Questo lavoro è stato supportato anche da finanziamenti della Fondazione per la Ricerca Sordità al Yc S e la NSF (IOS 0.930.096) alla KDB. KFB: NIH / NINDCD 2 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. Fare Elettrodi per elettroporazione <ol><li> Tagliare 75 micron diametro del filo di tungsteno (AM Systems, Inc. Carlsborg, WA) di lunghezza adeguata (circa 3-5 cm) </li><li> Mettere filo di tungsteno attraverso molex cavo connettore (pin ottone stampato) </li><li> Avvolgere i fili di tungsteno e il connettore del cavo molex con guaina termorestringente (SPC Technology, Chicago, IL) e al calore di becco Bunsen o pistola ad aria calda per sigillare il tubo riducendo al filo </li><li> Per affilare la punta del filo di tungsteno, immergere il filo in idrossido di sodio 1,0 N (Conrad et al. 1993) e, usando una graffetta come l&#39;altro elettrodo, elettrolizzare il filo con un Elettroporatore onda quadra. Le condizioni che usiamo sono 5 da 50 Volt impulsi di ciascuna della durata di 100 ms con 50 ms tra gli impulsi. Elettrodi deve essere affilato ad un diametro di 15-20 micron </li><li> Tenere i fili in un vassoio all&#39;interno di scanalature premuto in plastilina prima dell&#39;uso in elettroporazione </li></ol> 2. Configurazione Stazione di elettroporazione <ol><li> Nastro affilato 75μm elettrodi di tungsteno di micromanipolatori (strumenti di precisione World). </li><li> Micromanipolatori posto su entrambi i lati del palco microscopio da dissezione (Leica). </li><li> Collegare gli elettrodi ad un cuy-21 Proteggere Elettroporatore Modifica Onda quadra (Figura 1). </li><li> Accendere il elettroporatore e impostare i parametri per l&#39;elettroporazione, tra tensione, durata dell&#39;impulso e il numero di impulsi. I parametri più efficaci per questo esperimento sono risultati essere tre 13-volt impulsi che ogni durato 5.0ms, con 100 ms tra ogni impulso. Se elettroporazione crea bolle d&#39;aria nell&#39;embrione, ridurre la lunghezza degli impulsi. </li></ol> 3. Microiniezione di Morpholinos Into Zebrafish vescicole Otic <ol><li> Zebrafish embrioni raccolta da un serbatoio di allevamento. Incubare gli embrioni in embrione di sensibilizzazione di media con 0,3 PPM blu di metilene (Westerfield, 2005) a 28,5 ° C durante la notte. </li><li> Tirare aghi di vetro con un estrattore Sutter P-97 elettrodi </li><li> Caldo di un gel di base (1% agarosio in acqua i pesci in un piatto da mm 10 Petri) a 37 ° C in un bagno d&#39;acqua </li><li> Riscaldare 1% agarosio a basso punto di fusione (LMPA) in acqua i pesci fino al fuso e tenerlo caldo nel bagno d&#39;acqua a 37 ° C. </li><li> Riempire un ago di vetro con una soluzione morfolino (GeneTools, Corvallis, OR) e tagliare la punta di diametro adeguato. Montare l&#39;ago per l&#39;iniezione su un micromanipolatore (Kuhn). </li><li> Taglio punta dell&#39;ago a circa 10 micron ed iniettarlo in olio minerale per garantire che la punta permette di consegna sufficiente di soluzione morfolino. </li><li> Dechorinate embrioni postfertilization a 24 ore (HPF) e anestetizzare con MS222 (tricaine, Sigma) in 0,3 PPM acqua di metilene di pesce azzurro. </li><li> Allinea 3-5 embrioni anestetizzato sulla base di gel con il lato destro per comodo analisi uniforme </li><li> Mettete 1-2 gocce di 1% LMPA su ogni embrione e lasciate solidificare per fissare l&#39;embrione in posizione </li><li> Ruotare la piastra Petri in modo che la vescicola otica si trova sul lato destro </li><li> Posizionare l&#39;ago nel lume della vescicola otica e iniettare la soluzione morfolino con un pneumatico PV820 PicoPump (Strumenti di precisione mondiale) con un serbatoio di azoto allegato (Figura 2) </li></ol> 4. Procedura di elettroporazione <ol><li> Spostare gli embrioni montato la stazione elettroporazione immediatamente dopo l&#39;iniezione </li><li> Posizionare l&#39;elettrodo positivo sul tessuto appena posteriormente alla vescicola dell&#39;orecchio, ma non penetrano; inserire l&#39;elettrodo negativo nel cervello appena anteriormente alla vescicola otica </li><li> Applicare corrente utilizzando un pedale o premendo un interruttore. </li><li> Versare l&#39;acqua i pesci su agarosio e rimuovere gli embrioni di agarosio, rimestando e una pipetta di vetro. Fare attenzione a non danneggiare gli embrioni. Per gli embrioni che sono difficili da rimuovere, utilizzare un ago per rompere delicatamente via qualsiasi LMPA che circonda l&#39;embrione. </li><li> Embrioni posto in un piatto di pesce con l&#39;acqua di blu di metilene ed aumentare il embrioni a stadi desiderato per l&#39;analisi (morfologica, immunoistochimica, ibridazione in situ anlaysis, etc e microscopici). </li></ol> 5. Rappresentante dei risultati: <img src="/files/ftp_upload/2466/2466fig1.jpg" alt="Figura 1" /> Figura 1. Stazione di elettroporazione. Elettrodi sono stati realizzati con fili di tungsteno affilati 75μm e collegati da porta ad un cuy-21 onda Proteggere Modifica Piazza Elettroporatore. Questi elettrodi sono state registrate a micromanipolatori. <img src="/files/ftp_upload/2466/2466fig2.jpg" alt="Figura 2" /> Figura 2. Stazione di iniezione. Tutti gli embrioni sono stati iniettati nel orecchio destro a fini di normalizzazione. <img src="/files/ftp_upload/2466/2466fig3.jpg" alt="Figura 3" /> Figura 3. Colorazione falloidina di filamenti di actina con 3 embrioni dpf. (A) L&#39;embrione injecte 3 dpfd con controllo MO ha colorazione forte falloidina nella macula posteriore (pm). (B) L&#39;embrione che è stato iniettato con il controllo MO e poi elettroporate avevano livelli simili di colorazione in falloidina pm negli embrioni di iniezione unica. (C) Iniezione con MOS mif nella vescicola otica da solo non ha causato la riduzione di colorazione falloidina, mentre l&#39;iniezione di Mos mif con elettroporazione causato una drastica riduzione di colorazione falloidina nel pm (D). Sono, macula anteriore. <img src="/files/ftp_upload/2466/2466fig4.jpg" alt="Figura 4" /> Figura 4. Colorazione falloidina di filamenti di actina con 5 larve dpf. Non c&#39;era alcuna differenza tra l&#39;iniezione solo (A) e iniezione con elettroporazione, quando lo standard di controllo morfolino è stato utilizzato (B). Tuttavia, i 5 larve dpf che sono stati iniettati con mif Morpholinos e elettroporate hanno evidenziato una ridotta colorazione falloidina nella macula posteriore (pm) (D), anche se meno drammatico di 3 dpf, se confrontato con l&#39;embrione che è stato iniettato con mif Morpholinos solo (C ). Sono, macula anteriore. <img src="/files/ftp_upload/2466/2466fig5.jpg" alt="Figura 5" /> Figura 5. Colorazione tubulina acetilato di embrioni a 3 dpf. L&#39;embrione in (A) non ha ricevuto alcuna iniezione o elettroporazione. L&#39;embrione in (B) è stata elettroporate ma non ha ricevuto l&#39;iniezione. Ricevere embrioni a iniezione e elettroporazione (C, D) con mif Morpholinos era visibilmente diminuito tubulina colorazione rispetto ai controlli. (Pm, macula posteriore). <img src="/files/ftp_upload/2466/2466fig6.jpg" alt="Figura 6" /> Figura 6. Colorazione tubulina acetilato di embrioni a 3 dpf con controllo morfolino. (A) degli embrioni, senza alcun trattamento ha mostrato una forte colorazione tubulina acetilata nella macula posteriore (pm) e innervazione estesa. (B) degli embrioni iniettati con controllo morfolino. (C) degli embrioni trattati con elettroporazione solo. (D) embrione di controllo con il controllo morfolino iniettato e elettroporate ha livelli simili di tubulina acetilata nella macula posteriore e innervazione rispetto al non trattamento di controllo.

<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 produzione di questo articolo è stato sponsorizzato da Strumenti Gene, LLC che producono reagenti citati nel video e testo.

Noi abbiamo introdotto con successo MOS oligonucleotide antisenso nell&#39;orecchio dell&#39;embrione 24 zebrafish HPF. Embrioni che sono state sollevate dopo l&#39;iniezione ed elettroporazione sono stati vitali. Se gli embrioni sono sopravvissuti l&#39;elettroporazione, sono cresciuti a tassi normali rispetto agli embrioni che avevano ricevuto alcun trattamento, così come gli embrioni che erano stati soltanto iniettato e gli embrioni che era stato solo elettroporate. Abbiamo osservato gli effetti della commissione interbancaria multilaterale e mif-MOS, come dopo l&#39;iniezione orecchio ed elettroporazione attraverso l&#39;etichettatura con falloidina e tubulina acetilata. Colorazione falloidina di filamenti di actina in cellule ciliate sensoriali della macula posteriore indica che l&#39;elettroporazione di mif e mif-come MO al 24-HPF stadio causato cambiamenti nella cella dei capelli e / o numeri stereocilia rispetto agli embrioni che erano solo iniettato il MOS (Figura 3). Questa diminuzione del numero di cellule dei capelli è coerente con i risultati di Shen et al. (Inviato) che MOS a mif e mif simile a causare una riduzione del numero di cellule ciliate nella macula sacculare. La diminuzione del numero di cellule dei capelli in embrioni che sono stati iniettati con MOS e elettroporate dimostrare che elettroporazione può aiutare nello spostamento del MOS attraverso la membrana cellulare, aumentando così la portata degli effetti morfologici rispetto agli embrioni in cui la vescicola otica era solo iniettato. I cambiamenti nella morfologia del ganglio statoacoustic sono stati osservati attraverso la colorazione tubulina acetilata. Il grado di innervazione del ganglio statoacoustic ha risentito, come gli embrioni che sono stati iniettati e elettroporate mostrano diminuzione ramificazione dei neuriti (Figura 5). Ci possono anche essere ridotta di condensazione delle cellule gangliari e / o riduzione del numero di cellule gangliari, perché la colorazione tubulina acetilata è significativamente diminuito negli embrioni che sono stati entrambi iniettato e elettroporate. Perché mif ha dimostrato di essere fondamentale per lo sviluppo del sistema nervoso (Suzuki et al., 2004), i cambiamenti osservati dopo l&#39;elettroporazione può essere il risultato di trasfezione aumentato del MIF e mif-come MO soluzione nelle cellule neuroblasti. Elettroporazione di oligo antisenso Morpholinos direttamente in un tessuto di sviluppo è uno strumento utile per controllare l&#39;espressione di un gene durante lo sviluppo del tessuto che, sia spazialmente e temporalmente, nell&#39;embrione. Elettroporazione di mif e mif-MOS, come nei tessuti dell&#39;orecchio interno portato a morfologia anormale sia della macula posteriore e il ganglio statoacoustic. C&#39;è stata una riduzione del numero delle cellule ciliate sensoriali nella macula posteriore negli embrioni che erano stati iniettati e elettroporate rispetto a embrioni che non aveva ricevuto alcun trattamento o con embrioni che erano stati o solo iniettata o solo elettroporate. C&#39;era anche una diminuzione del grado di innervazione della patch posteriore sensoriali dal ganglio statoacoustic e la dimensione del SAG. L&#39;elettroporazione è un metodo prezioso per trasfezione macromolecole nei tessuti specifici, con il vantaggio di osservare gli effetti primari di quel particolare DNA, l&#39;RNA, o MO nel tessuto desiderato e discriminante da questi effetti secondari su altri tessuti, compreso il cervello, cresta neurale e mesenchima periotica sullo sviluppo dell&#39;orecchio interno (Barald e Kelley, 2004).

<ol><li> MOS: 0,3 mm di una combinazione di per MIF, mif-come MO1 e MO2 mif-come è stato utilizzato. Mif MO è gratuita per il codone di inizio di mRNA zebrafish mif: 5&#39;-acatcggcatgactgcgacagagat-3 &#39;. Due MOS sono stati utilizzati per mif-like mif-come MO1 è gratuita per il sito traslazionale inizio:. 5&#39;-GTTTCTATATTTATGAACGGCATGA-3 &#39;, mentre mif-come MO2 derivato da AS sequenza al confine intron1/exon 2: 5&#39;-GATTCATCCTCTGAAGACGTAAGCC -3 &#39;. Controllo MO era la sequenza nucleotidica strapazzate da Strumenti Gene: 5&#39;-CCTCTTACCTCAGTTACAATTTATA-3 &#39;. </li><li> blu di metilene (0,3 ppm) in acqua i pesci </li><li> MS222 (tricaine, Sigma; 0,64 mm in acqua i pesci) </li><li> Basso punto di fusione agarosio (LMPA; Roche) </li><li> rosso fenolo (Sigma) </li><li> Anti-acetilato tubulina (Sigma) </li><li> Alexa Fluor ® 488 capra anti-topo secondo anticorpo (sonde molecolari) </li><li> Texas rosso-falloidina (sonde molecolari) </li></ol>

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

Negli ultimi anni, elettroporazione è diventata una tecnica popolare per la transfezione in vivo di DNA, RNA, e Morpholinos in vari tessuti, tra cui l&#39;occhio, il cervello, e somiti di zebrafish. Il vantaggio di elettroporazione rispetto ad altri metodi di manipolazione genetica è che i tessuti specifici possono essere mirati, sia spazialmente e temporalmente, per l&#39;introduzione di macromolecole con l&#39;applicazione di corrente elettrica. Qui si descrive l&#39;utilizzo di elettroporazione per la trasfezione mif e mif-come Morpholinos nei tessuti dell&#39;orecchio interno lo sviluppo del pesce zebra. In studi precedenti, mif morfolino iniettate in embrioni a 1 - a stadio di 8 cellule portato a cambiamenti morfologici diffusi nel sistema nervoso e degli occhi, così come l&#39;orecchio. Prendendo di mira i tessuti dell&#39;orecchio interno nelle fasi successive dello sviluppo, possiamo determinare gli effetti primari di MIF nell&#39;orecchio sviluppo interno, al contrario di effetti secondari che possono derivare da l&#39;influenza di altri tessuti. Utilizzando tubulin falloidina e acetylated colorazione per studiare la morfologia dei neuroni, processi neuronali e cellule ciliate associati con la macula posteriore, siamo stati in grado di valutare l&#39;efficacia di elettroporazione come metodo per la trasfezione mirati nell&#39;orecchio zebrafish interno. Le vescicole otic di embrioni 24hpf sono stati iniettati con Morpholinos e elettroporate e sono stati poi confrontati con embrioni che avevano ricevuto alcun trattamento o erano stati solo iniettato o elettroporate. Embrioni che sono stati iniettati e elettroporate ha mostrato una diminuzione del numero delle cellule dei capelli, diminuzione della innervazione dal ganglio statoacoustic (SAG) e neuroni SAG meno rispetto ai gruppi di controllo. I nostri risultati hanno mostrato che la consegna diretta dei Morpholinos in otocysts nelle fasi successive evita la non specifico del sistema nervoso e gli effetti della cresta neurale Morpholinos consegnato in fase di 1-8 cellule. Consente inoltre l&#39;esame degli effetti che sono diretti per l&#39;orecchio interno e non effetti secondari su un orecchio da effetti primari sul cervello, cresta neurale o mesenchima periotica.

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

Un metodo per fornire Morpholinos direttamente nel otocyst pesce zebra a 24hpf è stato sviluppato. Mediante microiniezione di Morpholinos nel lume del vescicola otica ed elettroporazione di effettuare la penetrazione, siamo stati in grado di bypassare l&#39;effetto di Morpholinos sul cervello e ottenere effetti specifici per l&#39;orecchio interno.

Consegna diretta di Morpholinos MIF Into the Otocyst Zebrafish di iniezione e di elettroporazione influenza lo sviluppo dell&#39;orecchio interno

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 Views.  University of Wisconsin, Madison. Un metodo per fornire Morpholinos direttamente nel otocyst pesce zebra a 24hpf è stato sviluppato. Mediante microiniezione di Morpholinos nel lume del vescicola otica ed elettroporazione di effettuare la penetrazione, siamo stati in grado di bypassare l'effetto di Morpholinos sul cervello e ottenere effetti specifici per l'orecchio interno.

Video: 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.

Dissezione lordo e Arti di epiteli sensoriali dell&#39;orecchio interno in adulti 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 ...

Ricerca

Biologia

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 ...

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.

Iniezione intraperitoneale in Zebrafish adulti

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.

L&#39;interferenza di RNAi per iniezione in dsRNA Drosophila Embrioni

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.

Elettroporazione di Mesenchima craniofacciale

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.

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 ...

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.

In elettroporazione in vivo di Morpholinos nella Adult Regenerating 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.

Il trasferimento genico in embrioni di pollo da anziani<em&gt; Ex ovo</em&gt; Elettroporazione

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.

Uno studio comparato Drug metodi di consegna mirate per l&#39;orecchio interno mouse: Bullostomy<em&gt; Versus</em&gt; Iniezione transtimpanica

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.

Nuovo metodo di consegna plasmide DNA Urothelium della vescica del Mouse mediante elettroporazione

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