Bu çalışma KFB için hibe Sağırlık Araştırma Vakfı Yc S ve NSF (IOS 0.930.096) tarafından desteklenmiştir. 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 0.832.862, NSF IOS 0.930.096

1. Elektroporasyon için Elektrotlar yapma <ol><li> Kes uygun uzunluğu 75 mikron çaplı tungsten tel (AM Systems, Inc Carlsborg, WA) (yaklaşık 3-5 cm) </li><li> Molex kablo konektörü (pin damgalı pirinç) ile tungsten tel koyun </li><li> Sarın tungsten teller ve shrink boru (SPC Technology, Chicago, IL) ve Bunsen beki veya ısı tabancası ile daralan boru tel mühür ısı uygulamayın molex kablo konektörü </li><li> Tungsten tel ucu keskinleştirmek için, 1.0 N Sodyum hidroksit içine tel daldırma (Conrad ve ark 1993) ve diğer elektrot olarak bir ataş kullanarak, Kare Dalga Elektroporatör tel elektrolizi. Kullandığımız koşulları bakliyat arasında her biri 50 ms ile 100 ms 5 50 Volt bakliyat. Elektrotlar 15-20 mm çapa kadar bilenmiş olmalıdır </li><li> Elektroporasyon kullanmak için önce kil modelleme halinde preslenmiş oluklar içinde bir tepsi tel tutun </li></ol> 2. Elektroporasyon İstasyonu Kurma <ol><li> Teyp mikromanipülatörler (Dünya Hassas aletler) 75μm tungsten elektrot bilenmiş. </li><li> Diseksiyon mikroskobu aşamasında (Leica) her iki tarafında yerleştirin mikromanipülatörler. </li><li> Koru CUY-21 Düzenle Kare Dalga Elektroporatör (Şekil 1) elektrotlar bağlayın. </li><li> Elektroporatör açın ve elektroporasyon Parametreler, gerilim, darbe süresi ve bakliyat sayısı da dahil olmak üzere ayarlayabilirsiniz. Bu deneme için en etkili parametrelerin her biri Her darbe arasında 100ms ile, 5.0ms süren üç adet 13-volt bakliyat bulundu. Elektroporasyon embriyo hava kabarcıkları oluşturur, darbe süresini azaltır. </li></ol> 3. Zebra balığı Kulakla veziküllü içine Morpholinos Mikroenjeksiyon <ol><li> Hasat zebrafish embriyoların üreme tankından. Embriyolar embriyo-yükselterek ° C gecede 28.5 0.3 PPM metilen mavisi (Westerfield 2005) orta inkübe. </li><li> Sutter P-97 elektrot çektirmesi ile cam iğneler çekin </li><li> Bir agaroz jel tabanı (10 mm Petri kabında balık su içinde% 1 agaroz) - 37 ° C su banyosunda Isınma </li><li> Balık suda ısı% 1 düşük erime noktası agaroz (LMPA) kadar erimiş ve 37 ° C su banyosunda sıcak tutmak. </li><li> Bir morfolino çözüm (GeneTools, Corvallis, OR) ile doldurun ve bir bardak iğne ucu uygun çap kesmek. Bir iğne bir micromanipulator (Kuhn) üzerine monte edin. </li><li> Yaklaşık 10 mm iğne ucu kesin ve ucu morfolino çözüm yeterli teslim imkân vermesini sağlamak için madeni yağ içine enjekte edilir. </li><li> 24 saat postfertilization (hpf) embriyolar Dechorinate ve 0.3 PPM metilen mavisi balık su MS222 (tricaine Sigma) ile onları uyutmak. </li><li> Uygun üniforma analizi için sağ tarafında agaroz jel tabanı üzerine 3 ila 5 anestezi embriyolar hizalayın </li><li> Her embriyo üzerinde% 1 LMPA 1 ile 2 damla koyun ve izin yer embriyo düzeltmek için kuvvetlendirmek </li><li> Otik vezikül sağ tarafta böylece Petri çanağı </li><li> Otik vezikül lümen içine iğne yerleştirin ve bağlı bir nitrojen tankı (Şekil 2) ile morfolino çözüm PV820 Pnömatik PicoPump (Dünya Hassas Aletler) enjekte. </li></ol> 4. Elektroporasyon Prosedürü <ol><li> Hemen enjeksiyondan sonra elektroporasyon istasyona monte embriyolar Taşı </li><li> Otik vezikül sadece arka doku üzerine pozitif elektrot yerleştirin, fakat sadece otik vezikül ön beyin içine nüfuz etmez; negatif elektrot eklemek </li><li> Bir ayak pedalı veya bir anahtar basarak kullanarak uygulayın. </li><li> Agaroz balık su dökün ve dönen ve bir cam pipet ile agaroz embriyolar kaldırmak. Embriyoların zarar vermemek için dikkatli kullanın. Kaldırmak için zor olan embriyo, embriyonun çevresindeki herhangi bir LMPA hafifçe kurtulmak için bir iğne kullanın. </li><li> Embriyolar, metilen mavisi balık su ve analiz için istenen aşamaları (in situ hibridizasyon, etc ve mikroskopik anlaysis, immünohistokimyasal, morfolojik) embriyolar yükseltmek ile bir tabak koyun . </li></ol> 5. Temsilcisi Sonuçlar: <img src="/files/ftp_upload/2466/2466fig1.jpg" alt="Şekil 1" /> Şekil 1 Elektroporasyon istasyonu. Elektrotlar bilenmiş 75μm Tungsten teller kullanılarak yapılan ve bir koruyun CUY-21 Kare Dalga Düzenle Elektroporatör yol açar bağlı idi. Bu elektrotlar mikromanipülatörler bantlanmış. <img src="/files/ftp_upload/2466/2466fig2.jpg" alt="Şekil 2" /> Şekil 2. Enjeksiyon istasyonu. Tüm embriyolar standardizasyon amaçlar için sağ kulak içine enjekte edildi. <img src="/files/ftp_upload/2466/2466fig3.jpg" alt="Şekil 3" /> Şekil 3 3 dpf&#39;e embriyolar aktin filamentler Phalloidin boyama. (A), 3 dpf&#39;e embriyo injected kontrolü MO ile arka makula (pm) Phalloidin güçlü boyama vardır. (B) kontrolü MO ile enjekte edilir ve sonra electroporated oldu embriyo enjeksiyon sadece embriyolarda am Phalloidin boyama benzer seviyelerde vardı. Elektroporasyon birlikte mif katille enjeksiyon pm (D) Phalloidin boyama dramatik bir azalmaya neden olmuştur (C) yalnız otik vezikül mif katille Enjeksiyon, Phalloidin boyama azaltılması neden yoktu. am anterior makula. <img src="/files/ftp_upload/2466/2466fig4.jpg" alt="Şekil 4" /> Şekil 4, 5 dpf larvaları ile aktin filamentler Phalloidin boyama . Enjeksiyon arasında hiçbir fark sadece (A) ve standart kontrolü morfolino (B) elektroporasyon enjeksiyon. Ancak, mif morpholinos ve electroporated enjekte edilen 5 dpf larvaları sadece mif morpholinos (C enjekte edildi embriyo ile karşılaştırıldığında arka makula (pm) (D) 3 dpf&#39;e daha olsa daha az dramatik Phalloidin boyama, azaltılmış gösterdi .) am anterior makula. <img src="/files/ftp_upload/2466/2466fig5.jpg" alt="Şekil 5" /> Şekil 5 embriyoların 3 dpf&#39;e Asetillendirilmiş tubulin boyama. (A) embriyo herhangi bir enjeksiyon veya elektroporasyon almadı. (B) embriyo electroporated ama hiçbir enjeksiyon aldı. Embriyolar mif morpholinos enjeksiyon ve elektroporasyon (C, D) Her iki alan kontrol grubu ile karşılaştırıldığında gözle görülür bir şekilde azaldı tubulin boyama vardı. (Pm, arka makula). <img src="/files/ftp_upload/2466/2466fig6.jpg" alt="Şekil 6" /> Şekil 6 kontrolü morfolino 3 dpf&#39;e embriyoların Asetillendirilmiş tubulin boyama. (A) herhangi bir tedavi olmadan Embriyo arka makula (pm) ve geniş innervasyon güçlü asetillenmiş tubulin boyama gösterdi. (B) Embriyo kontrolü morfolino ile enjekte edilir. (C) sadece elektroporasyon ile tedavi Embriyo. (D) enjekte edilir ve electroporated kontrolü morfolino Kontrol embriyo hiçbir tedavi kontrolü ile karşılaştırıldığında arka makula ve innervasyon asetillenmiş tubulin benzer seviyesi vardır.

<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>

Maddenin üretim, video ve metin belirtilen reaktifler üreten Gen Araçlar, LLC tarafından sponsor oldu.

Biz başarıyla 24 hpf zebrafish embriyonun kulak içine antisens oligonükleotid katille girmiştik. Enjeksiyon ve elektroporasyon sonra toplandı Embriyolar yaşayabilir. Embriyoların elektroporasyon kurtuldu, onlar hiçbir tedavi yanı sıra, sadece enjekte edilen ve sadece electroporated olmuştu embriyolar olmuştu embriyolarda almıştı embriyolar göre normal fiyatla büyüdü. Biz Phalloidin ve asetillenmiş tubulin ile etiketleme yoluyla kulak enjeksiyon ve elektroporasyon sonra mif ve mif gibi katille bir etki gözlenmemiştir. Posterior makula duyusal saç hücreleri aktin filamentler Phalloidin boyama saç hücresi ve / veya stereocilia numaraları sadece katille enjekte edildi embriyolar ile kıyaslandığında değişikliğe neden mif ve 24-hpf aşamada mif gibi MO bu elektroporasyon gösterir (Şekil 3). Bu azalma saç hücre sayıları Shen ve arkadaşlarının bulguları ile tutarlıdır. Katille mif ve mif gibi sakküler makula saç hücrelerinin sayısında bir azalmaya neden olduğu (gönderildi). Pratisyenler ve electroporated enjekte edildi embriyolar, bu elektroporasyon göstermek saç hücre sayıları azalmaya ve böylece otik vezikül sadece enjekte edildiği embriyoların karşılaştırıldığında morfolojik etkileri ölçüde artırarak, hücre zarından katille hareketli yardımcı olabilir. Asetillenmiş tubulin boyama yoluyla statoacoustic ganglion morfolojisi değişiklikler gözlenmiştir. Enjekte gösterisi neurites (Şekil 5) dallanma azalma electroporated embriyolar olarak statoacoustic ganglion innervasyon ölçüde etkilendi. Asetillenmiş tubulin boyama embriyolarda enjekte ve electroporated her ikisi de önemli ölçüde azalmış olması nedeniyle de, ganglion hücreleri ve / veya ganglion hücrelerinin sayısı azalmış yoğuşma vardır azalma olabilir. Mif, sinir sistemi gelişimi (Suzuki ve ark, 2004) için kritik olduğu gösterilmiştir Çünkü, mif ve mif gibi neuroblast hücrelerin içine MO çözüm. Artış transfeksiyon elektroporasyon sonra görülen değişiklikler sonucu olabilir Doğrudan gelişmekte olan bir doku içine antisens oligo morpholinos Elektroporasyon embriyo doku gelişimi sırasında, hem de mekansal ve zamansal bir gen ifadesini kontrol etmek için yararlı bir araçtır. Mif ve benzeri iç kulak dokularına katille mif Elektroporasyon arka makula ve statoacoustic ganglion anormal morfoloji sonuçlandı. Embriyolar ile karşılaştırıldığında, ya sadece enjekte edilmiş ya da sadece electroporated embriyolarda ya da hiç tedavi almıştı enjekte electroporated olmuştu embriyolar arka makula duyusal saç hücrelerinin sayısında bir azalma vardı. Statoacoustic ganglion ve SAG boyutunu arka duyusal yama innervasyon derecesi bir azalma vardı. Elektroporasyon istenen dokuya, özellikle DNA, RNA, yada MO birincil etkilerinin gözlenmesi ve diğer dokular üzerindeki ikincil etkileri bu ayrımcı, beyin, nöral krest ve yararı, belirli dokulara makromoleküllerin transfecting için değerli bir yöntem iç kulak geliştirme periotic mezenşimin (Barald ve Kelley, 2004).

<ol><li> Pratisyenler: 0.3 mM, mif, mif gibi MO1 için bir kombinasyonu ve mif gibi MO2 kullanılmıştır. 5&#39;-acatcggcatgactgcgacagagat 3 &#39;mif MO zebrafish mif mRNA&#39;nın başlangıç ​​kodon ücretsiz. Iki katille mif gibi gibi MO1 mif translasyonel başlangıç ​​sitesine ücretsiz: gibi-MO2 mif intron1/exon 2 sınırında ise sırası AS türetilmiş, 5&#39;-GTTTCTATATTTATGAACGGCATGA-3 &#39;: 5&#39;-GATTCATCCTCTGAAGACGTAAGCC &#39;-3. 5&#39;-CCTCTTACCTCAGTTACAATTTATA-3 &#39;Gen Kontrol MO Araçlar şifreli nükleotid dizisi oldu. </li><li> balık suyu metilen mavisi (0.3 ppm) </li><li> MS222 (tricaine, Sigma, balık su içinde 0.64 mM) </li><li> Düşük erime noktası agaroz (LMPA; Roche) </li><li> fenol kırmızısı (Sigma) </li><li> Anti-asetillenmiş tubulin (Sigma) </li><li> Alexa Fluor ® 488 keçi anti-fare ikinci antikor (Moleküler probları) </li><li> Texas kırmızı Phalloidin (Moleküler probları) </li></ol>

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

Son yıllarda, elektroporasyon, göz, beyin, ve zebrafish, hücre grubu da dahil olmak üzere çeşitli dokular, içine, DNA, RNA ve morpholinos in vivo transfeksiyon için popüler bir yöntem haline gelmiştir . Elektroporasyon genetik manipülasyon diğer yöntemlere göre avantajı belirli dokuları, elektrik akımı uygulanması makromoleküllerin tanıtımı için, hem mekansal ve zamansal olarak, hedefli olabilir ki. Burada mif zebrafish gelişmekte olan iç kulağın dokularına ve mif gibi morpholinos transfecting elektroporasyon kullanımı açıklanmaktadır. Geçmiş çalışmalarda, mif morfolino 1 embriyolar içine enjekte - 8-hücreli aşamaya yaygın morfolojik değişikliklerin yanı sıra, sinir sistemi ve göz kulak sonuçlandı. Geliştirme sonraki aşamada iç kulak dokuları belirleyerek, diğer dokulara etkisi sonucu ikincil etkileri aksine, gelişmekte olan iç kulak MIF birincil etkileri belirleyebilirsiniz. Nöronlar, nöronal süreçleri ve arka makula ile ilişkili saç hücrelerinin morfolojisini incelemek için boyama Phalloidin ve asetillenmiş tubulin kullanarak, zebrafish iç kulak hedeflenen transfeksiyon için bir yöntem olarak elektroporasyon etkinliğini değerlendirmek mümkün. 24hpf embriyoların otik vezikül morpholinos ve electroporated enjekte edildi ve daha sonra hiçbir tedavi almış veya sadece enjekte veya electroporated olmuştu embriyoların karşılaştırıldı. Enjekte ve electroporated Embriyolar, saç hücre sayıları bir azalma olduğunu gösterdi statoacoustic ganglion (SAG) ve kontrol grupları ile karşılaştırıldığında daha az SAG nöronlar tarafından innervasyon azaldı. Sonuçları, daha sonraki aşamada otocysts içine morpholinos doğrudan teslim non-spesifik bir sinir sistemi ve nöral krest etkileri 1-8 hücre aşamasında teslim morpholinos önler olduğunu gösterdi. Ayrıca, iç kulak ve beyin, nöral krest veya periotic mezenşimin birincil etkileri kulak değil ikincil etkileri yönlendirilir etkilerinin incelenmesi.

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

24hpf zebrafish otocyst doğrudan morpholinos sunmak için bir yöntem geliştirilmiştir. Otik vezikül ve penetrasyon etkisi elektroporasyon lümenine morpholinos, mikroenjeksiyon, morpholinos beyin üzerinde etkisi atlayacak ve iç kulak özgü efektler elde etmek mümkün.

Enjeksiyon ve Elektroporasyon Zebra balığı Otocyst içine MIF Morpholinos Stoktan teslim İç Kulak Geliştirme etkiler

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 Görüntüleme.  University of Wisconsin, Madison. 24hpf zebrafish otocyst doğrudan morpholinos sunmak için bir yöntem geliştirilmiştir. Otik vezikül ve penetrasyon etkisi elektroporasyon lümenine morpholinos, mikroenjeksiyon, morpholinos beyin üzerinde etkisi atlayacak ve iç kulak özgü efektler elde etmek mümkün.

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.

Yetişkin Zebra balığı İç Kulak Duyu Epiteli Brüt ve Güzel Diseksiyon ( Danio rerio)

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

Biyoloji

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.

İlköğretim Kültür ve Corti murin Organ Plazmid Elektroporasyon.

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.

Yetişkin Zebra balığı içine intraperitoneal enjeksiyon

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.

DsRNA Enjeksiyon RNAi Girişim içine Drosophila Embriyolar

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.

Kraniofasial mezenşimin, Elektroporasyon

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.

Morpholinos in vivo Elektroporasyon Yetişkin Zebra balığı 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.

Yenileyici Yetişkin Zebra balığı Tail Fin içine Morpholinos in vivo olarak Elektroporasyon

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.

Tarafından Eski Tavuk Embriyolar içine Gen Transferi<em&gt; Ex ovo</em&gt; Elektroporasyon

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.

Fare İç Kulak Hedefli İlaç Dağıtım Yöntemleri Karşılaştırmalı İncelemesi: Bullostomy<em&gt; Karşı</em&gt; Transtimpanik Enjeksiyon

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.

Plazmid DNA teslimat için fare mesane Urothelium elektroporasyon tarafından roman yöntemi

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