Yazarlar, laboratuvar bu tekniği geliştirdi Laura Anne Lowery teşekkür etmek istiyorum. HS NIHMH70926 tarafından desteklenmektedir. Jennifer Gutzman CSBi / Merck, doktora sonrası burs tarafından desteklenmiştir.

Protokol Bölüm 1: mikroenjeksiyon için hazırlık <ol><li> Sutter Aletleri iğne çektirmenin kullanarak kılcal iğneler çekerek enjeksiyon için hazırlayın. </li><li> (Teksas Kırmızı Dextran&#39;ın gibi) floresan boya ile iğne doldurun. </li><li> Iğne mikromanipülatör ve mikroenjeksiyon cihazları monte edin. </li><li> Eğimli bir ipucu oluşturmak için uygun büyüklükte iğne bir açıyla kesin. </li><li> Damla boyutu ölçün ve petrol enjeksiyon başına 1-2nl arasında olduğunu kontrol edin. </li></ol> Bölüm 2: embriyoların hazırlanması <ol><li> Embriyoların ilgi aşamasında daha 30 dakika önce enjekte edilir. Örneğin, 24 saat sonra döllenme (hpf) ventriküllerin çalışma, 23.5 hpf embriyolar enjekte edilir. Sahne Kimmel ve ark göre, embriyolar. (4). </li><li> Stereomikroskop altında, forseps kullanarak embriyoların dikkatle koryon çıkarın. </li><li> % 1 agaroz ile kaplı plastik tabak, plastik 1-200 ul pipet ucu ile agaroz delik karıştırmak. Forseps kullanarak deliklerden agaroz fişler dikkatlice çıkarın. </li><li> Agaroz delikli çanak içine embriyolar embriyo medya aktarın. </li><li> Medya ekle tricaine Westerfield (5) göre yapılan embriyoların uyutmak ve deneme sırasında hareket etmesini önlemek için. </li><li> Deliğin içine embriyo kuyruk hafifçe aşağı gelecek şekilde yerleştirin ve böylece enjeksiyon cihazı embriyonun arka tarafında yönlendirmek bu. </li></ol> Bölüm 3: beyin ventrikül enjekte <ol><li> Beyin ventrikül enjekte etmek, iğne ucu, aynı alanda yüksek güç embriyo olarak böylece mikromanipülasyon kurulum düzenliyoruz. </li><li> Dikkatlice aşağıdaki beyin dokusu vurmadan r0/r1 hingepoint sadece arka Beyin ince roofplate iğne koymak. </li><li> Enjekte ventriküller tamamen doldurmak için yeterli boya. Embriyo aşamasına bağlı olarak, ventrikül boşluğu doldurmak için çeşitli enjeksiyonlar sürebilir. </li></ol> Bölüm 4: Görüntüleme <ol><li> Kullanarak, temiz bir% 1 agaroz kaplı çanak çanak yeni delikler karıştırmak, fişlerin kaldırmak ve embriyoların uyutmak ve hareket etmesini önlemek için tricaine ekleyin. </li><li> Ventrikül enjekte embriyo kuyruk, dorsal bir görüntü için delik ve konuma yerleştirin. </li><li> Iletilen ışık kullanarak bir resim çekin. </li><li> EMBRİYO VEYA MİKROSKOBU HAREKET çok kritik. </li><li> Mikroskop ayarları değiştirin ve floresan ışığı ile bir görüntü almak. </li><li> Iletilen ışık ve floresan ışığı ile yanal görüntüleri çekmek için embriyo konumlandırın. </li><li> Photoshop görüntü işleme için TIFF dosyaları olarak kaydedin. </li></ol> Bölüm 5: Görüntü İşleme <ol><li> Photoshop görüntüleri işlemek için, iletilen ışık ve floresan ışığı görüntüleri, aynı pozisyonda alınan aynı embriyo açın. Tüm ayarları her bir resim için aynı olduğundan emin olun. </li><li> Floresan görüntü iletilen ışık görüntü üzerine sürükleyin ve onları tam olarak hizaya. </li><li> Floresan görüntü seçildiğinde, &quot;Resim&quot;, &quot;Ayarlamalar, seçme&quot; ve ardından &quot;rengini değiştirme&quot; ve beyaz siyah arka plan değiştirmek. </li><li> Floresan görüntüler tekdüze renkli görünüyor kadar sağ &quot;rengini değiştirin&quot; penceresinde &quot;bulanıklık&quot; faktörü ayarlamak. </li><li> &quot;Katmanlar&quot; penceresinde, bindirme görüntüleri izlemek için &quot;Multiply&quot; seçeneğini seçin. Ventrikül alan görüntü tam olarak iletilen ışık görüntü uyumlu olmalıdır. </li><li> Bu dosya ve herhangi bir diğer görüntüler için tekrar kaydedin. </li></ol> Temsilcisi Sonuçlar / Sonuç Yanlış enjeksiyon görüntülerin bir örnek EH içinde gösterilmiştir Uygun ventrikül enjeksiyon görüntüleri, 1 panelleri AD Şekil gösterilmektedir. Uygun enjeksiyonları, keskin kenarları olmayan diffüz boya var. Iğne çok uzak ventrikül içine yerleştirilir ve beyin dokusu vurur altında olduğunda oluşur yanlış enjeksiyon, ventrikül alanı dışında görünür boya ve yumurta sarısında neden olabilir. <img src="/files/ftp_upload/1218/1218figure1.png" alt="Şekil 1" width="600" height="742" /> Şekil 1. 25 hpf vahşi tip embriyoların ventrikül enjeksiyonları Doğru enjeksiyon yöntemi (AD) ve (EH) yanlış enjeksiyon yöntemi örnek olarak gösterilmiştir. (A, E) Dorsal aydınlık görüntüleri (B, F) ve floresan ve aydınlık görüntülerin gösterildiği hem doğru ve yanlış enjeksiyon yöntemleri için kaplama (C, G) ilgili floresan görüntüler. Oklar, yanlış bir ventrikül enjeksiyon (&quot;Sorun Giderme&quot; bölümüne bakın) nedeniyle beyin ventrikül mekanın ötesinde boya gitti bölgelerde gösterir. Anterior tüm resimlerin sol. Sorun Giderme: <table border="1"><tbody><tr style="font-weight:bold; font-size:16px;"><td> Sorun </td&gt; <td> Neden </td><td> Çare </td></tr><tr><td> Iğne ile embriyo Ezici. </td><td> Iğne çok künt veya ucu çok büyük. </td><td> Yeni bir iğne ile başlayın ve bu kadar geri bozmazlar. Konik sizin iğne gibi keskin bir şırınga iğnesi yapmak. </td></tr><tr><td> Boya (bkz. Şekil 1E-H), embriyo ve yumurta sarısı boyunca. </td><td> İğne doku geçti ve ventrikül boşluk kalmadı. </td><td> Iğne yeteri kadar keskin olmayabilir, ponksiyon hızını kontrol etmek mümkün değildir. </td></tr><tr><td> Boya önbeyin çok zayıf ya da değildir. </td><td> Enjeksiyon için yeterince yüksek bir basınç kullanmayın, ya da iğne kadar boya enjekte edildiğinde önbeyin ulaşmak için yeterli ön değildi. </td><td> Enjeksiyon basıncı artırın ya da enjeksiyon sırasında iğne biraz daha ön yerleştirin. Daha ayrıntılı önbeyin gerekli ise orta beyin arka beyin sınır boyunca iğne koyabilirsiniz. Ayrıca, sadece ileri görüntüleme önce diffüz boya için biraz bekleyebilir. </td></tr><tr><td> Fluoresan görüntüler Photoshop renk değişiminden sonra kenarlarında koyu. </td><td> Renk değiştirme penceresinde &quot;bulanıklık&quot; ayarını değiştirmek için unuttum. </td><td> &quot;Bulanık&quot; renk değiştirme penceresi doğru şekilde hareket ettirin. </td></tr><tr><td> Iğne Beyin roofplate değil. </td><td> Iğne düzgün kesilmiş değildir. </td><td> Yeni bir iğne olun. Enjeksiyon yapıyoruz, mat gibi çeşitli iğneler gerekebilir. </td></tr></tbody></table>

<ol>
	<li>Gutzman, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+the+zebrafish+midbrain-hindbrain+boundary+constriction+requires+laminin-dependent+basal+constriction.">Formation of the zebrafish midbrain-hindbrain boundary constriction requires laminin-dependent basal constriction.</a> Mech. Dev. 125, 974-983 (2008).</li><li>Lowery, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+Classification+of+Zebrafish+Brain+Morphology+Mutants.">Characterization and Classification of Zebrafish Brain Morphology Mutants.</a> Anat. Rec. (Hoboken). 292, 94-106 (2009).</li><li>Lowery, L. A., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+formation+of+zebrafish+brain+ventricles+occurs+independently+of+circulation+and+requires+the+nagie+oko+and+snakehead/atp1a1a.1+gene+products.">Initial formation of zebrafish brain ventricles occurs independently of circulation and requires the nagie oko and snakehead/atp1a1a.1 gene products.</a> Development. 132, 2057-2067 (2005).</li><li>Kimmel, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev. Dyn. 203, 253-310 (1995).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish Book: A guide for the laboratory use of zebrafish. , (1995).</li></ol>

Bu video floresan boya (Texas-Kırmızı Dextran) Zebra balığı beyin ventriküller gelişmekte içine enjekte etmek için nasıl gösterilmektedir. Bu yöntem beynin ventrikül alanı çevreleyen neuroepithelium aksine görselleştirmek için kullanılan ve ventrikül alanı şekline yanı sıra çevredeki beyin dokusu şeklini belirlemek için son derece yararlı. Bu zaman içinde canlı embriyo beyin ventrikül oluşumu ve beyin morfolojilerinden süreci daha iyi anlamak için bize izin verir. Bu teknik, beyin vent...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Forceps</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>11252-20</td>
<td>&nbsp;</td>
<tr>
<td>Capillary Tubes (needles)</td>
<td>Tools</td>
<td>Frederick Haer and Co.</td>
<td>30-30-1</td>
<td>&nbsp;</td>
<tr>
<td>Agarose-Coated dishes</td>
<td>Tools</td>
<td>Agarose (Seakem GTG; Lonza) Dishes (Corning) </td>
<td>Agarose (50027) Dishes (430166) </td>
<td>Make agarose approximately 2-3 mm deep in the dish depending on the stage of embryo you are injecting</td>
</tr>
<tr>
<td>Dissecting Microscope</td>
<td>Microscope</td>
<td>Zeiss Stereomicroscope Stemi SV 6 </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Microscope Camera with Fluorescence capabilities and with image capture software</td>
<td>Tools</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1-200 &mu; pipette tips</td>
<td>Tools</td>
<td>Tip One, US Scientific</td>
<td>1111-0806</td>
<td>These are the perfect size to poke the holes in the agarose and have the embryos fit perfectly.</td>
</tr>
<tr>
<td>Photoshop</td>
<td>Image Analysis</td>
<td>Adobe</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Embryo Loop</td>
<td>Tools</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Make these yourself with a glass pipette and hair or fishing line and seal with beeswax.</td>
</tr>
<tr>
<td>Microinjector</td>
<td>Tools</td>
<td>Medical Systems Research Products Harvard Apparatus</td>
<td>PL1-100</td>
<td>&nbsp;</td>
<tr>
<td>Needle Puller</td>
<td>Tools</td>
<td>Sutter Instruments</td>
<td>&nbsp;</td>
<td>Settings (Heat 785, Pull 70, Velocity 40, Time 70).</td>
</tr>
<tr>
<td>Micromanipulator</td>
<td>Tools</td>
<td>Narishige</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescent dye Texas-Red Dextran 70,000MW</td>
<td>Reagent</td>
<td>Molecular Probes</td>
<td>D1830</td>
<td>Other Molecular weights are available if desired.</td>
</tr>
<tr>
<td>Tricaine (3-aminobenzoic acid ethyl ester)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A-5040</td>
<td>&nbsp;</td>
<tr>
<td>Embryo Media</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>According to Westerfield (5)</td>
</tr>		</tbody>
		</table>
		</div>

zebrafish brain ventricle injection

Uygun embriyonik beyin gelişimi sırasında beyin ventrikül oluşumu normal beyin fonksiyonu için gereklidir. Beyin ventriküller, beyin omurilik sıvısı ile dolu, beynin içinde son derece korunmuş boşluklar vardır. Zebra balığı olarak, nöral tüp oluşumu sonrası, beyin ventrikül oluşumu ile sonuçlanan bir sıvı ile doldurur neuroepithelium daralması ve kıvrımlarının bir dizi uğrar. Ventrikül oluşum süreci, aynı zamanda meydana gelen nöroepitelyal şekil değişiklikleri anlamak için, beyin dokusu ile karşılaştırıldığında ventrikül alanı görselleştirmek için bir yol gerekiyordu. Ancak, şeffaf zebrafish embriyoların doğası zor ventrikül uzaydan gelen doku ayırt etmek için yapar. Bu nedenle, ventrikül alan floresan boya ile doldurulmuş ve aydınlık ve floresan mikroskobu ile görüntülü bir beyin ventrikül enjeksiyon tekniği geliştirdi. Aydınlık ve floresan görüntüler daha sonra işlenmiş ve Photoshop bindirilmiş. Bu teknik, aydınlık görüntü neuroepithelium şekline göre, floresan boya ile ventrikül alanı görünüm için sağlar. Zebrafish Beyin ventrikül enjeksiyon erken larva aşamalardan 18 saat sonra döllenme ile istihdam edilebilirler. Biz yanı sıra beyin morfolojilerinden mutantlar (1-3) karakterize beyin ventrikül oluşumu ve morfolojilerinden çalışmalarımız yoğun bu tekniği kullanmıştır.

Watch this Scientific Journal Video about Zebrafish Brain Ventricle Injection at JoVE.com

Zebrafish Brain Ventricle Injection

Nöral tüp oluşumu sonra neuroepithelium daraltır ve tüp embriyonik beyin ventriküller oluşturmak için embriyonik beyin omurilik sıvısı (eCSF) ile doldurur katlanabilir. Biz daha iyi, canlı embriyo nöroepitelyal şekli aksine sıvı dolu boşluk görselleştirmek için bu ventrikül enjeksiyon tekniği geliştirdi.

Zebra balığı Beyin Ventrikül Enjeksiyon

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

Research

JoVE Journal

Biology

21.2K Görüntüleme.  Whitehead Institute for Biochemical Research. Nöral tüp oluşumu sonra neuroepithelium daraltır ve tüp embriyonik beyin ventriküller oluşturmak için embriyonik beyin omurilik sıvısı (eCSF) ile doldurur katlanabilir. Biz daha iyi, canlı embriyo nöroepitelyal şekli aksine sıvı dolu boşluk görselleştirmek için bu ventrikül enjeksiyon tekniği geliştirdi.

Video: Zebrafish Brain Ventricle Injection

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

Konfokal Mikroskop Zebra balığı Embriyonik Beyin Görüntüleme Canlı

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Biyoloji

Retro-orbital Injection in Adult Zebrafish

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is often the most effective and noninvasive form of delivery of an agent of interest. In the zebrafish (Danio rerio), IV injection of drugs has long been a challenge because of the small vessel diameter. This has also proved a significant hurdle for the injection of cells during hematopoeitic stem cell transplantation. Historically, injections into the bloodstream were done directly through the heart. However, this intra-cardiac procedure has a very high mortality rate as the heart is often punctured during injection leaving the fish prone to infection, massive blood loss or fatal organ damage. Drawing on our experience with the mouse, we have developed a new injection procedure in the zebrafish in which the injection site is behind the eye and into the retro-orbital venous sinus. This retro-orbital (RO) injection technique has been successfully employed in both the injection of drugs in the adult fish as well as transplantation of whole kidney marrow cells. RO injection has a much lower mortality rate than traditional intra-cardiac injection. Fish that are injected retro-orbitally tend to bleed less following injection and are at a much lower risk of injury to a major organ like the heart. Further, when performed properly, injected cells and/or drugs quickly enter the bloodstream allowing compounds to exert their effect on the whole fish and kidney cells to easily home to their niche. Thus, this new injection technique minimizes mortality while allowing efficient delivery of material into the bloodstream of adult fish. Here we exemplify this technique by retro-orbital injection of Tg(globin:GFP) cells into adult casper fish as well as injection of a red fluorescent dye (dextran, Texas Red ) into adult casper fish. We then visualize successful injections by whole animal fluorescence microscopy.

Here we show how to do retro-orbital injection in adult zebrafish.

Yetişkin Zebra balığı retro-orbital Enjeksiyon

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

Transplantasyon GFP of-Kimerik Zebra balığı Embriyolar Retina ve Beyin Gelişimi Canlı Görüntüleme blastomer ifade

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

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, ...

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

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the eye, brain, and somites of zebrafish. The advantage of electroporation over other methods of genetic manipulation is that specific tissues can be targeted, both spatially and temporally, for the introduction of macromolecules by the application of electrical current. Here we describe the use of electroporation for transfecting mif and mif-like morpholinos into the tissues of the developing inner ear of the zebrafish. In past studies, mif morpholino injected into embryos at the 1- to 8-cell stage resulted in widespread morphological changes in the nervous system and eye, as well as the ear. By targeting the tissues of the inner ear at later stages in development, we can determine the primary effects of MIF in the developing inner ear, as opposed to secondary effects that may result from the influence of other tissues. By using phalloidin and acetylated tubulin staining to study the morphology of neurons, neuronal processes, and hair cells associated with the posterior macula, we were able to assess the efficacy of electroporation as a method for targeted transfection in the zebrafish inner ear. The otic vesicles of 24hpf embryos were injected with morpholinos and electroporated and were then compared to embryos that had received no treatment or had been only injected or electroporated. Embryos that were injected and electroporated showed a decrease in hair cell numbers, decreased innervation by the statoacoustic ganglion (SAG) and fewer SAG neurons compared with control groups. Our results showed that direct delivery of morpholinos into otocysts at later stages avoids the non-specific nervous system and neural crest effects of morpholinos delivered at the 1-8 cell stage. It also allows examination of effects that are directed to the inner ear and not secondary effects on the ear from primary effects on the brain, neural crest or periotic mesenchyme.

A method to deliver morpholinos directly into the zebrafish otocyst at 24hpf has been developed. Using microinjection of morpholinos into the lumen of otic vesicle and electroporation to effect penetration, we were able to bypass the effect of morpholinos on the brain and obtain effects specific to the inner ear.

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 ...

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 ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Yetişkin Zebra balığı Gavaging

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to employ reverse genetics have not yet been adequately developed to assess gene function in regeneration or tissue homeostasis during larval stages after zebrafish embryogenesis, and several tissues within the zebrafish larva are difficult to target. Intraventricular injections of gene-specific morpholinos offer an alternative method for the current inability to genomically target zebrafish genes in a temporally controlled manner at these stages. This method allows for complete dispersion and subsequent incorporation of the morpholino into various tissues throughout the body, including structures that were formerly impossible to reach such as those in the larval caudal fin, a structure often used to noninvasively research tissue regeneration. Several genes activated during larval finfold regeneration are also present in regenerating adult vertebrate tissues, so the larva is a useful model to understand regeneration in adults. This morpholino dispersion method allows for the quick and easy identification of genes required for the regeneration of larval tissues as well as other physiological phenomena regulating tissue homeostasis after embryogenesis. Therefore, this delivery method provides a currently needed strategy for temporal control to the evaluation of gene function after embryogenesis.&#160;

An adaptable reverse genetic method for zebrafish to assess gene function during later stages of development and physiological homeostasis such as tissue regeneration using intraventricular injections of gene-specific morpholinos.

Zebra balığı Larva Çoklu Zor-hedef dokular geçiştirilmesi için Kardiyak Ventriküler Injection kullanarak Genetik Morfolino Yaklaşım ters

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Zebra balığı Nephrotoxin Mikroenjeksiyon Akut Böbrek Yaralanması Model

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...