Bu çalışma Deutsche Forschungsgemeinschaft (DFG) tarafından desteklenen, biz teknik destek için Ayele Tsedeke Taddese teşekkür etmek istiyorum.

1.. Cam İğneler hazırlanması (Şekil 1) <ol><li> Enjeksiyon iğneleri (Şekil 1A) hazırlamak için bir 0.75 mm çapa sahip bir cam kılcal kullanın. </li><li> Bir iğne çektirmenin içine cam kılcal yerleştirin ve aşağıdaki parametre ile iğneyi çekin: ısıtma çevrimi değeri: 463; döngüsü değerini çekerek: 230; hız: 150 ms; süresi: 150 msn (Şekil 1B). </li><li> Bir mikrometre mercek ile Stereoskop altında 20 mikron çaplı bir iğne üretmek için saatçi cımbızla çekti cam kılcal kırın. </li><li> Iğne keskinleştirmek ve 20 mikron eğimi (Şekil 1C ve 1D) üretmek için, ıslatılmış kauçuk çıkrık ile bir torna kullanın. Not: Sharp, eğimli iğneler ventrikül içine yerleştirilmesi kolaylaştıracak ve böylece doku hasarı en aza indirmek ve kas delikli iğnenin (Şekiller 1 E-G) çıkarılmasından sonra yeniden mühürlemek için izin verir. </li></ol> 2. PreparMorfolino Çözüm ation <ol><li> 7.5 mM&#39;lik nihai bir konsantrasyona kadar 1x Fosfat Tamponlu Salin (PBS) &#39;de liyofilize morfolino çözülmesiyle morfolino stoku hazırlayın. [Detaylar için üreticinin talimatlarına (Malzemeler tablo) bakın]. </li><li> 3.5 mM ve 0.5 mM morfolino Endo-Porter nihai konsantrasyonu için 2.8 ul Endo-Porter stok çözeltisi (1 mM) (Malzemeler tabloya bakınız) 2.5 ul morfolino stok çözeltisi (7.5 mM) karıştırılarak morfolino enjeksiyon çözeltisi hazırlayın. </li></ol> 3.. Morfolino enjeksiyon preparasyonu (Şekil 2) <ol><li> 10 ul Microloader pipet ucu ile bir mikropipet kullanılarak, bu çözeltinin 5 ul kesik cam iğne yerleştirin. </li><li> Pnömatik pico pompanın (Şekil 2A-C) bağlı mikromanipülatör iğne tutucuya cam iğne takın. </li><li> Sonraki mikroskop iğne tutucu yerleştirin iğne sadece ihtiyaçları böylecelarva (Şekil 2A), kardiyak ventrikül içine eklemek için bir düzlemsel yönde taşınacak. </li><li> Yaklaşık 45 ° enjeksiyonlar için açısını ayarlayın. </li><li> Tutma basıncı aşağıdaki gibidir: mikroenjektör değerlerini ayarlayın inç kare (psi) başına 20 £; ejeksiyon basıncı: 15 psi; Kapılanamaması 100 msn aralığı 1.9 dönemi değeri (10.9 msn karşılık gelir). </li><li> % 1.5 ile 20 ml (w / v) jeli oluşturmak için standart bir mikrodalga kullanılarak 1 x PBS içinde agaroz eritin. , 10 cm Petri kabı içine erimiş jel dökün. Sertleştikten sonra, agaroz sedasyon larva 11 (Şekil 2D ve 2E) yerleştirileceği oluklar içinde olacak, böylece sıcak bir yivli agaroz enjeksiyon formu yerleştirin. </li></ol> 4. Enjeksiyonları (Şekil 3) <ol><li> 20 mg / L tricaine (L-Etil-m-amino-benzoat-metan sülfonat) onlar dokunmak yanıt vermemesine kadar akvaryum suyu 100 ml larva anestezisi. </li><li> Plastik bir Pasteur pipett kullanınventral yan oluk (Şekil 3A) dikey agaroz duvara bakacak şekilde e dikkatle ıslak agaroz kalıbın bir oluğuna sedasyon larva aktarmak için kullanılır. </li><li> Ventrikül uzak enjeksiyon iğnesi (Şekil 3A) bakacak şekilde stereomikroskop altında agaroz plakası yerleştirin. </li><li> Kalbin karıncık içine eklemek için iğne indirin. Sadece 1-2 mikron çok derin iğne (Şekil 3B ve 3C) takmamaya özen ventrikül içine iğne takın. </li><li> Iğne takıldıktan sonra, kalbin (Şekil 3D ve 3E) temizlenmesini sağlamak için aralıklarla bekleyen, 4-6 bakliyat, çözümün her sağlayan 3 nl ile ventrikül içine morfolino çözüm enjekte. </li><li> Enjeksiyondan sonra, iğne (ekleme düzlemine paralel) kaldırmak ve daha sonra dikkatli bir şekilde E3 orta BAC dolu plastik bir Pasteur pipeti kullanarak larva aktarmataze E3 ortamı içeren bir Petri kabı içine k. </li><li> Enjekte edilen larva aktarırken iğnenin kurumasını önlemek için 1x PBS içeren bir petri kabı içine iğne yerleştirin. </li><li> Tekrar 4,1-4,6 deney süresince her 12-24 saat adımları tekrarlayın. Not: enjeksiyon Yinelenen hücrelerdeki morfolino alımını ve bakım sağlar. </li></ol> 5. Enjeksiyonları Analizleri (Şekil 4) <ol><li> Onlar dokunmak yanıt vermemesine kadar 20 mg / L tricaine akvaryum su 100 ml balık anestezisi. </li><li> Düz ıslak% 1.5 üzerinde yanal olarak larva yerleştirin E3 ortam maddesi ile kaplanmış (ağ / hac) agaroz. </li><li> Aydınlık ve floresan görüntüleme ile stereomikroskopta kullanarak görüntü larva. Not: larvalar şeffaf olduğu için, floresein-etiketli morfolino doğrudan enjeksiyondan sonra hayvan boyunca kan damarlarında floresan yeşili gibi görünür olmalıdır. Bu floresan ayrıca aşağıdaki diğer vaskülarize doku içine dağılacaktır15 dakika (Şekil 4A-4C). </li></ol> Rejeneratif kıbet 6. Değerlendirilmesi <ol><li> Fiji Resim J özgür yazılım (http://fiji.sc/Fiji) kullanılarak çekilen görüntüleri değerlendirmek. Rejenerasyon araştırmak için, kontrol ve morfolinodur enjekte larva oluştu rejeneratif büyüme miktarını belirlemek için hat-izleme aracını kullanın. </li><li> Görüntüleri kalibre etmek için, sürükleyerek ve ana Fiji penceresinin içine dosyayı bırakarak Fiji orijinal görüntü dosyalarından birini açın. </li><li> Aşağıdaki tüm görüntüler için ölçeğini ayarlamak için, ana menüde &quot;Analiz&quot; gidin. </li><li> &quot;Set ölçekte&quot; menüsünü tıklatın Açılır. </li><li> Bu pencerede, onay kutusunu tıklatarak &quot;Küresel&quot; seçeneğini açın. </li><li> Şimdi sürükle ve bırak (adım 6.1) tekrar rejeneratif büyüme miktarını ölçmek için görüntüyü açın. </li><li> Araç çubuğunda &quot;serbest seçimler&quot; aracını seçin. </li><li> Ölçülecek alanı (Fi kuşatmakdeerlerini 4J-4L). </li><li> Ctrl + M. Bu kare piksel çevrili alanın değerini gösteren yeni bir &quot;Sonuçlar&quot; penceresi açılacaktır. </li></ol>

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Yazarlar ifşa hiçbir şey yok.

Intraventriküler enjeksiyon Embriyogenez sırasında gen fonksiyonlarını etkilemeden gelişim veya vücut homeostasis ileriki aşamalarında gen fonksiyon testi için hızlı ve güvenilir bir değerlendirme yöntemi sağlar. Bu tekniğin başarı sağlamak için, bir dört kritik noktaların farkında olmalıdır: iğneden 1) iğne boyutu, 2) kurutma, 3) hacmini minimize ve sedasyon çözeltide 4) minimal maruz kalma süresi. Çok büyük iğneler ventrikül zarar ve aşırı kanamaya neden olurken çok küçük iğ...

Organ ve ekleri rejenerasyon yaşam ve spor için temelde önemli olan; Ancak, insan dahil birçok omurgalı rejeneratif yetenekleri sınırlıdır. Çeşitli hayvan modellerde kapsamlı rejeneratif kapasiteye sahip olduğunu mevcut olmakla birlikte, organ ve apendiks rejenerasyon sırasında gen fonksiyonunu değerlendirmek için genetik teknikler ters çok sınırlı ya da varolmayan kalır. Bu nedenle, yeni yaklaşımlar bu model organizmaların yenilenme moleküler biyoloji incelemek gerekmektedir. Zebra balığı birkaç transgenik balık hatları hücreleri izlemek için var, çünkü hücre ve nedeniyle önemli çoklu organ, doku ve uzantıları yeniden yeteneği, ama aynı zamanda değil tek organ ve apendiksinde rejenerasyon 1, moleküler biyoloji anlamak için iyi kurulmuş bir modeldir ve genini aşırı ifade eden 2, 3, oluşturur. Bununla birlikte, larva Zebrafish gen inhibisyonu esas olarak overexpre sınırlıdırolan transgen ürünü genin endojen aktivitesi yansıtmamaktadır kazanç fonksiyon-etkilerini elde etmek için her ilgi genleri ya da mevcut değildir dominant-negatif yapılar, salgılanması. Bu nedenle, özel olarak nakavt veya demonte gen ifadesini ortadan kaldırmak için alternatif bir yöntem, bu sorunların üstesinden gelmek için gereklidir. TALENS gen fonksiyonu nakavt için ters genetik aracı olarak kullanan mevcut gen hedefleme özgü; genin ilk gereksinimleri embriyonik gelişimin ilerleyişi önlemek çünkü ancak, bu nakavt strateji, çok sık erken embriyonik dönemde fonksiyonel değerlendirmelere sınırlıdır. Dolayısıyla, bu tür Talens kullanarak geliştirme sonra rejenerasyon veya organ homeostasisin sonraları olayları inceleyerek, 5 4 engellenir. Bu nedenle, alternatif bir gen çıkarma stratejisi tamamen oluşmuş organ ve yapıların gen gereksinimlerini değerlendirmek için erken gelişme sonrası gen işlevini hedefleyen ihtiyaç duyulmaktadır. </p&gt; Morfolino enjeksiyon birkaç yetişkin organlarında genleri hedef ve yetişkin fin 6-8 rejenere etkili olduğu gösterilmiştir, ancak bu yöntemler elektroporasyon gerektiren ve çok iç organları nedeniyle konuma veya dolayı elektrik duyarlılıklarına ya electroporate zordur bozulması. Doğrudan enjeksiyon yapısal bütünlüğünü bozabilir, çünkü ya da boyut sınırlayıcı olduğu için Dahası, larva bazı dokular doğrudan enjekte etmek zordur. Finfold içine direkt enjeksiyon mümkün değil çünkü larva kuyruk yüzgeci, böyle bir yapıdır. Bu nedenle, elektroporasyon ve direkt enjeksiyon için alternatif ya da enjekte etmek için çok küçük ya da elektroporasyona edilemez dokularda genlerin hedeflenmesi için gerekli idi. Larva kuyruk yüzgeci rejenerasyon sırasında belirli genlerin işlevini inhibe etmek ve hedef amacıyla, mevcut morfolino sağlayan teknolojiler değiştirilmiş olan birGeç-aşamalı larva kuyruk yüzgeci rejenerasyon sırasında gen fonksiyonu ssessment. Bu yöntem, bir arada Endo-Porter transfeksiyon reaktifi 10, fluorescein-etiketli morpholinos intraventriküler teslim 9 kullanmaktadır. Bir kez ventriküle, morfolino-Endo-Porter karışımı hızla damar yoluyla larva yayılır ve hedef önceden imkansız olmuştur dokuları girer. Bu enjeksiyon yöntemi özel dokularda genlerin hedeflenmesi için modifiye edilebilir ve büyük ihtimalle halen gen fonksiyonu inhibe etmek için ters genetik yöntemler eksikliği diğer hayvan modelleri olarak uygulanabilir. Böylece, hemen larva aşamasında genel organı homeostasis ve rejenerasyon sırasında gen işlevini incelemek için geniş bir yelpazede kullanım potansiyeli ile hızlı ve kolay bir yöntem sunar.

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			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:171px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Reagent/Material</td>
			<td style="width:171px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
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			<td height="42" style="height:42px;width:216px;">Morpholino with 3&#180;fluorescein tag</td>
			<td style="width:171px;">Gene Tools Inc.&#160;</td>
			<td style="width:119px;">Customized</td>
			<td style="width:167px;">More information on www.genetools.com</td>
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			<td height="42" style="height:42px;width:216px;">Endo-Porter</td>
			<td style="width:171px;">Gene Tools Inc.&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">More information on www.genetools.com</td>
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			<td height="63" style="height:63px;width:216px;">Agarose</td>
			<td style="width:171px;">Serva</td>
			<td style="width:119px;">120623</td>
			<td style="width:167px;">For pouring plates to place the fish during injections and imaging</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tricane (L-Ethyl-m-amino-benzate-methane sulfonate)</td>
			<td style="width:171px;">Applichem</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">For anesthesia</td>
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		<tr height="63">
			<td height="63" style="height:63px;width:216px;">E3 medium (5 mM NaCl; 0.17 mM KCl; 0.33 mM CaCl2; 0.33 mM MgSo4)</td>
			<td style="width:171px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">For larval husbandry</td>
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			<td height="63" style="height:63px;width:216px;">Petri Dishes</td>
			<td style="width:171px;">Sarstedt</td>
			<td style="width:119px;">821.472</td>
			<td style="width:167px;">For placing the fish during injections and imaging</td>
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			<td height="21" style="height:21px;width:216px;">&#160;Equipment</td>
			<td style="width:171px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Thin Wall Glass Capillaries</td>
			<td style="width:171px;">World Precision Instruments, Inc.&#160;</td>
			<td style="width:119px;">TW100F-3</td>
			<td style="width:167px;">For preparing injection needles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microloader pipette tips</td>
			<td style="width:171px;">Eppendorf</td>
			<td style="width:119px;">5,242,956,003</td>
			<td style="width:167px;">For loading the needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pasteur Pipettes (3 ml/1 ml)</td>
			<td style="width:171px;">Brand</td>
			<td style="width:119px;">747760/ 74775</td>
			<td style="width:167px;">For transfering larva</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Flaming / Brown&#160; needle puller&#160;</td>
			<td style="width:171px;">Sutter</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">More information on www.sutter.com</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Watchmaker (Dumont) Tweezers (size 5)</td>
			<td style="width:171px;">World Precision Instruments, Inc.&#160;</td>
			<td style="width:119px;">501985</td>
			<td style="width:167px;">To brake the needle before sharpening</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dissecting microscope</td>
			<td style="width:171px;">Olympus, Leica, Zeiss</td>
			<td style="width:119px;">Varies with the manufacturer</td>
			<td style="width:167px;">Part of the whole injection setup</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Fluorescence camera</td>
			<td style="width:171px;">Olympus, Leica, Zeiss</td>
			<td style="width:119px;">Varies with the manufacturer</td>
			<td style="width:167px;">To image the fluorescence after injeciton</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PicoNozzle kit (microinjector holder)</td>
			<td style="width:171px;">World Precision Instruments</td>
			<td style="width:119px;">5430-12</td>
			<td style="width:167px;">For microinjections</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pneumatic PicoPump (microinjector)</td>
			<td style="width:171px;">World Precision Instruments</td>
			<td style="width:119px;">SYS-PV820</td>
			<td style="width:167px;">For microinjections</td>
		</tr>
	</tbody>
</table>

reverse genetic morpholino approach using cardiac ventricular injection to transfect multiple difficult-to-target tissues in the zebrafish larva

Zebra balığı hücre ve organ ve apendiks yenilenme moleküler biyolojisini anlamak için önemli bir modeldir. Ancak, ters genetik istihdam moleküler stratejiler henüz yeterince Zebrafish embriyogenezin sonra larva dönemlerinde rejenerasyon veya doku homeostazisindeki gen fonksiyonunu değerlendirmek için geliştirilmiş değil, ve Zebra balığı larva içinde çeşitli dokuları hedeflemek için zordur. Gene özgü morpholinos intraventriküler enjeksiyonlar genomik bu aşamalarda, bir zamansal olarak kontrollü bir şekilde zebra balığı genlerin hedef mevcut yetersizlik için alternatif bir yöntem sunar. Bu yöntem, daha önce bu tür larva kuyruk kanadı, genellikle, invaziv olmayan doku yenilenmesini aratmak için kullanılan bir yapı gibi ulaşmak mümkün olmayan yapılar dahil olmak üzere, vücutta çeşitli dokular içine tam dağılım ve morfolino sonraki dahil edilmesine imkan verir. Larva finfold rejenerasyon sırasında aktive edilen çeşitli genler de Presen ediliryetişkin omurgalı doku yenileyici t, yani larva erişkinlerde rejenerasyon anlamak için bir modeldir. Bu yöntem, morfolino dağılım larva doku tamiri gibi embriyojenez sonra doku homeostazını düzenleyen diğer fizyolojik olayları için gerekli genlerin hızlı ve kolay tanımlanması için gereklidir. Bu nedenle, bu dağıtım yöntemi embriyojenez sonra gen fonksiyonunun değerlendirilmesi için zamansal kontrol için şu anda ihtiyaç strateji sağlar.

(Şekil 3A) dorsal yaklaştı keskin, eğimli enjeksiyon iğnesi kolayca Zebra balığı larva kalp ventrikül yerleştirilir. Kalp iğne (Şekil 3B ve 3C) varlığına rağmen devam eder ve pompalama kan akışı muhafaza edilmektedir. Dikkatli enjeksiyonları kalp (Şekil 3E) içine morfolino enjeksiyonu rağmen ventrikül veya kalp kasılmaları (Şekil 3D) morfolojisini bozmayacak. Birkaç dakika içinde...

Watch this Scientific Journal Video about Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva at JoVE.com

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

Bu tür gen-spesifik morpholinos intraventriküler enjeksiyonları kullanarak doku rejenerasyonu gibi gelişme ve fizyolojik homeostasis sonraki aşamalarında gen fonksiyonunu değerlendirmek için zebrabalıkları için uyarlanabilir ters genetik bir yöntem.

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 ...

reverse-genetic-morpholino-approach-using-cardiac-ventricular

Research

JoVE Journal

Biology

10.5K Görüntüleme.  Technische Universität Dresden.  Bu tür gen-spesifik morpholinos intraventriküler enjeksiyonları kullanarak doku rejenerasyonu gibi gelişme ve fizyolojik homeostasis sonraki aşamalarında gen fonksiyonunu değerlendirmek için zebrabalıkları için uyarlanabilir ters genetik bir yöntem. 

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

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

Zebra balığı Embriyolar mRNA ve morfolino Antisens Oligonükleotidler Mikroenjeksiyon.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

Biyoloji

Zebrafish Brain Ventricle Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid.  In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation.  In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue.  However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space.  Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy.  The brightfield and the fluorescent images are then processed and superimposed in Photoshop.  This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image.  Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages.  We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

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 ...

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 ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Yüksek Verimli Primer Hücre Transfect Gen Verici MXcell Elektroporasyon Sistemi

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

Genetik Đndüklenebilir Kader Haritalama Pratik Bir Yaklaşım: Hücreler Mark ve Track Görsel Bir Rehberi In Vivo</em

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

Embriyo sırasında Fonksiyonel Artıklık Test Ters Genetik Yaklaşım

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

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, ...

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 ...

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 ...

A Multi-hole Cryovial Eliminates Freezing Artifacts when Muscle Tissues are Directly Immersed in Liquid Nitrogen

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible cytoplasmic compartments. Another hurdle is the tight apposition of myofibers to the surrounding tissues. Because the process of tissue fixation and paraffin embedding leads to the shrinkage of muscle fibers, freezing is an optimal means of hardening muscle tissue for sectioning. However, a commonly encountered issue, the formation of ice crystals, occurs during the preparation of frozen sections because of the high water content of muscle. The protocol presented here first describes a simple and efficient method for properly freezing muscle tissues by immersing them in liquid nitrogen. The problem with using liquid nitrogen alone is that it causes the formation of a nitrogen gas barrier next to the tissue, which acts as an insulator and inhibits the cooling of the tissues. To avoid this "vapor blanket" effect, a new cryovial was designed to increase the speed of liquid flow around the tissue surface. This was achieved by punching a total of 14 inlet holes in the wall of the vial. According to bubble dynamics, a higher rate of liquid flow results in smaller bubbles and fewer chances to form a gas barrier. When liquid nitrogen flows into the cryovial through the inlet holes, the flow velocity around the tissue is fast enough to eliminate the gas barrier. Compared to the method of freezing muscle tissues using pre-chilled isopentane, this protocol is simpler and more efficient and can be used to freeze muscle in a throughput manner. Furthermore, this method is optimal for institutions that do not have access to isopentane, which is extremely flammable at room temperature.

This protocol describes a procedure to freeze muscle tissues by plunging them directly into liquid nitrogen. This protocol also highlights a new cryovial that can avoid the &#34;blanket effect&#34; of nitrogen gas when liquid nitrogen contacts the tissue surface of a specimen.

Kas Dokular doğrudan sıvı azot içine girdikleri zaman Artifacts Donma bir çoklu-delikli dondurarak saklamaya uygun vialler ortadan kaldırır

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible ...