Ce travail a été soutenu par des subventions de la Fondation de recherche sur la surdité à Yc S et la NSF (IOS 0930096) à la KFB. KFB: NIH / 2 NINDCD RO1 DC04184, <a href="https://commons.era.nih.gov/commons/genericStatus.do?actionRole=nonPI&amp;applID=7854446&amp;uhf-token=Ugf7JvudZTCbTUW9jSAR1snMUgg%3D">3R01DC004184-08W1</a> , NSF DBI 0832862, 0930096 NSF IOS

1. Fabrication d&#39;électrodes pour l&#39;électroporation <ol><li> Couper 75 fils de tungstène um de diamètre (PM Systems, Inc Carlsborg, WA) de longueur appropriée (environ 3-5 cm) </li><li> Passez le fil de tungstène grâce molex câble connecteur (broche en laiton estampé) </li><li> Envelopper et des fils de tungstène connecteur du câble molex avec gaine thermorétractable (CPS Technology, Chicago, IL) et appliquer de la chaleur avec brûleur Bunsen ou pistolet à air chaud pour sceller le tube rétrécit au fil </li><li> Pour affûter la pointe de fil de tungstène, tremper le fil dans l&#39;hydroxyde de sodium 1,0 N (Conrad et al. 1993) et, à l&#39;aide d&#39;un trombone que l&#39;autre électrode, électrolyse le fil avec un Electroporateur onde carrée. Les conditions que nous utilisons sont de 5 à 50 volts impulsions d&#39;une durée de 100 ms avec 50 ms entre les impulsions. Les électrodes doivent être affûtées à un diamètre de 15 à 20 um </li><li> Gardez les fils dans un plateau dans les rainures enfoncé dans la pâte à modeler avant l&#39;utilisation dans l&#39;électroporation </li></ol> 2. Set Up station électroporation <ol><li> Ruban aiguisé 75μm électrodes de tungstène pour micromanipulateurs (instruments de précision du monde). </li><li> Micromanipulateurs place de chaque côté de la platine du microscope disséquant (Leica). </li><li> Brancher les électrodes à un Protéger CUY-21 Electroporateur Modifier Place des vagues (figure 1). </li><li> Allumez le électroporateur et mettre en place des paramètres pour l&#39;électroporation, y compris la tension, durée d&#39;impulsion, et le nombre d&#39;impulsions. Les paramètres les plus efficaces pour cette expérience ont été trouvés être de trois 13 volts impulsions qui ont duré chacune des 5.0ms, avec 100ms entre chaque impulsion. Si l&#39;électroporation crée des bulles d&#39;air dans l&#39;embryon, de réduire la durée d&#39;impulsion. </li></ol> 3. Microinjection d&#39;morpholinos Into vésicule otique Zebrafish <ol><li> Embryons de poissons zèbres récolte à partir d&#39;un réservoir d&#39;élevage. Incuber les embryons dans l&#39;embryon de conscience moyenne, avec 0,3 PPM méthylène bleu (Westerfield, 2005) à 28,5 ° C pendant la nuit. </li><li> Tirez aiguilles de verre avec un extracteur Sutter P-97 électrodes </li><li> Warm up une base de gel d&#39;agarose (1% d&#39;agarose dans de l&#39;eau du poisson dans un plat de Pétri de 10 mm) à 37 ° C dans un bain d&#39;eau </li><li> Chauffer 1% d&#39;agarose à faible point de fusion (EPMT) dans les poissons d&#39;eau jusqu&#39;à ce qu&#39;il fonde et le garder au chaud dans le bain à 37 ° C. </li><li> Remplir une aiguille de verre avec une solution morpholino (GeneTools, Corvallis, OR) et couper l&#39;extrémité d&#39;un diamètre approprié. Montez l&#39;aiguille d&#39;injection sur un micromanipulateur (Kuhn). </li><li> Couper pointe de l&#39;aiguille d&#39;environ 10 um et les injecter dans l&#39;huile minérale pour s&#39;assurer que la pointe permet une livraison suffisante de solution morpholino. </li><li> Dechorinate embryons à 24 heures post-fécondation (HPF) et les anesthésier avec MS222 (tricaïne, Sigma) dans 0,3 ppm d&#39;eau de méthylène poisson bleu. </li><li> Alignez 3 à 5 embryons anesthésiés sur la base de gel d&#39;agarose avec le côté droit pour analyse uniforme commodes </li><li> Mettre 1 à 2 gouttes d&#39;EPMT 1% sur chaque embryon et laissez solidifier à fixer l&#39;embryon en place </li><li> Tournez la boîte de Pétri de telle sorte que la vésicule otique est sur le côté droit </li><li> Placez l&#39;aiguille dans la lumière de la vésicule otique et injecter la solution avec un morpholino PicoPump PV820 pneumatique (instruments de précision du monde) avec un réservoir d&#39;azote fixé (figure 2) </li></ol> 4. Procédure d&#39;électroporation <ol><li> Déplacer les embryons montée à la station de électroporation immédiatement après l&#39;injection </li><li> Placer l&#39;électrode positive sur le tissu juste derrière la vésicule otique, mais ne pénètrent pas, insérer l&#39;électrode négative dans le cerveau juste en avant la vésicule otique </li><li> Appliquer en cours en utilisant une pédale ou en appuyant sur un interrupteur. </li><li> Versez de l&#39;eau du poisson sur agarose et retirez embryons à partir d&#39;agarose tourbillonnant et une pipette en verre. Faites preuve de prudence pour ne pas endommager l&#39;embryon. Pour les embryons qui sont difficiles à enlever, utiliser une aiguille doucement rompre toute EPMT entourant l&#39;embryon. </li><li> Embryons Placer dans un plat avec de l&#39;eau du poisson bleu de méthylène et d&#39;élever les embryons à des stades souhaités pour l&#39;analyse (morphologique, immunohistochimique, dans anlaysis hybridation, etc et microscopiques in situ). </li></ol> 5. Les résultats représentatifs: <img src="/files/ftp_upload/2466/2466fig1.jpg" alt="Figure 1" /> Figure 1. Station de l&#39;électroporation. Les électrodes ont été faites en utilisant des fils de tungstène affûté 75μm et reliés par un conduit Protéger CUY-21 Electroporateur Modifier onde carrée. Ces électrodes ont été enregistrées au micromanipulateurs. <img src="/files/ftp_upload/2466/2466fig2.jpg" alt="Figure 2" /> Figure 2. Station d&#39;injection. Tous les embryons ont été injectés dans l&#39;oreille droite à des fins de normalisation. <img src="/files/ftp_upload/2466/2466fig3.jpg" alt="Figure 3" /> Figure 3. Coloration phalloïdine des filaments d&#39;actine avec 3 embryons DPF. (A) L&#39;embryon injecte 3 dpfD avec contrôle MO a une forte coloration de la phalloïdine dans la macula postérieur (pm). (B) L&#39;embryon qui a été injecté avec un contrôle MO puis électroporées avaient des niveaux similaires de coloration phalloïdine dans h dans les embryons d&#39;injection seulement. (C) l&#39;injection avec MOS mif dans la vésicule otique seul ne cause pas de réduction de la coloration phalloïdine, tandis que l&#39;injection d&#39;OM mif avec électroporation a provoqué une réduction spectaculaire de la coloration de la phalloïdine h (D). h, la macula antérieure. <img src="/files/ftp_upload/2466/2466fig4.jpg" alt="Figure 4" /> Figure 4. Coloration phalloïdine des filaments d&#39;actine avec 5 larves DPF. Il n&#39;y avait aucune différence entre l&#39;injection seulement (A) et l&#39;injection de l&#39;électroporation, lorsque la norme de contrôle a été utilisé morpholino (B). Cependant, les larves 5 dpf qui ont été injectés avec mif morpholinos et électroporé montré réduite coloration phalloïdine dans la macula postérieure (h) (D), bien que moins spectaculaire que trois DPF, en comparaison avec l&#39;embryon qui a été injecté avec mif morpholinos seulement (C ). h, la macula antérieure. <img src="/files/ftp_upload/2466/2466fig5.jpg" alt="Figure 5" /> Figure 5. Coloration tubuline acétylée d&#39;embryons à 3 dpf. L&#39;embryon en (A) n&#39;a pas reçu d&#39;injection ou l&#39;électroporation. L&#39;embryon en (B) a été électroporé mais n&#39;a reçu aucune injection. Embryons recevoir l&#39;injection et l&#39;électroporation (C, D) avec mif morpholinos avaient visiblement coloration tubuline diminué par rapport aux contrôles. (H, macula postérieur). <img src="/files/ftp_upload/2466/2466fig6.jpg" alt="Figure 6" /> Figure 6. Coloration tubuline acétylée d&#39;embryons à 3 dpf avec contrôle morpholino. (A) Embryon sans aucun traitement a montré une forte coloration tubuline acétylée dans la macula postérieur (pm) et l&#39;innervation étendue. (B) avec contrôle de l&#39;embryon injecté morpholino. (C) Embryon traités avec électroporation seulement. (D) d&#39;embryon commande avec morpholino injecté et électroporées a des niveaux similaires de la tubuline acétylée dans la macula et l&#39;innervation postérieure par rapport au témoin un traitement non.

<ol>
	<li>Conrad, G. W., Bee, J. A., Roche, S. M., Teillet, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+microscalpels+by+electrolysis+of+tungsten+wire+in+a+meniscus.">Fabrication of microscalpels by electrolysis of tungsten wire in a meniscus.</a> J Neurosci Methods. 50, 123-127 (1993).</li><li>Suzuki, M., Takamura, Y., Maeno, M., Tochinai, S., Iyaguchi, D., Tanaka, I., Nishihira, J., Ishibashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+laevis+macrophage+migration+inhibitory+factor+is+essential+for+axis+formation+and+neural+development.">Xenopus laevis macrophage migration inhibitory factor is essential for axis formation and neural development.</a> J Biol Chem. 279, 21406-21414 (2004).</li><li>Barald, K. F., Kelley, M. W., W, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+placode+to+polarization:+new+tunes+in+inner+ear+development.">From placode to polarization: new tunes in inner ear development.</a> Development. 131, 4119-4135 (2004).</li></ol>

La production de l&#39;article a été parrainé par Gene Tools, LLC qui produisent les réactifs mentionnés dans la vidéo et du texte.

Nous avons introduit avec succès OM oligonucléotide antisens dans l&#39;oreille de l&#39;embryon de poisson zèbre 24 HPF. Les embryons qui ont été soulevées après l&#39;injection et l&#39;électroporation étaient viables. Si les embryons ont survécu à l&#39;électroporation, ils ont progressé à des taux normaux par rapport à des embryons qui n&#39;avaient reçu aucun traitement, ainsi que des embryons qui avaient seulement été injectés et les embryons qui avaient seulement été électroporé. Nous avons observé les effets de la MIF et MIF, comme OM après l&#39;injection d&#39;oreille et l&#39;électroporation grâce à l&#39;étiquetage et la tubuline avec la phalloïdine acétylés. Phalloïdine coloration des filaments d&#39;actine dans les cellules ciliées sensorielles de la macula postérieure indique que l&#39;électroporation de MIF et MIF tels que MO au stade 24-HPF a provoqué des changements dans la cellule des cheveux et / ou des numéros de stéréocils par rapport aux embryons qui ont été injectés avec la seule OM (Figure 3). Cette diminution du nombre de cellules ciliées est cohérent avec les conclusions de Shen et al. (Soumis) que OM à mif mif-like et de provoquer une réduction du nombre de cellules ciliées dans la macula sacculaire. La diminution du nombre de cellules de cheveux chez les embryons qui ont été injectés avec les OM et électroporées démontrer que l&#39;électroporation peut aider à déplacer l&#39;OM à travers la membrane cellulaire, ce qui augmente l&#39;étendue des effets morphologiques par rapport aux embryons dans lequel la vésicule otique était seulement injecté. Les changements dans la morphologie du ganglion statoacoustic ont été observées grâce à la tubuline acétylée coloration. L&#39;étendue de l&#39;innervation par le ganglion statoacoustic a été affectée, comme les embryons qui ont été injectés et électroporées montrent une diminution de branchement des neurites (figure 5). Il peut également être diminué de condensation des cellules ganglionnaires et / ou diminution du nombre de cellules ganglionnaires, parce que la coloration tubuline acétylée est significativement diminué chez les embryons qui ont été injectés à la fois et électroporé. Parce que MIF a été montré pour être critique pour le développement du système nerveux (Suzuki et al., 2004), les changements observés après l&#39;électroporation peut être le résultat de la transfection accrue de la MIF et MIF, comme solution de MO dans les cellules neuroblastes. L&#39;électroporation des antisens morpholinos oligo directement dans un tissu en développement est un outil utile pour contrôler l&#39;expression d&#39;un gène au cours du développement que le tissu, à la fois spatialement et temporellement, dans l&#39;embryon. L&#39;électroporation de MIF et MIF-like OM dans les tissus de l&#39;oreille interne conduit à une morphologie anormale des deux macula postérieur et le ganglion statoacoustic. Il y avait une réduction du nombre de cellules ciliées sensorielles dans la macula postérieure des embryons qui avaient été injectés et électroporées en comparaison avec les embryons qui n&#39;avaient reçu aucun traitement ou avec des embryons qui avaient été soit seulement injecté ou seulement électroporé. Il y avait aussi une diminution du degré d&#39;innervation du patch postérieure sensorielle par le ganglion statoacoustic et la taille de la SAG. L&#39;électroporation est une méthode précieuse pour la transfection des macromolécules dans des tissus spécifiques, avec l&#39;avantage d&#39;observer les effets primaires de l&#39;ADN notamment, de l&#39;ARN, ou MO dans le tissu désiré et discriminant de ces d&#39;effets secondaires sur d&#39;autres tissus, y compris le cerveau, de la crête neurale et des mésenchyme périotique sur le développement oreille interne (Barald et Kelley, 2004).

<ol><li> OM: 0,3 mm d&#39;une combinaison de des mif, mif-comme MO1 et MO2 mif-like a été utilisé. Mif MO est gratuit au codon d&#39;initiation de l&#39;ARNm de poisson zèbre MIF: 5&#39;-acatcggcatgactgcgacagagat-3 &#39;. Deux OM ont été utilisés pour mif mif-comme-comme MO1 est gratuit sur ​​le site de début de traduction:. 5&#39;-GTTTCTATATTTATGAACGGCATGA-3 &#39;, tandis que le MIF, comme MO2 dérivé de la séquence à l&#39;AS intron1/exon deux limites: 5&#39;-GATTCATCCTCTGAAGACGTAAGCC -3 &#39;. Contrôle MO a été la séquence nucléotidique du brouillés Outils Gene: 5&#39;-CCTCTTACCTCAGTTACAATTTATA-3 &#39;. </li><li> bleu de méthylène (0,3 ppm) dans les poissons d&#39;eau </li><li> MS222 (tricaïne, Sigma; 0,64 mM dans l&#39;eau du poisson) </li><li> Bas point de fusion d&#39;agarose (EPMT; Roche) </li><li> rouge de phénol (Sigma) </li><li> Anti-acétylés tubuline (Sigma) </li><li> Alexa Fluor ® 488 de chèvre anti-souris second anticorps (Molecular Probes) </li><li> Texas Red-phalloïdine (Molecular Probes) </li></ol>

direct delivery of mif morpholinos into the zebrafish otocyst by injection and electroporation affects inner ear development

Ces dernières années, l&#39;électroporation est devenue une technique populaire pour la transfection in vivo de l&#39;ADN, l&#39;ARN et morpholinos dans les tissus divers, y compris l&#39;œil, du cerveau et des somites poisson zèbre. L&#39;avantage de l&#39;électroporation rapport aux autres méthodes de manipulation génétique, c&#39;est que certains tissus peuvent être ciblées, à la fois spatialement et temporellement, pour l&#39;introduction de macromolécules par l&#39;application de courant électrique. Nous décrivons ici l&#39;utilisation de l&#39;électroporation pour la transfection et la mif mif-like morpholinos dans les tissus de l&#39;oreille interne de l&#39;élaboration du poisson zèbre. Dans les études antérieures, mif morpholino injectées dans des embryons à la une - au stade de 8 cellules a entraîné des modifications morphologiques très répandue dans le système nerveux et des yeux, ainsi que l&#39;oreille. En ciblant les tissus de l&#39;oreille interne à des stades ultérieurs de développement, nous pouvons déterminer les principaux effets de MIF dans l&#39;oreille de développement interne, par opposition aux effets secondaires qui peuvent résulter de l&#39;influence des autres tissus. En utilisant la tubuline acétylée phalloïdine et coloration pour étudier la morphologie des neurones, les processus neuronaux, et les cellules ciliées associées à la macula postérieure, nous avons été en mesure d&#39;évaluer l&#39;efficacité de l&#39;électroporation comme une méthode pour la transfection ciblée dans l&#39;oreille le poisson-zèbre intérieure. Les vésicules otiques d&#39;embryons ont été injectés avec 24hpf morpholinos et électroporé et ont ensuite été comparés aux embryons qui n&#39;avaient reçu aucun traitement ou avaient été injectés ou ne électroporé. Les embryons qui ont été injectés et électroporées ont montré une diminution du nombre de cellules de cheveux, une diminution de l&#39;innervation par le ganglion statoacoustic (SAG) et les neurones en moins par rapport SAG avec des groupes de contrôle. Nos résultats ont montré que la prestation directe de morpholinos en otocystes à des stades ultérieurs du système évite non spécifiques nerveux et les effets de la crête neurale morpholinos livré au stade 1-8 cellules. Il permet également l&#39;examen des effets qui sont dirigées vers l&#39;oreille interne et non pas des effets secondaires sur l&#39;oreille d&#39;effets primaires sur le cerveau, de la crête neurale ou mésenchyme périotique.

Watch this Scientific Journal Video about Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development at JoVE.com

Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development

Une méthode pour livrer morpholinos directement dans le poisson-zèbre à l&#39;otocyste 24hpf a été développé. L&#39;utilisation de micro-injection d&#39;morpholinos dans la lumière de la vésicule otique et l&#39;électroporation à effet de pénétration, nous avons été en mesure de contourner l&#39;effet de morpholinos sur le cerveau et obtenir des effets spécifiques de l&#39;oreille interne.

Livraison directe de morpholinos MIF Into the otocyste Zebrafish par injection et électroporation affecte le développement oreille interne

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the ...

direct-delivery-mif-morpholinos-into-zebrafish-otocyst-injection

Research

JoVE Journal

Biology

14.2K vues.  University of Wisconsin, Madison. Une méthode pour livrer morpholinos directement dans le poisson-zèbre à l'otocyste 24hpf a été développé. L'utilisation de micro-injection d'morpholinos dans la lumière de la vésicule otique et l'électroporation à effet de pénétration, nous avons été en mesure de contourner l'effet de morpholinos sur le cerveau et obtenir des effets spécifiques de l'oreille interne.

Vidéo: Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.
Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

Dissection fine et globale de l&#39;épithélium sensoriel de l&#39;oreille interne adultes poisson zèbre ( Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the ...

Recherche

Biologie

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.

Culture primaire et l&#39;électroporation plasmidique de l&#39;Organe de Corti murin.

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 ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

Injection intrapéritonéale en Zebrafish adultes

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

RNAi Interference by dsRNA Injection into Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.
Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.

Interférences ARNi par injection dans les ARNdb Drosophile Embryons

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

Electroporation of Craniofacial Mesenchyme

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse 1-10. However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme.
When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis 11-13. In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation.
Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers 14. Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (&tilde;100 million years closer) 15. Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.

Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo.

Electroporation de Mesenchyme craniofaciale

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

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.

En électroporation in vivo de morpholinos dans la rétine de poisson zèbre adulte

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

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

En électroporation in vivo de morpholinos dans la régénération des adultes Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos1. Different structures such as DNA plasmids encoding genes2-4, small interfering RNA (siRNA) plasmids5, small synthetic RNA oligos6, and morpholino antisense oligonucleotides7 can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20 - according to Hamburg and Hamilton)8 and there are some disadvantages for its application in embryos at later stages (older than stage HH22 - approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell.
In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes9 and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos10-12.

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.

Transfert de gènes dans des embryons de poulet âgés par<em&gt; Ex ovo</em&gt; Électroporation

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

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. The first procedure consists of perforation of the tympanic bulla, termed bullostomy; the second one is a transtympanic injection. Both emulate human clinical intratympanic procedures.
Chitosan-glycerophosphate (CGP) and Ringer&#180;s Lactate buffer (RL) were used as biocompatible vehicles for local drug delivery. CGP is a nontoxic biodegradable polymer widely used in pharmaceutical applications. It is a viscous liquid at RT&#160;but it congeals to a semi solid phase at body temperature. RL is an isotonic solution used for intravenous administrations in humans. A small volume of this vehicle is precisely placed on the Round Window (RW) niche by means of a bullostomy. A transtympanic injection fills the middle ear and allows less control but broader access to the inner ear.
The safety profiles of both techniques were studied and compared by using functional and morphological tests. Hearing was evaluated by registering the Auditory Brainstem Response (ABR) before and several times after microsurgery. The cytoarchitecture and preservation level of cochlear structures were studied by conventional histological techniques in paraformaldehyde-fixed and decalcified cochlear samples. In parallel, unfixed cochlear samples were taken and immediately frozen to analyze gene expression profiles of inflammatory markers by quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR).
Both procedures are suitable as drug delivery methods into the mouse middle ear, although transtympanic injection proved to be less invasive compared to bullostomy.

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

Two microsurgery approaches for local drug delivery to the inner ear are described here and compared in terms of impact on hearing parameters, cochlear cytoarchitecture and expression of inflammatory markers.

Une étude comparative des médicaments Méthodes de livraison ciblés à l&#39;oreille de la souris intérieure: Bullostomy<em&gt; Versus</em&gt; Transtympaniques Injection

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. ...

Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms of human diseases such as cancer. Transgenic and conditional knockout mice have been frequently used for gene overexpression or ablation in specific tissues or cell types in vivo. However, generating germline mouse models can be time-consuming and costly. Recent advancements in gene editing technologies and the feasibility of delivering DNA plasmids by viral infection have enabled rapid generation of non-germline autochthonous mouse cancer models for several organs. The bladder is an organ that has been difficult for viral vectors to access, due to the presence of a glycosaminoglycan layer covering the urothelium. Here, we describe a novel method developed in lab for efficient delivery of DNA plasmids into the mouse bladder urothelium in vivo. Through intravesical instillation of pCAG-GFP DNA plasmid and electroporation of surgically exposed bladder, we show that the DNA plasmid can be delivered specifically into the bladder urothelial cells for transient expression. Our method provides a fast and convenient way for overexpression and knockdown of genes in the mouse bladder, and can be applied to building GEMMs of bladder cancer and other urological diseases.

We describe a novel method for the delivery of DNA plasmid into the urothelial cells of mouse bladder in vivo through urethra catheterization and electroporation. It offers a fast and convenient way for generating autochthonous mouse models of bladder diseases.

Nouvelle méthode de livraison ADN plasmidique aux souris vessie urothélium par électroporation

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms ...