the embryo.  Next, the embryo is visualized with a dilute solution of India ink injected below the embryo.  A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator.  Finally, the egg is sealed with tape and placed back into an incubator for further development.  Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.

Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo. Common applications of this technique include functional analysis of genes and putative enhancer elements. This video demonstrates neural tube electroporation in HH 10 chick embryos. Injection technique and proper egg handling are discussed.

In Electroporations Ovo della Fase HH 10 embrioni di pollo

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of ...

Ricerca

Biologia

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

Metodo per la colorazione intero anticorpi Monte a Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

Test di induzione neurale nell&#39;embrione Chick

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.
For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used1-4, no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay5,6, (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye7-9, (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM10-12, (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice13 as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM14.
In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications. 
Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study.

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

The chick chorioallantoic membrane (CAM) is a unique, naturally immunodeficient supportive culture environment to study angiogenesis and tumorigenesis. This video article demonstrates the different steps in chick ex ovo culture, application of potentially angiogenic substances and successful inoculation of tumor cells and tissues on the surface of the CAM.

Pulcino Ex ovo Cultura e Ex ovo CAM saggio: Come funziona veramente

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

Preparazione di embrioni per la microscopia elettronica del Drosophila Tubo cuore embrionale

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

Cultura primaria e elettroporazione plasmidi dell&#39;Organo murini di Corti.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking axonal projection of spinal interneurons in vertebrates, germ line-targeted reporter genes yield bilaterally symmetric labeling. Therefore, it is hard to distinguish between the ipsi- and contra-laterally projecting axons. Unilateral electroporation into the chick neural tube provides a useful means to restrict expression of a reporter gene to one side of the central nervous system, and to follow axonal projection on both sides 1 ,2-5. This video demonstrates first how to handle the eggs prior to injection. At HH stage 18-20, DNA is injected into the sacral level of the neural tube, then tungsten electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator. The egg is sealed with tape and placed back into an incubator for further development. Three days later (E6) the spinal cord is removed as an open book preparation from embryo, fixed, and processed for whole mount antibody staining. The stained spinal cord is mounted on slide and visualized using confocal microscopy.

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

This video demonstrates how to visualize axonal pathways of genetically defined groups of neurons in the embryonic chick spinal cord utilizing in ovo electroporation of reporter genes under the control of specific enhancer elements.

Decifrare Percorsi assonale di gruppi geneticamente definito di neuroni nel tubo neurale Chick Utilizzando In ovo Elettroporazione

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Video Bioinformatica Analisi delle embrionali umane crescita Colony cellule staminali

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.

Cella Transfection singola in embrioni di pollo

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

In elettroporazione in vivo dei Morpholinos nella retina Zebrafish per adulti

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

Methods Article

In elettroporazione Ovo in Retina Chick Embryonic