Diese Arbeit wurde durch Zuschüsse der Taubheit Research Foundation zu Yc S und der NSF (IOS 0930096) zu KFB unterstützt. 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, NSF IOS 0930096

1. Herstellung von Elektroden für die Elektroporation <ol><li> Cut 75 um Durchmesser Wolframdraht (AM Systems, Inc. Carlsborg, WA) von geeigneter Länge (ca. 3-5 cm) </li><li> Put Wolframdraht durch Molex Stecker (Pin gestempelt Messing) </li><li> Wrap Wolframdrähten und Molex Stecker mit Schrumpfschlauch (SPC Technology, Chicago, IL) und erwärmt mit Bunsenbrenner oder Heißluftpistole zum Schrumpfen Schlauch an den Drahtplombe </li><li> Zum Schärfen der Spitze der Wolfram-Draht-, Tauch-den Draht in 1,0 N Natriumhydroxid (Conrad et al. 1993) und mit Hilfe einer Büroklammer als die andere Elektrode, Elektrolyse der Draht mit einer Rechteck Electroporator. Die Bedingungen, die wir benutzen sind 5 50-Volt Impulse einer Dauer von jeweils 100 ms mit 50 ms in zwischen den Impulsen. Die Elektroden sollten zu einem Durchmesser von 15-20 um geschärft werden </li><li> Halten Sie die Drähte in einem Fach in Nuten in Modelliermasse vor in Elektroporation verwenden gedrückt </li></ol> 2. Set Up Elektroporation Bahnhof <ol><li> Band geschärft 75 um Wolfram Elektroden Mikromanipulatoren (World Precision Instruments). </li><li> Legen Sie Mikromanipulatoren auf beiden Seiten des Präpariermikroskop Stufe (Leica). </li><li> Verbinden Sie die Elektroden mit einem Protect CUY-21 Bearbeiten Square Wave Electroporator (Abbildung 1). </li><li> Schalten Sie den Elektroporator und SET UP-Parameter für die Elektroporation, einschließlich Spannung, Pulsdauer und die Anzahl der Impulse. Die effektivste Parameter für dieses Experiment wurde festgestellt, dass drei 13-Volt-Impulse, die jeweils für 5.0ms dauerte, mit 100ms zwischen jedem Puls werden. Wenn Elektroporation erzeugt Luftblasen im Embryo, reduzieren Sie die Impulslänge. </li></ol> 3. Mikroinjektion von Morpholinos in Zebrafische Otic Vesicle <ol><li> Ernte Zebrafischembryonen aus einem Aufzuchtbecken. Inkubieren Sie die Embryonen in Embryo-raising-Medium mit 0,3 ppm Methylenblau (Westerfield, 2005) bei 28,5 ° C über Nacht. </li><li> Pull Glasnadeln mit einem Sutter P-97-Elektrode puller </li><li> Warm up ein Agarosegel Basis (1% Agarose in Fisch Wasser in einer 10 mm Petrischale) auf 37 ° C im Wasserbad </li><li> Heat 1% Agarose für niedrigen Schmelzpunkt (LMPA) in Fisch Wasser, bis sie geschmolzen und warm halten in der 37 ° C Wasserbad. </li><li> Füllen Sie ein Glas Nadel mit einem Morpholino-Lösung (GeneTools, Corvallis, OR) und schneiden Sie die Spitze zu entsprechenden Durchmesser. Montieren Sie die Injektionsnadel auf einem Mikromanipulator (Kuhn). </li><li> Cut Nadelspitze zu etwa 10 um und injizieren in Mineralöl, um sicherzustellen, dass die Spitze reicht Lieferung von Morpholino-Lösung ermöglicht. </li><li> Dechorinate Embryonen nach 24 Stunden der Befruchtung (HPF) und betäuben sie mit MS222 (Tricaine, Sigma) in 0,3 ppm Methylenblau Fisch Wasser. </li><li> Richten Sie 3 bis 5 betäubt Embryonen auf das Agarosegel Basis mit der rechten Seite bis zur komfortablen einheitliche Analyse </li><li> Setzen Sie 1 bis 2 Tropfen 1% LMPA über jeden Embryo und lassen Sie sich verfestigen, um den Embryo zu fixieren </li><li> Drehen Sie die Petrischale, so dass die otic Vesikel ist auf der rechten Seite </li><li> Setzen Sie die Nadel in das Lumen des otic Vesikel und injizieren Sie die Morpholino-Lösung mit einem PV820 Pneumatische PicoPump (World Precision Instruments) mit einer angehängten Stickstofftank (Abbildung 2) </li></ol> 4. Elektroporation Procedure <ol><li> Bewegen Sie den montierten Embryonen in die Elektroporation Station unmittelbar nach der Injektion </li><li> Legen Sie die positive Elektrode auf das Gewebe direkt hinter dem Ohr-Vesikel, aber nicht durchdringen; legen Sie die negative Elektrode in das Gehirn unmittelbar vor dem otic Vesikel </li><li> Wenden Sie muss mit einem Fußpedal oder durch Drücken eines Schalters. </li><li> Gießen Fisch Wasser auf Agarose und entfernen Embryonen aus Agarose mit wirbelnden und einer Glaspipette. Seien Sie vorsichtig, nicht auf die Embryonen Schaden. Bei Embryonen, die nur schwer zu entfernen sind, mit einer Nadel vorsichtig brechen alle LMPA Umgebung des Embryos. </li><li> Legen Embryonen in einem Teller mit Methylenblau Fisch Wasser und erhöhen die Embryonen zu wünschen Stadien für die Analyse (morphologische, immunhistochemische, in-situ-Hybridisierung, etc und mikroskopische anlaysis). </li></ol> 5. Repräsentative Ergebnisse: <img src="/files/ftp_upload/2466/2466fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Elektroporation Station. Elektroden wurden mit geschärften 75 Mikrometern Wolframdrähte und verbunden durch führt zu einer Protect CUY-21 Bearbeiten Square Wave Electroporator. Diese Elektroden wurden Mikromanipulatoren abgeklebt. <img src="/files/ftp_upload/2466/2466fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Injection-Station. Alle Embryonen wurden in das rechte Ohr für die Standardisierung Zwecke injiziert. <img src="/files/ftp_upload/2466/2466fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Phalloidin-Färbung von Aktin-Filamenten mit 3 dpf Embryonen. (A) Die 3 dpf Embryo injected mit Kontroll-MO hat eine starke Färbung von Phalloidin in der hinteren Makula (pm). (B) Der Embryo, die mit Kontroll-MO injiziert wurde und dann elektroporiert hatte ähnliches Niveau Phalloidin-Färbung in pm in der Injektion nur Embryonen. (C) Injektion mit mif MOs in die otic Vesikel allein nicht die Ursache für Reduzierung der Phalloidin-Färbung, während die Injektion von mif MOs zusammen mit Elektroporation führte zu einer dramatischen Reduzierung der Phalloidin-Färbung in der Uhr (D). bin, anterior Makula. <img src="/files/ftp_upload/2466/2466fig4.jpg" alt="Abbildung 4" /> Abbildung 4. Phalloidin-Färbung von Aktin-Filamenten mit 5 dpf Larven. Es gab keinen Unterschied zwischen Injektion nur (A) und Injektion mit Elektroporation, wenn die Standard-Steuerung Morpholino verwendet wurde (B). Jedoch zeigte die 5 dpf Larven, die mit mif Morpholinos und elektroporiert injiziert wurden Phalloidin-Färbung in der hinteren Makula (pm) (D), wenn auch weniger dramatisch als 3 dpf, wenn sie mit dem Embryo, der mit mif Morpholinos nur (C injiziert wurde im Vergleich reduziert ). bin, anterior Makula. <img src="/files/ftp_upload/2466/2466fig5.jpg" alt="Abbildung 5" /> Abbildung 5. Acetylierte Tubulin-Färbung von Embryonen bei 3 dpf. Der Embryo in (A) erhielten keine Injektion oder Elektroporation. Der Embryo in (B) wurde durch Elektroporation, erhielt aber keine Injektion. Embryonen erhalten sowohl der Injektion und Elektroporation (C, D) mit mif Morpholinos hatte sichtlich abgenommen Tubulin-Färbung im Vergleich zur Kontrollgruppe. (Pm, posterior Makula). <img src="/files/ftp_upload/2466/2466fig6.jpg" alt="Abbildung 6" /> Abbildung 6. Acetylierte Tubulin-Färbung von Embryonen bei 3 dpf mit Kontroll-Morpholino. (A) Embryo ohne Behandlung zeigte eine starke acetyliertes Tubulin-Färbung in der hinteren Makula (pm) und umfangreiche Innervation. (B) Embryo mit Kontroll-Morpholino injiziert. (C) Embryo mit Elektroporation nur behandelt. (D) Control Embryo mit Kontrolle Morpholino injiziert und Elektroporation hat ähnliche Ebenen der acetylierten Tubulin in den hinteren Makula und Innervation im Vergleich zu den nicht behandelten Kontrollgruppe.

<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>

Die Produktion der Artikel wurde von Gene Tools, LLC, die Reagenzien in der Video-und Text erwähnt produzieren gesponsert.

Wir haben erfolgreich Antisense-Oligonukleotid MOs in das Ohr des 24 hpf Zebrafischembryo eingeführt. Embryonen, die nach der Injektion und Elektroporation erhöht wurden lebensfähig. Wenn die Embryonen überlebten die Elektroporation, wuchsen sie zu normalen Tarifen im Vergleich zu Embryonen, die keine Behandlung, sowie Embryonen, die nur injiziert worden war und Embryonen, die erst seit elektroporiert erhalten hatte. Wir beobachteten die Wirkung von MIF und MIF-like MOs nach Ohr Injektion und Elektroporation durch die Kennzeichnung mit Phalloidin und acetyliertes Tubulin. Phalloidin-Färbung von Aktin-Filamenten in sensorischen Haarzellen des hinteren Makula zeigt an, dass die Elektroporation von MIF und MIF-like MO an der 24-hpf Bühne verursacht Veränderungen in Haarzellen und / oder Stereozilien Zahlen, wenn Embryonen, die nur mit der MOs injiziert wurden im Vergleich (Abbildung 3). Dieser Rückgang in Haarzellen Zahlen stimmen mit den Ergebnissen von Shen et al. (Submitted), die MOs zu mif und mif-wie eine Reduzierung der Anzahl der Haarzellen in der sackförmigen Makula verursachen. Der Rückgang der Haare Zellzahlen in Embryonen, die mit MOs und elektroporiert injiziert wurden zeigen, dass der Elektroporation kann bei der Bewegung der MOs durch die Zellmembran Hilfe, wodurch das Ausmaß der morphologischen Auswirkungen bei Embryonen, in denen die otic Vesikel nur injiziert wurde verglichen. Änderungen in der Morphologie der statoacoustic Ganglion wurden durch acetyliertes Tubulin-Färbung beobachtet. Das Ausmaß der Innervation durch die statoacoustic ganglion betroffen war, als Embryonen, die injiziert und wurden elektroporiert zeigen verringerte Verzweigung der Neuriten (Abbildung 5). Es kann auch Kondensation der Ganglienzellen und / oder eine verringerte Zahl von Ganglienzellen verringert werden, weil die acetylierten Tubulin-Färbung ist deutlich in Embryonen, die sowohl injiziert wurden und Elektroporation verringert. Da mif gezeigt worden ist, kritisch zu sein für die Entwicklung des Nervensystems (Suzuki et al., 2004), können die Änderungen nach der Elektroporation gesehen das Ergebnis der erhöhten Transfektion der mif und mif-like MO-Lösung in den Neuroblasten Zellen werden. Elektroporation von Antisense-Oligo Morpholinos direkt in ein Entwicklungsland Gewebe ist ein nützliches Werkzeug für die Steuerung der Expression eines Gens in das Gewebe der Entwicklung, sowohl räumlich als auch zeitlich, in den Embryo. Elektroporation von MIF und MIF-like MOs in das Gewebe des Innenohrs ergab anormale Morphologie der beiden hinteren Makula und die statoacoustic Ganglion. Es wurde eine Reduktion in der Anzahl der Haarzellen in der hinteren Makula in Embryonen, die injiziert worden war und im Vergleich mit Embryonen, die keine Behandlung erhalten hatten oder mit Embryonen, die entweder war nur gespritzt oder nur elektroporiert elektroporiert. Es gab auch eine Abnahme der Grad der Innervation der hinteren sensorischen Patch von der statoacoustic Ganglion und die Größe der SAG. Elektroporation ist eine wertvolle Methode zur Transfektion von Makromolekülen in bestimmten Geweben, mit dem Vorteil der Beobachtung der primären Auswirkungen des jeweiligen DNA, RNA, oder MO in das gewünschte Gewebe und diskriminierende diese von Nebenwirkungen auf andere Gewebe einschließlich des Gehirns, Neuralleiste und periotischen Mesenchym am Innenohr Entwicklung (Barald und Kelley, 2004).

<ol><li> MOs: 0,3 mM aus einer Kombination von für mif, mif-like MO1 und mif-like MO2 verwendet wurde. Mif MO ist komplementär zu dem Startcodon des Zebrafisch mif mRNA: 5&#39;-acatcggcatgactgcgacagagat-3 &#39;. Zwei MOs wurden mif-like verwendet mif-like MO1 ist kostenlos, die translationale Startstelle:. 5&#39;-GTTTCTATATTTATGAACGGCATGA-3 &#39;, während mif-like MO2 aus AS-Sequenz am intron1/exon 2 Grenze abgeleitet: 5&#39;-GATTCATCCTCTGAAGACGTAAGCC -3 &#39;. Steuerung MO war die verschlüsselten Nukleotidsequenz von Gene Tools: 5&#39;-CCTCTTACCTCAGTTACAATTTATA-3 &#39;. </li><li> Methylenblau (0,3 ppm) in Fisch Wasser </li><li> MS222 (Tricaine, Sigma, 0,64 mm in Fisch Wasser) </li><li> Low-Schmelzpunkt Agarose (LMPA; Roche) </li><li> Phenolrot (Sigma) </li><li> Anti-acetyliertes Tubulin (Sigma) </li><li> Alexa Fluor ® 488 Ziege anti-Maus Zweitantikörper (Molecular Probes) </li><li> Texas-Rot-Phalloidin (Molecular Probes) </li></ol>

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

In den letzten Jahren hat Elektroporation zu einer beliebten Methode zur in-vivo-Transfektion von DNA, RNA und Morpholinos in verschiedenen Geweben, einschließlich der Augen, des Gehirns und Somiten der Zebrafisch. Der Vorteil der Elektroporation gegenüber anderen Methoden der genetischen Manipulation ist, dass bestimmte Gewebe gezielt können, sowohl räumlich als auch zeitlich, denn die Einführung von Makromolekülen durch die Anwendung von elektrischem Strom. Hier beschreiben wir den Einsatz der Elektroporation zur Transfektion von MIF und MIF-like Morpholinos in das Gewebe des sich entwickelnden Innenohr des Zebrafisch. In früheren Studien, mif Morpholino in Embryonen im 1 eingespritzt - bis 8-Zell-Stadium führte zu weit verbreiteten morphologischen Veränderungen im Nervensystem und Auge sowie das Ohr. Durch die Ausrichtung der Gewebe des Innenohrs in späteren Phasen in der Entwicklung, können wir die primären Auswirkungen von MIF in den Entwicklungsländern Innenohr zu bestimmen, um sekundäre Effekte, die sich aus dem Einfluss von anderen Geweben führen kann entgegengesetzt. Durch die Verwendung von Phalloidin und acetyliertes Tubulin-Färbung, um die Morphologie von Neuronen, neuronale Prozesse und Haarzellen mit der hinteren Makula verbunden Studie konnten wir die Wirksamkeit der Elektroporation als Methode zur gezielten Transfektion in der Zebrafisch Innenohr zu beurteilen. Die otic Vesikel 24hpf Embryonen wurden mit Morpholinos und elektroporiert injiziert und wurden dann zu Embryonen, die keine Behandlung erhalten hatten oder bisher nur gespritzt oder elektroporiert verglichen. Embryonen, die injiziert und wurden elektroporiert zeigten eine Abnahme der Haarzelle Zahlen, verringerte Innervation durch die statoacoustic Ganglion (SAG) und weniger SAG Neuronen im Vergleich mit Kontrollgruppen. Unsere Ergebnisse zeigten, dass die direkte Lieferung von Morpholinos in otocysts in späteren Stadien der unspezifischen Nervensystem und Neuralleiste Auswirkungen Morpholinos am 1-8 Zell-Stadium geliefert vermeidet. Darüber hinaus ermöglicht die Untersuchung von Effekten, die an das Innenohr und nicht sekundäre Effekte auf das Ohr von primären Auswirkungen auf das Gehirn, Neuralleiste oder periotischen Mesenchym gerichtet sind.

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

Eine Methode, um Morpholinos direkt liefern in der Zebrafisch otocyst bei 24hpf entwickelt worden. Mit Mikroinjektion von Morpholinos in das Lumen des otic Vesikel und Elektroporation, um das Eindringen Effekt, konnten wir die Wirkung von Morpholinos auf das Gehirn zu umgehen und erhalten Wirkungen, die spezifisch an das Innenohr.

Direkte Lieferung von MIF Morpholinos Into the Zebrafisch Otocyst durch Injektion und Elektroporation Beeinflusst Inner Ear Entwicklung

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 Aufrufe.  University of Wisconsin, Madison. Eine Methode, um Morpholinos direkt liefern in der Zebrafisch otocyst bei 24hpf entwickelt worden. Mit Mikroinjektion von Morpholinos in das Lumen des otic Vesikel und Elektroporation, um das Eindringen Effekt, konnten wir die Wirkung von Morpholinos auf das Gehirn zu umgehen und erhalten Wirkungen, die spezifisch an das Innenohr.

Serie: 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.

Gross und Fine Dissection of Inner Ear Sinnesepithelien in Adult Zebrafisch ( Danio rerio)

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

Forschung

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.

Primäre Kultur und Plasmid Elektroporation der Murine Corti-Organ.

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.

Intraperitoneale Injektion in Adult Zebrafisch

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.

RNAi-Interferenz durch dsRNA Injektion in 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 ...

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.

Elektroporation von Craniofacial Mesenchym

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-vivo-Elektroporation von Morpholinos in die Adult Zebrafisch Retina

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-vivo-Elektroporation von Morpholinos in das Regenerations Adult Zebrafisch Leitwerk

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.

Gentransfer in Hühnerembryonen durch Ältere<em&gt; Ex ovo</em&gt; Die Elektroporation

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.

Eine vergleichende Studie von Drug-Delivery-Methoden Gezielte an die Maus Inner Ear: Bullostomy<em&gt; Versus</em&gt; Transtympanale 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.

Neuartige Methode der Transport der Plasmid-DNA zu Maus Blase Urothel durch Elektroporation

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