We would like to acknowledge David O'Brien for his support with data analysis, and all the members of the Canto-Soler lab for their critical discussions. This work was supported by NIH grants EY004859 and EY022631 (MVCS), Core Grant EY1765, and an unrestricted departmental grant from Research to Prevent Blindness, Inc.

Bu çalışmada tarif edilen tüm prosedürler, Johns Hopkins Üniversitesi Hayvan Bakımı ve Kullanımı Komitesi tarafından kurallara göre gerçekleştirilmiştir. Öncesinde Zaman: 1. Araçlar, Reaktifler ve Yemekleri Hazırlama <ol><li> Yumurtalar hazırlanması: Bir yumurta kuluçka makinesi içinde 5 gün 37,5 ° C ve% 60 bağıl nemde döllenmiş Beyaz Legorn tavuk yumurtası inkübe edin. NOT: standartlaştırılması ve embriyonik gelişim senkronizasyonu için yararlı olabilir kapasitesi sallanan, ama sallanan bir inkübatör bu protokol sonucu için kesinlikle gerekli değildir. </li><li> Plazmid hazırlanması: <ol><li> Gerektiğinde tasarım plasmid (RNAi teknolojisinde 1,34 kullanımı yoluyla örneğin) geni aşırı ekspresyonu veya aşağı doğru düzenleme için yapılırlar. Mümkün olan her yapı içinde bir raportör gen (örneğin, EGFP, RFP veya LacZ gibi) içerir. Not: CMV gibi bazı yaygın olarak kullanılan promotörler, bir süre sonra susturulması hale gelebilir. CAG promoter (tavuk beta-aktin promoteri, CMV artırıcı ile) ilgi 35-37 genin etkin, uzun vadeli ifade sağlayan, çok iyi bir alternatiftir. </li><li> Şekilde steril PBS içerisinde 1.5 ug / ul bir konsantrasyonda bir plazmid çözeltisi hazırlayın. İSTEĞE BAĞLI: vaktinden plazmid çözüm hazırlayın ve dondurulmuş Kullanıma hazır olana kadar saklayın. NOT: Yüksek plazmid konsantrasyonları kullanılarak oysa alt plazmid konsantrasyonları transfeksiyon verimliliği 1 düşük, verimlilik önemli bir artışa yol açmaz. </li></ol></li><li> Elektrotlar: <ol><li> Anot kullanımı için kalın bir altın (Belirli Malzemeler / Ekipman Tablo) elektrodu uçlu. % 70 etanol ile silin. Not: altın İzolasyon maruz kalan yaklaşık 0,5 mm bırakarak, elektrot uçlu, elektrik akımının sızmasını en aza indirmek için tavsiye edilir. Yalıtım malzemesi için bilgiler Özgül Materyallerin Tablo bulunabilir. </li><li> 2.5 mm kare b dışında bir katot yapın(normalde Mikropipet Elcikler kullanılan) 4.5 mm genişliğinde öküz filament. Forseps yardımı ile, kare kutu geri almak ve uzun bir şerit haline filamanın düzeltin. Daha sonra, diseksiyon mikroskobu ve bir kılavuz olarak bir cetvel kullanarak altında, tabanda 3 mm uzunluğunda kare &quot;U&quot; şekline filamanın bükülmesi ve diğer tarafta filament kalan uzunluğu (Şekil 1 ile birlikte, bir yandan 2 mm yükseklikte C - D). Forseps kullanarak,% 96 etanol ve alev elektrot sterilize etmek batırın. Sonra elektrot uzun koluna küçük bir timsah klibi ile bir elektrik kurşun takın. </li></ol></li><li> Elektroporasyon Odası Kurulumu: <ol><li> 100 mm Petri kabı kullanarak bant (Şekil 1E) için, aşağı, düz tarafı bunu steril bir 1.5 ml mikrosantrifüj tüp kapağı kesme ve yapıştırılması ile elektroporasyon odasını hazırlayın. Not: işe benzer büyüklükte bir oyuk şeklinde diğer bir kap; Ancak, bir mikrosantrifüj tüp kapağı kolayca erişilebilir ve otoklavlanabilir. Kuyuplazmid çözümün bir atık sonuçlanması bir civciv göz ve katot elektrot evine kadar büyük, ama çok büyük olmamalıdır. </li></ol></li></ol> 2. Ex ovo Elektroporasyon Prosedürü <ol><li> Araçların Hazırlanması: <ol><li> Mikro-diseksiyon araçları sterilize batırarak (yumurta kabukları açılması ve embriyolar, RPE kaldırmak için Dumont cımbız kavisli ince ucu 2 çift dışarı karıştırıyordu için büyük kavisli Moloney forseps 2 çift, Bonn bir çift kaldırma lens ve vitreus için forseps dişli) % 96 etanol ve alevli ipuçları. % 70 etanol batırılmış mendil kullanarak temizleyin diseksiyon kapsamı. Bir 37 ° C su banyosu içinde, steril Kalsiyum-Magnezyum içermeyen Hank Dengeli Tuz Çözeltisi (CMF-HBSS) bir şişe ısıtın. Sıcak CMF-HBSS ile tam 3/4 100 mm steril Petri kapları doldurun. </li><li> Başka bir temiz diseksiyon mikroskobu altında elektroporasyon odasına yerleştirin ve plazmid çözüm 120 ul ile doldurun. Elektroporasyon aparatı elektrotları geç yapmaEmin ısmarlama elektrot negatif kutbuna bağlı olan ve altın pozitif kutup elektrodu uçlu. </li></ol></li><li> Embriyo Koleksiyon: <ol><li> % 70 etanol içinde ıslatılmış mendillere kabukları ile silerek yumurta temizleyin. Kültürleri kirlenmesini önlemek için taşıma protokolün geri kalanı boyunca steril prosedür koruyun. Büyük kavisli Moloney forseps kullanarak dikkatlice yavaşça (hava bölmesinin üstünde) bir yumurta yuvarlak ucunda dokunarak ve ardından sürükleyici ve kabuk parçaları çekerek yumurta kabuğunda bir delik açın. </li><li> Forseps başka çift membranlar kaldırmak ve yumurta dışarı karıştırıyordu embriyo toplamak ile (dikkatli olmak embriyo zarar vermemek için). CMF-HBSS ile bir tabak koyun. NOT: 2.4.4 belirtildiği gibi elektroporasyon için retina bardak istenen sayısını elde etmek için gerektiği kadar embriyolar toplayın </li><li> KRİTİK ADIM: Hamburger ve Hamilton 38 göre embriyolar Sahne. NOT: Optimum verimlilik embriyolar elde etmek içinevre 27 (Şekil 2A) olmalıdır. </li><li> Moloney forseps kullanarak kafaları kesilmek suretiyle embriyolar Euthanize. </li></ol></li><li> Retina Kupaları edinme: <ol><li> İnce Dumont cımbız kullanarak, dikkatle gözleri enükleasyon ve yeni bir CMF-HBSS çanak aktarın. </li><li> Optik sinir başı yakınında her göz, arka kısmından başlayarak, iki ince Dumont cımbız kullanarak, nöral zarar vermemeye RPE dikkatli olmak teşrih nöral retina ve RPE ve dikkatle arasındaki cımbız her çiftin bir ipucu tanıtmak Retina (Şekil 2B). NOT: RPE yardımcı olacaktır çözeltide Ca 2 + ve Mg 2+ eksikliği daha kolay nöral retina ayırmak. </li></ol></li><li> Elektroporasyon: <ol><li> 15 Volt, 50 msn süreli ve 950 msn aralığının 5 kare darbeleri teslim Electroporator parametreleri ayarlayın. Yer &#39;U&#39; elektroporasyon odası (mikrosantrifüj kapak dolgu içinde negatif elektrot şeklindeplazmid içeren çözeltinin 120 ul) ile ed. </li><li> Kullanımı kavisli Dumont cımbız (değil karıştırıyordu basarak) elektroporasyon odasına tek retina fincan aktarmak ve elektrodun alt ve yukarı bakacak objektif yönüne retinanın arka kısmı ile, &#39;U&#39; şekilli elektrodun içine yerleştirin ( Şekil 2B). </li><li> Bir sonraki lens, gözün ön kısmını dokunur ve Electroporator ayak pedalı elektrik atımları teslim kullanarak böylece anot yerleştirin. </li><li> CMF-HBSS ile yeni bir Petri kabı elektropore retina fincan aktarın ve kalan retina bardak her biri ile elektroporasyon prosedürü tekrarlayın. Not: 6 retina bardak kadar transfeksiyon verimi önemli bir azalma olmadan aynı plazmid çözeltisi ile elektropore edilebilir. </li><li> Bonn dişli forseps ile onları kavrama ve yavaşça t içinden çekerek her electroporated retina fincan lens ve vitreus vücuda kaldırkavisli Dumont cımbız arkası ile yerinde gözü tutarken o açılış öğrenciye. </li><li> Standart prosedürler izlenerek Electroporated retina hücreleri ayrışma ve kültür geçin. NOT: civciv retina hücreleri ayrışma ve kültürü üzerinde detaylı bir protokol için Vergara ve Canto-Soler, 2012, Ek dosyaya 4 21 bakın Bu protokolde hücreler poliornitin kaplı 6-kuyucuğu veya 35 düşük yoğunlukta numaralı seribaşı. -mm yemekleri (6 x 10 5 hücre / 35 mm çaplı kuyu veya çanak). Kullanılan ortam penisilin / glutamin, 10% fetal buzağı serumu (FCS) ve 100 ug / ml&#39;de, linoleik asit-BSA ile 199 ortamıdır. Kültürler,% 5 CO2 ile nemlendirilmiş bir 37 ° C kuluçka makinesi içinde muhafaza edilir. Bu prosedürü kullanarak, 5-6 x 10 6 hücre genellikle her aşama 27 Electroporated göz başına elde edilebilir. NOT: İdeal olarak, en fazla 3 saat embriyo toplama ve hücre kaplama arasında geçmelidir, iyi hücre hayatta kalmasını sağlamak içinve stresi en aza indirmek. Gözlem ve daha fazla analiz için tipik bir zamanlama bu noktada hücreler, iyi bir morfolojik ve moleküler farklılaşma elde ettik ve hayatta kalma hala yüksek olduğu için, kültür içinde 4 gün sonra olur. </li></ol></li></ol>

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+robust+system+for+RNA+interference+in+the+chicken+using+a+modified+microRNA+operon.">A robust system for RNA interference in the chicken using a modified microRNA operon.</a> Dev Biol. 294 (2), 554-563 (2006).</li><li>Nguyen, A. T., Dow, A. C., Kupiec-Weglinski, J., Busuttil, R. W., Lipshutz, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+gene+promoters+for+liver+expression+by+hydrodynamic+gene+transfer.">Evaluation of gene promoters for liver expression by hydrodynamic gene transfer.</a> J Surg Res. 148 (1), 60-66 (2008).</li><li>Xu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CMV-beta-actin+promoter+directs+higher+expression+from+an+adeno-associated+viral+vector+in+the+liver+than+the+cytomegalovirus+or+elongation+factor+1+alpha+promoter+and+results+in+therapeutic+levels+of+human+factor+X+in+mice.">CMV-beta-actin promoter directs higher expression from an adeno-associated viral vector in the liver than the cytomegalovirus or elongation factor 1 alpha promoter and results in therapeutic levels of human factor X in mice.</a> Hum Gene Ther. 12 (5), 563-573 (2001).</li><li>You, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hybrid+promoter-containing+vector+for+direct+cloning+and+enhanced+expression+of+PCR-amplified+ORFs+in+mammalian+cells.">A hybrid promoter-containing vector for direct cloning and enhanced expression of PCR-amplified ORFs in mammalian cells.</a> Mol Biol Rep. 37 (6), 2757-2765 (2009).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> Dev Dyn. 195 (4), 231-272 (1992).</li></ol>

The authors declare that they have no competing financial interests or other conflicts of interest.

Bu protokolün başarısı için en kritik adım embriyoların uygun sahne seçmektir. Önceki yayınlarda, embriyonik gelişim evrelerinin bir dizi tipik kuluçka veya embriyonik gün gün (ED) tarafından tanımlanan, bu kültürlerin verilir; Böylece genellikle ED 6 embriyolar ED 5 kullanarak eşdeğer sonuçlar verecektir varsayılmaktadır. Bununla birlikte, yukarıda belirtildiği gibi sahne 27 (ED 5) üzerine, transfeksiyon verimi, toplam hücre nüfusunun yaklaşık% 22 olacak olduğunu bulmuşlardır; Henüz v...

Embriyonik civciv retina gelen ayrışmış hücre kültürleri yaygın olarak kendi yaşam 2-9, farklılaşma 10-12, neurite akıbet 13, ve daha fazlasını içeren fotoreseptör hücre biyolojisi çeşitli yönlerini incelemek için kullanılmıştır. Ruben Adler ve işbirlikçileri tarafından 1980&#39;li yıllarda geliştirilen ve onun ve diğer gruplar 14-20 tarafından mükemmelleştirilmiş bu sistemin avantajları, bir hayvan modelinde 21 olarak civciv içsel özelliklerinin bulunması. Hatta embriyonik aşamada civciv gözün büyük boy, kültürlerin için malzeme büyük miktarlarda sağlar. Kültürler embriyonik gün (ED) 5 kullanılarak yapılır Dahası, - 6 retinalar, 55 - Bu hayvanda fotoreseptör yaklaşık% 86 olduğu için kendi progenitör hücrelerin% 80 Fotoreseptörlerin 14,15,18,22,23 olarak ayırt ve koniler 24, bu kültürler, bu hücre tipi ilgili çalışma için özellikle uygundur. Biz son zamanlarda develo varped ve böylece, genetik missexpression çalışmaları 1 kolaylaştırarak bu sistem kullanışlılığı genişletilmesi, bu kültürlerde hücre yüksek verimli plazmid transfeksiyon sağlayan basit bir teknik ile karakterize edilir. Bu tekniğin geliştirilmesi, bir hücre bağımsız bir şekilde gen fonksiyonlarının araştırılmasında izin vermek için transfeksiyon kabul edilebilir bir düzeyde sağlayacak yöntemler bilimsel literatürde bir boşluk kaynaklanmıştır. Primer nöronal kültürlerin 25,26 transfect herkesin bildiği zor, çünkü bu bölümünde yer almaktadır. Bu amaç için daha önce mevcut olan en yaygın olarak kullanılan tekniklerin bazıları 3-5% sırasına verim neden olur ve ciddi yan etkilere 27-32 uygulayabilir gibi lipofeksiyon veya kalsiyum fosfat aracılı transfeksiyon gibi kimyasal nakil yöntemlerini, dahildir. Enzimatik muhabir sistemi ile Plazmidlerin kullanılması sinyalini yükselterek yoksul transfeksiyon verimlilik sorunu aşmak olsa bile, onlar değilHücre-spesifik etkileri ayırt ve bunların sonuçları bütünün temsilcisi olmayabilir küçük bir hücre popülasyonu dayanmaktadır. Civciv Yaygın olarak kullanılan bir yöntem olup, RCAS virüsü enfeksiyonu, çoğalan hücreler için geçerlidir, bu primer retina kültür sistemine 33 dolayısıyla uygun değildir. Mevcut protokolde civciv embriyosu gözler evre 27 (ED 5), retinal pigment epitel (RPE) çıkarılır ve retina kap bir plasmid ihtiva eden çözelti ile doldurulmuş bir elektroporasyon odasına yerleştirilen en çıkarılmış ve kullanılarak, elektroporasyon bakımından güçlü ölçüye standart teknikler kullanılarak retina ayrışması 21 ve kültür izledi elektrotlar. Bu prosedürü optimize sonra hayatta kalma ve farklılaşma özelliklerini ödün vermeden sürekli, kültür hücreleri ve yalnız fotoreseptör nüfus içinde% 25 toplam sayısının% 22 mertebesinde transfeksiyon verimliliği elde etmek mümkün olmuşturKültürler 1. Burada bu tekniğin başarı ve yeniden üretilebilirlik sağlamak için, bu prosedür tüm önemli adımları gösteren bir detaylı bir protokol sağlar.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>ECM 830 Electro Square Porator</td>
			<td>BTX/ Harvard Apparatus</td>
			<td>45-0052</td>
			<td></td>
		</tr>
		<tr>
			<td>Genetrode, L-Shaped, 5 mm Gold Tip</td>
			<td>BTX/ Harvard Apparatus</td>
			<td>45-0115 model 512</td>
			<td>Gold tipped electrode used as anode</td>
		</tr>
		<tr>
			<td>Polyimide Tubing</td>
			<td>Vention Medical</td>
			<td>custom made</td>
			<td>Internal Diameter: 0.5mm / wall thickness: 0.15-0.2mm. Used for insulating gold tiped electrode</td>
		</tr>
		<tr>
			<td>2.5mm square box filament, 4.5mm wide</td>
			<td>Sutter Instrument Company</td>
			<td>FB245B</td>
			<td>Used to make cathode electrode</td>
		</tr>
		<tr>
			<td>HBSS, no calcium, no magnesium, no phenol red</td>
			<td>Gibco - Life Technologies</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr>
			<td>Moloney forceps</td>
			<td>Roboz</td>
			<td>RS-8254</td>
			<td>Serrated; Slight Curve; 4.5&#34; Length</td>
		</tr>
		<tr>
			<td>Dumont tweezers 5/45</td>
			<td>Roboz</td>
			<td>RS-5058</td>
			<td>Pattern #5, 45 Degree Angle; .10 X .06mm Tip Size; 109mm Length</td>
		</tr>
		<tr>
			<td>Bonn micro forceps, 1x2 teeth</td>
			<td>Roboz</td>
			<td>RS-5172</td>
			<td>Tying Platform; 1X2 Teeth, 0.12mm Teeth; 3.75&#34; Length; .3mm Tip Width</td>
		</tr>
	</tbody>
</table>

efficient gene transfer in chick retinas for primary cell culture studies: an ex-ovo electroporation approach

Embriyonik civciv retina elde edilen koni fotoreseptör zenginleştirilmiş kültürler retinal nöronların, özellikle fotoreseptör biyoloji okuyan dünyadaki araştırmacılar için vazgeçilmez bir araç haline gelmiştir. Kolayca ilaç gelişimi için yüksek verimli teknolojileri için adapte edilebilir, bu sistemin uygulamalar temel araştırma ötesine geçmektedir. Bununla birlikte, bu kültürlerde retina fotoreseptörlerinin genetik manipülasyon sisteminin yararlılığını önemli bir sınırlandırma getirmeden, çok zor olduğu kanıtlanmıştır. Biz son zamanlarda geliştirilen ve diğer mevcut protokoller 1 ile karşılaştırıldığında beş kat bu kültürlerde retina hücrelerinin transfeksiyon hızını artırır plazmid elektroporasyon tekniği ovo bir eski valide. Bu yöntemde, civciv embriyosu gözler RPE çıkarılır, evre 27 de enükle edilir ve retina kap bir plasmid ihtiva eden bir çözeltiye yerleştirilmiş ve kolaylıkla inşa alışılagelmiş kullanılarak elektroporasyonayapılan elektrotlar. Retinalar sonra ayrışmış ve kültürlü standart prosedürleri kullanıyor. Bu teknik genel olarak, fotoreseptör nüfusunun% 25 transgen ekspresyonunu elde plazmit odaklı RNAi teknolojisinde kullanımı ile, örneğin, fazla sentezlenme çalışmaları yanı sıra, gen ekspresyonunda azalma uygulanabilir. Mevcut yayın video formatı primer retina kültürlerde gen fonksiyonu çalışma sağlayan, alanında araştırmacılara bu teknoloji kolayca erişilebilir hale getirecektir. Biz de başarılı bir sonuç ve tekrarlanabilirlik için bu prosedürün kritik adımlar ayrıntılı açıklamalar dahil ettik.

Burada daha sonraki ayrışmış hücre kültürü için çıkartılmış civciv retina bardak içine plazmid transfeksiyon için basit bir protokol mevcut. Transfeksiyon özel elektrotlar (- 2 Şekil 1) hazırlanması kolaydır kullanılarak elektroporasyon ile elde edilmektedir. Bu protokol açıklanan parametreler% 20 ila 27 (Şekil 3D) (% 22 ortalama ile) aralığında transfeksiyon verimliliği elde etmek için optimize edilmiştir. Yukarıda belirtilen nedenlerden dolayı, bu sonu...

Watch this Scientific Journal Video about Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach at JoVE.com

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Embriyonik civciv retinal hücre kültürleri fotoreseptör biyoloji çalışma için değerli araçlar oluşturmaktadır. Biz kültür öncesinde retina plazmid elektroporasyon ovo ex dayalı etkin gen transferi tekniğini geliştirdi. Bu teknik önemli ölçüde bu sistem için uygun genetik manipülasyon yaparak, mevcut protokoller üzerinde transfeksiyon verimliliği artırır.

Primer Hücre Kültürü Araştırmaları Chick retina Verimli Gen Transferi: Bir<em&gt; Eski-ovo</em&gt; Elektroporasyon Yaklaşımı

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

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Research

JoVE Journal

Developmental Biology

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

8.1K Görüntüleme.  The Johns Hopkins University School of Medicine.  Embriyonik civciv retinal hücre kültürleri fotoreseptör biyoloji çalışma için değerli araçlar oluşturmaktadır. Biz kültür öncesinde retina plazmid elektroporasyon ovo ex dayalı etkin gen transferi tekniğini geliştirdi. Bu teknik önemli ölçüde bu sistem için uygun genetik manipülasyon yaparak, mevcut protokoller üzerinde transfeksiyon verimliliği artırır. 

Video: Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

Optimize<em&gt; Ex-ovo</em&gt; Kalkınma Gelişmiş Aşamaları Chick Embriyolar ekimi

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos ...

Gelişim Biyolojisi

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 ...

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

Bir Co-kültür Sistemde nöronal bağlantıları Oluşumunda değerlendirilmesi için bir optogenetic Yaklaşım

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &#8212; A High-throughput Screen in ES Cells

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely limits its potential. We describe here the use of mouse embryonic stem (ES) cells as surrogate cells to screen for a phenotype of interest and subsequently introduce these cells into a host embryo to develop into a living mouse carrying the phenotype. This method provides (1) a cost effective, high-throughput platform for genetic screen in mammalian cells; (2) a rapid way to identify the mutated genes and verify causality; and (3) a short-cut to develop mouse mutants directly from these selected ES cells for whole animal studies. We demonstrated the use of paraquat (PQ) to select resistant mutants and identify mutations that confer oxidative stress resistance. Other stressors or cytotoxic compounds may also be used to screen for resistant mutants to uncover novel genetic determinants of a variety of cellular stress resistance.

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &amp;#8212; A High-throughput Screen in ES Cells

Stress resistance is one of the hallmarks for longevity and is known to be genetically governed. Here, we developed an unbiased high-throughput method to screen for mutations that confer stress resistance in ES cells with which to develop mouse models for longevity studies.

İleri Genetik Yaklaşım Farelerde Stres Direnci Genler ortaya çıkarmak için - ES Hücrelerde bir Yüksek verim Ekranı

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely ...

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer is the site of the largest transit amplification in the developing brain. Here, we present a protocol to target genetic modification to this layer at the peak of proliferation using ex vivo electroporation and culture of cerebellar slices from embryonic Day 14 chick embryos.

Ex Vivo Chick Serebellar Dilimleri Kültür ve Mekansal Elektroporasyon granül hücre öncülerinin Hedefli

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying ...

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

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2&#180;-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr&#160;after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

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

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.

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

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

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and molecular level. To alter intracardiac hemodynamics, fertilized chicken eggs are incubated in a humidified chamber to obtain embryos of the desired stage (HH17). Once this developmental stage is achieved, the embryo is maintained ex ovo and hemodynamics in the embryonic heart are altered by partially constricting the outflow tract (OFT) with a surgical suture at the junction of the OFT and ventricle (OVJ). Control embryos are also cultured ex ovo but are not subjected to the surgical intervention. Banded and control embryos are then incubated in a humidified incubator for the desired period of time, after which 2D ultrasound is employed to analyze the change in blood flow velocity at the OVJ as a result of OFT banding. Once embryos are maintained ex ovo, it is important to ensure adequate hydration in the incubation chamber so as to prevent drying and eventually embryo death. Using this new banded model, it is now possible to perform analyses of changes in the expression of key players involved in valve development and to understand the role of hemodynamics on cellular responses in vivo, which could not be achieved previously.

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

For the first time we present here a reproducible banding procedure to alter hemodynamics in the developing heart ex ovo. This is achieved by partially constricting the outflow tract (OFT).

Bir roman<em&gt; Ex Ovo</em&gt; Yapıştırma Tekniği bir Embriyonik tavuk Sistemde İntrakardiyak hemodinamiği Alter

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and ...

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture.
The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Here, we present a new technique to culture chick embryos ex ovo for time-lapse imaging using transmitted light and fluorescence microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique1 that allows for the culturing of chick embryos fully-submerged in a liquid culture medium.

Uzun vadede bir Batık Filtre Kağıdı Sandviç<em&gt; Ex Ovo</emErken Civciv Embriyolar&gt; Time-lapse Görüntüleme

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of ...

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors of the diseases. To validate the contribution of a dysfunction of the mutated genes to disease development, the generation of animal models carrying the mutations is one obvious approach. While germline genetically engineered mouse models (GEMMs) are popular biological tools and exhibit reproducible results, it is restricted by time and costs. Meanwhile, non-germline GEMMs often enable exploring gene function in a more feasible manner. Since some brain diseases (e.g., brain tumors) appear to result from somatic but not germline mutations, non-germline chimeric mouse models, in which normal and abnormal cells coexist, could be helpful for disease-relevant analysis. In this study, we report a method for the induction of CRISPR-mediated somatic mutations in the cerebellum. Specifically, we utilized conditional knock-in mice, in which Cas9 and GFP are chronically activated by the CAG (CMV enhancer/chicken &#223;-actin) promoter after Cre-mediated recombination of the genome. The self-designed single-guide RNAs (sgRNAs) and the Cre recombinase sequence, both encoded in a single plasmid construct, were delivered into cerebellar stem/progenitor cells at an embryonic stage using in utero electroporation. Consequently, transfected cells and their daughter cells were labeled with green fluorescent protein (GFP), thus facilitating further phenotypic analyses. Hence, this method is not only showing electroporation-based gene delivery into embryonic cerebellar cells but also proposing a novel quantitative approach to assess CRISPR-mediated loss-of-function phenotypes.

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Conventional loss-of-function studies of genes using knockout animals have often been costly and time-consuming. Electroporation-based CRISPR-mediated somatic mutagenesis is a powerful tool to understand gene functions in vivo. Here, we report a method to analyze knockout phenotypes in proliferating cells of the cerebellum.

CRISPR-aracılı kaybı serebellar granül işlev analizinin gen transferi Elektroporasyon tabanlı rahim içinde kullanarak hücreleri

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors ...

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 ...