Biz ifade plazmaları ve in situ sondaları Dr. Doris K. Wu teşekkür Duyu Biyoloji görüntüleme tesisi için Johns Hopkins Üniversitesi Merkezi ve İşitme ve Denge Merkezi. Bu çalışma LE NIDCD Grant T32 DC000023 tarafından desteklenen

yumurta ve çıkmamış embriyolar için bakım ve etik ve insanca muamele edilir. çıkmamış embriyo kullanımı için tüm protokoller Tıp Baltimore, Maryland Johns Hopkins School Hayvan Bakımı ve Kullanımı Komitesi tarafından kabul edildi. 1. Yumurta ve ekspresyon yapıları hazırlanması <ol><li> Yumurtalar: <ol><li> 3 inkübe - 4 düzine döllenmiş tavuk yumurtası (Gallus gallus) 37 ° C&#39;de onların yüzüne düz yalan. <ol><li> inkübasyon 24 saat sonra yumurtanın yuvarlak arka ucundan bir şırınga ile albümin 3 cc çekin ve şeffaf bir bantla delik mühür. Bu yumurta 19 üstünde erişim için pencere keserken kabuk yapışmasını embriyo olmadan embriyo erişim kolaylığı sağlayan, embriyo ve yumurta kabuğu arasında bir hava boşluğu oluşturur. </li><li> İnkübasyon süresi 117 saatte, Hamburger ve Hamilton 20 gelişim aşamalarına göre embriyolar sahneE4 (HH24-25) de (Şekil 1D bakınız). </li></ol></li></ol></li><li> İfade oluşturur: <ol><li> reaktifler kullanılarak plazmid DNA ve bir ticari olarak temin edilebilir preparat kitinin üretici tarafından yapılan protokoller hazırlayın. hazırlama son aşamasında, 500 ul TE (Tris-EDTA), 1.5 ml tüp içine kitte sağlanan tampon içine DNA elüte. </li><li> Etanol 3 M sodyum asetat ve 50 ul ve boru 100,% 1 mL etanol ekleyerek konsantre DNA hızlandırabilir. -20 ° CO / N tüp yerleştirin. </li><li> 4 ° C&#39;de 14,000 rpm&#39;de 30 dakika boyunca tüp santrifüjleyin. Süpernatantı ve oda sıcaklığında 10 dakika boyunca pelet hava-kurutun. ~ 4 ug / ul konsantrasyon elde edilmelidir, 15 ul TE tampon maddesi içinde pelet, eritin. Not: Burada kullanılan kontrol tanım yapılan bir tavuk β-aktin promotör tarafından yönlendirilen PMES IRES-GFP 10, ya da pCI-H2B-IRES-RFP vardır <sCMV promoteri tarafından tahrik edilir&gt; 10 kadar. </li></ol></li></ol> Ovo Mikro elektroporasyon ve Ovo Hücre Proliferasyon Tahlili 2 tavuk <ol><li> İfade Construct ve Mikro-elektroporasyon Ovo Mikro enjeksiyon: <ol><li> 2.5 x 2.5 mm Kutu Platin Isıtma Filament için aşağıdaki optimize parametreler ile bir mikro-pipet çektirmenin kullanarak ince iğneler içine cam kılcal tüpler çekin: Basınç = 500, Isı = 600 ° C, Velocity = 105 ve Zaman = 64 çekin = 150 milisaniye (bakınız Tablo 1). </li><li> Sol- ve mikroskop yan Sağ el mikro-manipülatör aşamalarında kurmak (Şekil 1A bakınız). Sağ el mikro-manipülatör cam kılcal iğne ve Solaklar mikro-manipülatör elektrotlar (Şekil 1A) tutar tutar. </li><li> keserek mikro-enjeksiyon için ince çekti cam kapiller iğne hazırlayınBir forseps ile iğne ucu mikroskop altında çalışırken. </li><li> Mikroskop altında çalışırken% 0.1 Hızlı Yeşil ile renklendirilmiş plazmid DNA 2.5 ul mikro-manipülatör kullanarak elle manuel olarak iğne doldurun. </li><li> (Şekil 1A bakınız) bir kalıp / yumurta tutma cihaz içinde yan yatan yumurta yerleştirin. Makas 19 (Şekil 1A) kullanarak yumurtanın üstünde bir yuvarlak pencere kesti. </li><li> Mikroskop altında çalışırken, dikkatle forseps kullanarak embriyo kaplayan iki membranlar açın. </li><li> Mikroskop altında çalışırken, elle mikro-manipülatör kullanarak ve mikro-elektro-sağ elinizi kullanarak mikro-enjeksiyon sağ kulak vezikül lümene plazmid DNA yaklaşık ~ 0.5 ul teslim. <ol><li> Aynı anda sol ışığımdır kullanırken bu yüzden, Sağ mikro-manipülatör (Şekil 1B-D) DNA ile sağ kulak vezikül lümen mikro-enjekte yapmakEmbriyonun baş aşaması E4 dips çünkü ga forcep, embriyonun baş sakinleştirmeye. Bu otik vezikül zarar verecektir çünkü, otik vezikül lümeni geçmiş mikro-enjekte etmeyin. </li><li> Hemen mikro-enjeksiyon sonrasında, mikroskop altında çalışırken, pozitif (anot) platin elektrot ön-ventral sağ kulak vezikül ve paralel olarak negatif 2 mm elektrot (katot) ile 2 mm yerleştirmek için Sol mikro-manipülatör kullanın 1 mm aralıklı (Şekil 1 C). 100 milisaniye süre ve 200 msn aralıkla 12 V 4 darbeleri sunun. sol kulak vezikül bir iç işlenmemiş kontrol olarak hizmet vermektedir. NOT: 1x PBS batırılmış bir pamuk uçlu çubukla electroporations arasında elektrotlar temizleyin. </li></ol></li></ol></li><li> Ovo Hücre Proliferasyon Tahlili: <ol><li> Hemen elektroporasyon sonra el bütün üzerine 50 ul Edu çözüm bırakarak 0.25 mg 50 ul ekleyin / ml edu 1x PBS içindeBir pipet kullanarak ovo embriyo. bant ile yumurta Seal ve 18 için inkübatör dönmek - 96 saat. </li><li> ( &#39;Şekil 1 EE bakınız) standart floresan mikroskop kullanılarak 24 saat - dikkatle bandı çıkarın ve 18 sonrası otik vezikül içinde floresan GFP veya RFP sinyali embriyolar kontrol edin. Floresan sinyal varsa, ( &#39;&#39; Şekil 1 FP bakınız) bu noktada analizler için embriyolar hasat veya bant ile yeniden mühürlemek ve hasat için inkübatör iade ve sonraki aşamalarda analiz eder. NOT: - 7 saat elektroporasyon 10 sonra PMES IRES-GFP yapı ile GFP sinyali Anlatım 6 açıktır. </li></ol></li></ol> Yerinde hibridizasyon ve immünohistokimya RNA 3. Embriyo Hasat ve Doku İşleme <ol><li> forseps kullanarak embriyolar hasat. Soğuk 1x PBS embriyo durulayın. onun boynundan başını keserek, embriyoların başkanları kaldır4 ° C &#39;de% 4 paraformaldehid O / N olarak kafaları forseps kullanılarak ve yerleştirin. kafasının içinde% 4 paraformaldehit girmesine izin vermek üzere, bir forsepsle kullanarak beyin delikler. </li><li> 4 ° C&#39;de O / N (1X PBS)% 30 sukroz içinde kafaları kurutmak. </li><li> kuru buz ve metilbütan (2-metilbütan) içindeki bir bulamacı içinde hızlı dondurulması ile kriyokoruyucu ortam içinde kafa monte edin. Alternatif olarak, hızlı sıvı azot ile kriyokoruyucu ortamda başlarını dondurma. -80 ° C de monte kafaları saklayın. </li><li> 12 mikron kalınlığında doku bölüme başlarını cryosection ve superfrosted mikroskop cam slaytlar üzerine tüm iç kulak içeren doku bölümleri toplamak. </li><li> Daha önce 4,21 açıklandığı gibi in situ hibridizasyon ve immünohistokimya standart RNA gerçekleştirin ve kit üreticisi tarafından sağlanan protokol izlenerek edu hücre çoğalması için analiz edin. NOT: Saç hücreleri tavşan poliklonal kullanılarak tespit edilebilir a-MyosinVIIa (Myo7a) birtibody (1: 1,000, Proteus Biosciences) ile ve floresan etiketli ikincil antikor (1: 250, keçi anti-tavşan AlexaFluor 488; Invitrogen). </li></ol> 4. Görüntü Yakalama ve İşleme <ol><li> dijital görüntüleme ekipmanları ile görüntü alabilir. Standart bir floresan mikroskop ve (5x gelen büyütme 40x arasında değişen) standart geniş alan mikroskobu kullanarak in situ sonuçları ile görüntü immünhistokimya sonuçları. </li><li> Görüntü işleme yazılımı kullanarak görüntüleri işlemek. </li></ol>

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The authors have nothing to disclose.

Ovo mikro-elektroporasyon burada anlatılan yöntem geliştirilmesi işitsel organ haline gen transferi için optimize edilmiştir. Tipik olarak, gen fonksiyonu / ifade işlemek için kullanılan plazmid DNA bazlı ekspresyon vektörleri ile uyumludur. E4 elektroporasyon zamanlaması işitsel organında HC geliştirme odaklanmak araştırmalar için en uygunudur. En kritik adımlar iğne ile çok derin gidiyor ve otik vezikül zarar vermeden otik vezikül lümenine DNA mikro-enjekte otik vezikül ön-ventral al...

Memeli ve kuş işitsel duyu organı arasındaki morfoloji farklılıkları ve hücresel desenlendirme rağmen, moleküler faktörler ve duyusal HC geliştirmeden sorumlu yollar evrimsel 1-3 korunmuş olduğu düşünülmektedir. işitsel HCS ve çevrelerindeki SC&#39;ler evler baziler papilla, iç kulak otocyst outpocketing olarak gelişir. Erken HC ve SC atalarıdır bir havuz otik plakod / otik fincan sinir-duyu yetkili etki alanı (NSDAP) içinde belirtilir. Kader-haritalama verileri nöronal ve duyusal soylar bağlantılı ve otik fincan antero-ventral bölgesinde bulunan ya da fareler ve tavuk 4,5 yılında otocyst NSDAP kaynaklanan olduğunu kanıtlar sunmaktadır. İlk olarak, Nöroblastlar sonunda işitsel ve vestibüler ganglionlar bölünmüş işitsel-vestibüler ganglion, nöronlar yol vermek için NSDAP dan tabakalara. Bu nöronlar beyin sapı iç kulak ve çekirdeklerin duyusal HCS innerve. th hücreleriNSDAP kalır at mekano nakleden duyusal HCS ve bunların ilişkili AVM oluşan işitsel duyu organı dahil olmak üzere çeşitli duyusal yamalar, yol düşünülmektedir. civciv ve sıçangil hem de, işitme organı duyusal progenitör hücre döngüsü çıkış ve HC farklılaşma degradeler karşı meydana gelir. Civciv, işitsel progenitör hücre döngüsü çıkış ~ E5 etrafında başlar ve tabandan tepeye doğru ilerler ve merkezden çevreye 6. Bir gün sonra, HC farklılaşma apeks başlar ve tabanına 7 ilerler. Fonksiyonel mekanizmalar karmaşık ameliyatları gerektirir diğer model sistemler uterus, embriyolar manipüle aksine ovo göreli kolaylıkla araştırılabilir olarak tavuk iç kulak gelişiminin incelenmesi birçok teknik avantajlar sağlamaktadır. Burada tarif edilen yöntem, olası Basila ilgi genleri hedef ovo mikro elektroporasyon kullananÖzellikle E4 otik vezikül r papilla alanı ön-ventral. Ovo Elektroporasyon iyi kurulmuş ve genel olarak 8-14 kullanılan bir tekniktir. Elektroporasyon tekniği ilkesi nükleotidleri (örneğin, plazmid DNA, sentetik DNA ya da RNA oligonükleotidleri) negatif yüklü gerçeğine dayanır. DNA, ilgi konusu doku içine enjekte edilir. bir elektrik akımı doku boyunca yerleştirildiğinde, mevcut hücre duvarlarının geçici gözenekleri açılır ve negatif yüklü DNA, pozitif elektrot (anot) doğru akarken, DNA alımı için izin verir. kazanç fonksiyon-deneyleri için, ilgi konusu gen genellikle yeşil flüoresan protein (GFP) veya kırmızı flöresanlı proteindir (RFP) için uygun ekspresyon kasetleri içeren ifade plazmidleri alt klonlanır. kayıp-fonksiyon deneyleri plazmid tabanlı dominant negatif represör yapıları veya RNAi veya morfolino yapıları için c vardırommonly 15,16 kullanılır. Bazlı plazmid tipik mikroRNA (MiRNA) işlevi miRNA&#39;lar sünger yapıları, inhibe etmek için, 17 kullanılabilir. Ovo mikro elektroporasyon yöntemi kolayca tüm embriyo edu ekleyerek ovo hücre çoğalma tahlilinde birleştirilebilir burada tarif edilen yöntem olup, işitme organı proliferasyonu ve farklılaşmasını kontrol eden molekül mekanizmalarının incelenmesi için izin verir. elektroporasyon ve Edu eklenmesi bir gün işitsel organında hücre döngüsü çıkış başlangıcından önce olduğu otik vezikül içinde E4 gerçekleştirilir. tipik olarak 18 gerçekleştirilen bir işitsel organ Analizi - (HC farklılaşma sırasında) E5 (duyusal progenitör hücre döngüsü çıkış başlangıcı), E6 (HC farklılaşma başlangıcı) ve E7 elektroporasyon sonra 96 ​​saat; ve daha sonra ~ E9 (HC farklılaşma sonuna) kadar. Gen transferi bu elektroporasyon yöntemi olup, ~ 4 gün yaklaşık süren geçicidir becauSE ilgili genler, genomu içine entegre değildir, ancak yöntem, Tol2-aracılı gen transferi 11 gibi genomuna entegre yeteneğine sahip yapmak için, uygun plazmid DNA bazlı ekspresyon vektörleri ile kullanılmak için uygundur. Bu yöntemle sağlam GFP veya RFP ifade sürer ~ ile sinyalleri işaret solmaya, ancak işitsel organ karmaşık gelişimini incelemek için geniş bir zaman pencere sağlamak sonra kokleadaki 4 gün. Ovo gen aktarım yöntemi Bu roman ve özellikle işitme organı HC geliştirme odaklanmak araştırmalar için en uygun olan E4 olası işitsel organı, hedef sağlar. Bu çok daha genç gelişim aşamalarında 11,12 ya da in vitro baziler papilla eksplant kültürleri 18 kullanımı ile karşılaştırıldığında en elektroporasyona alternatif yöntemler, iyi bir yer.

<table><tbody><tr><td>Sterile 1x PBS pH 7.4</td><td>gibco</td><td>10010-023</td><td></td></tr><tr><td>Fast Green FCF Powder</td><td>Sigma</td><td>F7252-5G</td><td></td></tr><tr><td>EdU Powder</td><td>Invitrogen</td><td>E10187</td><td></td></tr><tr><td>Click-IT EdU Alexa Fluor 555 Imaging Kit</td><td>Invitrogen</td><td>C10338</td><td></td></tr><tr><td>HiSpeed Plasmid Midi Kit (25)</td><td>Qiagen</td><td>12643</td><td></td></tr><tr><td>ECM 830 ElectroSquarePorator&amp;nbsp;</td><td>BTX Harvard Apparatus</td><td></td><td></td></tr><tr><td>Banana to Micrograbber Cable Kit</td><td>BTX Harvard Apparatus</td><td>45-0216</td><td></td></tr><tr><td>Right Handed &amp;amp; Left Handed Micromanipulators</td><td>World Precision Instruments Inc.&amp;nbsp;</td><td>M3301R &amp;amp; M3301L</td><td></td></tr><tr><td>Two 12 mm Magnetic Holding Device Stages with 7 inch vertical posts</td><td>World Precision Instruments Inc.&amp;nbsp;</td><td>M10</td><td></td></tr><tr><td>Metal Steel Base Plate 12x24 inch</td><td>World Precision Instruments Inc.&amp;nbsp;</td><td>5479</td><td></td></tr><tr><td>Scissors for Eggs 12 cm long curved, 12 mm extrafine blades; Spring Scissors</td><td>World Precision Instruments Inc.&amp;nbsp;</td><td>14120</td><td></td></tr><tr><td>Micropippette Puller for pulling needles</td><td>Sutter Instrument Co.</td><td>P-97</td><td></td></tr><tr><td>2.5x2.5 mm Box Platinum Heating Filament</td><td>Sutter Instrument Co.</td><td></td><td></td></tr><tr><td>Glass Capillary Tubes/Needles/No Fiber/Borosil 1 mm</td><td>FHC</td><td>27-30-0</td><td></td></tr><tr><td>Hamilton Glass Syringe 100 &amp;mu;l</td><td>Hamilton</td><td>80601 Model 710LT</td><td></td></tr><tr><td>Mineral Oil (Heavy) for Hamilton Glass Syringe</td><td>Fisher Scientific</td><td>O122-1</td><td></td></tr><tr><td>Polyethylene Tubing for connecting the Glass Syringe and Glass Cappillary Needles/ Non Toxic</td><td>Becton Dickinson and Company (BD)</td><td>427420 Intramedic Clay Adams Brand</td><td></td></tr><tr><td>3 cc Disposable Syringes</td><td>Becton Dickinson and Company (BD)</td><td>309657</td><td></td></tr><tr><td>Disposable 21 Gauge Needles</td><td>Becton Dickinson and Company (BD)</td><td>305122</td><td></td></tr><tr><td>One pair of 2 mm Platinum Electrodes</td><td>Bulldog Bio. / Nepagene</td><td>CUY611P3-2</td><td></td></tr><tr><td>Electrode Holder</td><td>Bulldog Bio. / Nepagene</td><td>CUY580</td><td></td></tr><tr><td>One pair of Dumont fine forceps number 5</td><td>Fine Science Tools (FST)</td><td></td><td></td></tr><tr><td>Matte finish invisible tape for sealing eggs</td><td>Office Depot</td><td>520-928</td><td></td></tr><tr><td>Cotton-Tipped Swabs</td><td>Fisher Scientific</td><td>23-400-101</td><td></td></tr><tr><td>Sterile filter tips</td><td></td><td></td><td></td></tr></tbody></table>

gene transfer into the chicken auditory organ by in ovo micro-electroporation

Tavuk embriyo gen fonksiyonunun manipülasyonlar ovo göreli kolaylıkla yapılabilir embriyonik gelişme çalışmak için ideal bir model sistemleridir. iç kulak işitme duyu organı duymak yeteneğimizi için kritik öneme sahiptir. Bu destek hücreleri (SC&#39;ler) glial gibi son derece uzmanlaşmış duyusal (HCS) mekano nakleden saç hücrelerden oluşur epiteli ve çevresindeki evler. Işitsel organlarda yapısal farklılıklara rağmen, işitsel organ gelişimini düzenleyen moleküler mekanizmalar evrimsel memeliler ve kuşların arasında muhafaza edilir ovo elektroporasyon E1 erken aşamalarında büyük ölçüde sınırlıdır -. E3. Nedeniyle E5 işitsel organ göreli geç gelişmesine, E3 geçmiş ovo elektroporasyon ile işitsel organın manipülasyonlar nedeniyle daha sonraki aşamalarda tavuk embriyo ileri gelişimine zordur. Burada sunulan yöntem de ilgi genleri hedef için geçici bir gen transferi yöntemisahne E4 - ovo mikro-elektroporasyon yoluyla gelişmekte olan tavuk işitsel duyu organ E4.5. Bu yöntem, gain- ve-kaybı fonksiyonları bilinen plazmid DNA bazlı ekspresyon vektörleri ile uygulanabilir ve en bütün embriyoya edu (5-etinil-2&#39;-deoksiuridin) eklenerek ovo hücre çoğalma tahlilinde birleştirilebilir elektroporasyon süresi. yeşil ya da kırmızı floresan proteininin (GFP veya RFP) ifade plazmaları kullanımı deneyci hızla elektroporasyon başarıyla gelişen iç kulağın işitme bölümünü hedef belirlemek için izin verir. Bu yöntem yazıda, GFP Electroporated örneklerin temsili örnekleri gösterilmiştir; elektroporasyon sonra 96 saat ve işitsel organ yanlısı duyusal alana GFP hedefleyen in situ hibridizasyon RNA tarafından doğrulandı - embriyolar 18 toplandı. yöntemi kağıt da CellProlif analiz etmek edu analog timidin kullanımı için optimize edilmiş bir protokol sağlardurmasýna; immüno-etiketlenmesi birleştiren edu Click kimyası eğitsel göre hücre çoğalma tahlilinin bir örneği temin edilmiştir.

yeşil floresan protein içeren bu yöntem, kağıt plazmid DNA içinde (GFP) ifade kasetleri ~ bir sonuçta 12 V ve 4 100 milisaniye darbe süresi ile bakliyat ve 200 milisaniye aralıklarla optimize parametreleri ile gelişmekte olan tavuk baziler papilla (BP) içine hedef aldı % 50 embriyo sağkalım oranı ve BP içine hedefleme plazmid-DNA etkinliği. (Beyaz ok Şekil 1, EE &#39;) ve GFP tanımı bir süre boyunca takip edilebilir embriyoların geliştirilmesinde a...

Watch this Scientific Journal Video about Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation at JoVE.com

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Tavuk İşitsel Organ gen Transferi tarafından<em&gt; Ovo olarak</em&gt; Mikro-elektroporasyon

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

gene-transfer-into-chicken-auditory-organ-ovo-micro

Research

JoVE Journal

Developmental Biology

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

9.4K Görüntüleme.  The Johns Hopkins University, School of Medicine. The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ. 

Video: Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

Elektroporasyon tarafından Embriyonik Fare Koklear Eksplantlarından Kültür ve Gen Transferi

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

Gelişim Biyolojisi

In Ovo Electroporations of HH Stage 10 Chicken Embryos

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of molecular biological techniques, the chick has become a useful system in which to study gene regulation and function.  By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function.  Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements.  Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive.  This video demonstrates first how to make a window in the eggshell to manipulate the embryo.  Next, the embryo is visualized with a dilute solution of India ink injected below the embryo.  A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator.  Finally, the egg is sealed with tape and placed back into an incubator for further development.  Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.

Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo. Common applications of this technique include functional analysis of genes and putative enhancer elements. This video demonstrates neural tube electroporation in HH 10 chick embryos. Injection technique and proper egg handling are discussed.

HH Sahne 10 Tavuk Embriyolar Ovo Electroporations

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of ...

Biyoloji

In Ovo Electroporation in the Chicken Auditory Brainstem

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that the chicken embryo is an effective research model for studying basic biological functions of auditory system development. More recently, the chicken embryo has become particularly valuable in studying gene expression, regulation and function associated with hearing. In ovo electroporation can be used to target auditory brainstem regions responsible for highly specialized auditory functions. These regions include the chicken nucleus magnocellularis (NM) and nucleus laminaris (NL). NM and NL neurons arise from distinct precursors of rhombomeres 5 and 6 (R5/R6). Here, we present in ovo electroporation of plasmid-encoded genes to study gene-related properties in these regions. We show a method for spatial and temporal control of gene expression that promote either gain or loss of functional phenotypes. By targeting auditory neural progenitor regions associated with R5/R6, we show plasmid transfection in NM and NL. Temporal regulation of gene expression can be achieved by adopting a tet-on vector system. This is a drug inducible procedure that expresses the genes of interest in the presence of doxycycline (Dox). The in ovo electroporation technique &#8211; together with either biochemical, pharmacological, and or in vivo functional assays &#8211; provides an innovative approach to study auditory neuron development and associated pathophysiological phenomena.

In Ovo Electroporation in the Chicken Auditory Brainstem

Auditory brainstem neurons of avians and mammals are specialized for fast neural encoding, a fundamental process for normal hearing functions. These neurons arise from distinct precursors of embryonic hindbrain. We present techniques utilizing electroporation to express genes in the hindbrain of chicken embryos to study gene function during auditory development.

Tavuk İşitsel Beyinsapındaki Ovo Elektroporasyonunda

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that ...

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6.
The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. 
We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

Tarafından geliştirilmesi Fare İç Kulak için Gen Transferi<em&gt; In Vivo</em&gt; Elektroporasyon

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

Nörobilim

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

Dopaminerjik Nöron Geliştirme Gen Fonksiyonunun Çalışılması için Chick orta beyin içinde ovo Elektroporasyon yılında

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial and temporal control of ectopic microRNA expression. However, ventral brain structures like ventral midbrain are rather difficult to reach for any manipulations. Here, we demonstrate an efficient way to electroporate miRNA into ventral midbrain using thin platinum electrodes. This method offers a reliable way to transfect specific areas of the midbrain and a useful tool for in vivo studies.

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Ectopic expression is one technique to elucidate the microRNAs role in brain development. However, targeting specific areas using in ovo electroporation is challenging. Here, we show an efficient way to selectively electroporate ventral and dorsal midbrain regions.

Ventral Chick orta beyin içinde MikroRNA ovo İfade

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as the hair cell. Basic research in the inner ear has led to a deep understanding of how the hair cell functions, and how dysregulation can lead to hearing loss and vertigo. For this research, the mouse has been the pre-eminent model system. However, mice, like all mammals, have lost the ability to replace hair cells. Thus, when trying to understand cellular therapies for restoring inner ear function, complementary studies in other vertebrate species could provide further insights. The auditory epithelium of birds, the basilar papilla (BP), is a sheet of epithelium composed of mechanosensory hair cells (HCs) intercalated by supporting cells (SCs). Although the anatomical architecture of the basilar papilla and the mammalian cochlea differ, the molecular mechanisms of inner ear development and hearing are similar. This makes the basilar papilla a useful system for not only comparative studies but also to understand regeneration. Here, we describe dissection and manipulation techniques for the chicken inner ear. The technique shows genetic and small molecule inhibition methods, which offer a potent tool for studying the molecular mechanisms of inner ear development. In this paper, we discuss in ovo electroporation techniques to genetically perturb the basilar papilla using CRIPSR-Cas9 deletions, followed by dissection of the basilar papilla. We also demonstrate the BP organ culture and optimal use of culture matrices, to observe the development of the epithelium and the hair cells.

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The chick is a cost-effective, accessible, and widely available model organism for a variety of studies. Here, a series of protocols is detailed to understand the molecular mechanisms underlying avian inner ear development and regeneration.

Ovo ve Ex Ovo'da Kuş İç Kulak Gelişimini İnceleme Yöntemleri

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as ...

In Ovo Electroporation in Embryonic Chick Retina

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is comparatively very easy to manipulate the chick embryonic retina using recombinant DNA/RNA technology. Electroporation of DNA/RNA constructs into the embryonic retina have a great advantage to study gene regulation in retinal stem/progenitor cells during retinal development. Different type of assays such as reporter gene assay, gene over-expression, gene knock down (shRNA) etc. can be performed using the electroporation technique. This video demonstrates targeted retinal injection and in ovo electroporation into the embryonic chick retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). Here we show a rapid and convenient in ovo electroporation technique whereby a plasmid DNA that expresses green fluorescent protein (GFP) as a marker is directly delivered into the chick embryonic subretinal space and followed by electric pulses to facilitate DNA uptake by retinal stem/progenitor cells. The new method of retinal injection and electroporation at E4 allows the visualization of all retinal cell types, including the late-born neurons1, which has been difficult with the conventional method of injection and electroporation at E1.52.

In Ovo Electroporation in Embryonic Chick Retina

The overall goal of this video is to show how to perform targeted retinal injection and in ovo electroporation of DNA/RNA constructs into the chick embryonic retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). This technique is very useful to study gene expression, gene regulation, and morphological change in developing chick retina.

Embriyonik Chick Retina Ovo Elektroporasyon

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is ...

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 ...

Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation

Fragile X mental retardation protein (FMRP) is an mRNA-binding protein that regulates local protein translation. FMRP loss or dysfunction leads to aberrant neuronal and synaptic activities in fragile X syndrome (FXS), which is characterized by intellectual disability, sensory abnormalities, and social communication problems. Studies of FMRP function and FXS pathogenesis have primarily been conducted with Fmr1 (the gene encoding FMRP) knockout in transgenic animals. Here we report an in vivo method for determining the cell-autonomous function of FMRP during the period of circuit assembly and synaptic formation using chicken embryos. This method employs stage-, site-, and direction-specific electroporation of a drug-inducible vector system containing Fmr1 small hairpin RNA (shRNA) and an EGFP reporter. With this method, we achieved selective FMRP knockdown in the auditory ganglion (AG) and in one of its brainstem targets, the nucleus magnocellularis (NM), thus providing a component-specific manipulation within the AG-NM circuit. Additionally, the mosaic pattern of the transfection allows within-animal controls and neighboring neuron/fiber comparisons for enhanced reliability and sensitivity in data analyzing. The inducible vector system provides temporal control of gene editing onset to minimize accumulating developmental effects. The combination of these strategies provides an innovative tool to dissect the cell-autonomous function of FMRP in synaptic and circuit development.

Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation

Using in ovo electroporation, we devised a method to selectively transfect the auditory inner ear and cochlear nucleus in chicken embryos to achieve a cell-group-specific knockdown of fragile X mental retardation protein during discrete periods of circuit assembly.

Tavuk İşitsel Organ gen Transferi tarafından

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