Yazarlar Math1, Ngn1 Jane Johnson teşekkür ve Ptf1a antikorlar ve pCAG için Connie Cepko :: GFP plazmid olacaktır. Bu çalışma NIH R15 1R15HD059922-01 tarafından finanse edildi.

1. Elektroporasyon öncesinde hazırlıklar <ol><li> Bir maxi hazırlık (Prime-It or Qiagen) tarafından elektroporasyon için DNA Amplify. DNA konsantrasyonu etkin alımı için 1 mg / mL &#39;lik bir az olmalıdır. </li><li> Bir mikrosantrifüj tüp içinde 1 X PBS (fosfat tamponlu tuz)% 0.01 &#39;Fast Green 5 ul DNA ve karışımı 495 uL çıkarın. </li></ol> 2. Embriyonik Hasat <ol><li> CD-1 fareleri (Harlan) ile zamanlanmış çiftleşmelerin oluşturulması. Vajinal fişler varlığı için kontrol edin ve vajinal fiş embriyonik gün (E) 0.5 olarak tespit edildiği tarihten görüyoruz. Embriyolar 11 gün fişi (E11.5) görselleştirmek sonra hasat edilir. </li><li> % 70 etanol içinde sterilize araçları yerleştirin. Kullanmadan önce en az bir saat için UV ışığı ile laminar akım kaput davranın. Sprayhood, diseksiyon tepsi, ve% 70 etanol ile kapsamı diseksiyon. 37 Öncesi ısı 1 X PBS ° C </li><li> E11.5 On Enstitüsü tarafından onaylanmış koşullarına göre kadın euthanizeHayvan Bakımı ve Kullanımı (ICCUA) için ional Komitesi. Laminer akış kaputu diseksiyon tepsiye yerleştirin. % 70 etanol ile kadın karın aşağı püskürtün. </li><li> Periton boşluğuna açın ve diseksiyon tepsiye duvarlara takılmalıdır. Bu periton boşluğu içinde duracak şekilde laparotomi çekin. </li><li> Dikkatle uterin horn duvarı açın kesti. Hafifçe uzak plasenta yolk kesesi içinde embriyonun kaldırmak için bir 20 mm kaşık (Güzel Bilimsel Araçları) kullanın. 100 mm doku kültürü kabına Yeri embriyolar 2.2 ön ısıtmaya edildi steril 1 X PBS 10 mL doldurulmuş. </li><li> Mevcut tüm embriyolar için tekrarlayın. </li></ol> 3. E11.5 Embryolarının Elektroporasyon (Şekil 1) <ol><li> Bir 20 mm kaşık kullanılarak, 37 ısıtılmış edildi steril 1XPBS 10 mL ile doldurulmuş yeni bir 100 mm çanağı ° C&#39;ye kadar birinci embriyo transferi </li><li> Dikkatli bir şekilde tekrar bir 0.05 x 0.02 mm uç (Güzel Bilimsel Araçları) ile 11 cm forseps kullanmataşımak ve yolk kesesi atın. </li><li> Pozisyon embriyo yüzden onun sırt tarafında Şekil 1A karikatür benzer şekilde yukarı baktığından emin. Yavaşça embriyo için 7 mm elektrot kürekler (Harvard Apparatus) kullanın. Elektrotlar ardbeyin dördüncü ventrikül seviyesinde sinir tüpün her iki tarafında konumlandırılmış edilmelidir. Ventrikül çıplak gözle ancak bir diseksiyon mikroskobu kullanarak doğru kürek yerleştirme kolaylaştırabilir. Pedallarının Konumlandırma electroporated DNA aldığı bölgenin belirlenmesi için çok önemlidir. Dorsal embriyonik ardbeyin alt eşkenar dudak (LRL) isteniyorsa, kürek doğrudan dördüncü ventrikül açıklığı olan en geniş kısmını taarruzcu olduğu gibi yer olmalıdır. </li><li> % 0.01 Fast Green ile karıştırılır plazmid DNA hazırlamak için 1 cc şırınga kullanın. Karışım 500 uL, en azından 8-10 embriyosu (3,5 bakınız) elektroporasyon için yeterli olmalıdır. </li><li> Yavaşça ponksiyon roofplate ov1 cc enjektöre bağlanmış 25G 5/8 tüberkülin iğnesi ile dördüncü ventriküle erlying ve ventrikül içine DNA-boya karışımı enjekte edilir. Başarılı enjeksiyonlar tamamını ventriküler sistemi (Şekil 1C) dolum DNA-boya karışımı ile karakterize edilir. Tipik olarak enjekte DNA-boya karışımının miktarı en az 50 uL olup. Karışımın bir kısmı embriyonun çevresindeki PBS içine ventrikül taþmasýna eğilimi kesin miktarda embriyoları arasında değişkendir. DNA-boya karışımı teslim alternatif demektir Ortabeyin örten zar delinmesiyle ventriküler sisteme erişen yoluyla olacaktır. Yine, başarılı bir enjeksiyon tamamını ventriküler sistemi dolum DNA-boya karışımı ile karakterize edilir. </li><li> Elektrikli bir darbe jeneratörü (BTX) ve 7 mm elektrot kürekler kullanarak beş kare bakliyat sunun. Her darbe, her darbe arasında 500 ms puls başına 50 V ömürlü 5 ms. Pozitif yüklü elektrot en yakın doku tak sonra olacakplazmid kadar e. </li></ol> 4. Embriyolar Kültür <ol><li> Bir laminar akış davlumbaz olarak,% 10 fetal bovin serumu,% 5 equine serumu,% 1 glutamin,% 1 penisilin / streptomisin ısıtılmış edildi ile desteklenmiş DMEM/F12 ortam 2 ml ile 12 bölümlü kültür çanağı dış kuyu dolgu 37 ° C Kültür koşullarında de Diego ve arkadaşları uyarlandı. 15 </li><li> Forseps ile midsection (kalp aşağıda) laminer akış kaput tutam embriyo ve embriyonun arka kısmını kaldırın. 12 oyuklu kültür kabının doldurulması kuyuları birine ön kısmına yerleştirin. </li><li> Tüm embriyolar için tekrarlayın. Kültürlerin kirlenmesini önlemek için 12-iyi plaka sadece dış kuyuları doldurun. </li><li> % 5 CO2 içeren bir inkübatör 37 ° C de kültüre embriyosu. Electroporated ekspresyon plazmidi 24 saat içinde gözlemlenebilir edilmelidir. </li><li> Artık kültür kez istenen olmalı, 2 mL ile yeni bir 12 de plaka Kuyulardan doldurunOrtam 4,1 kullanılır. Steril bir kaşık ile yeni plaka bir kuyuya embryo transfer. 37 ° C inkübatör içine geri yerleştirin. 48 saat için Kültür mümkündür. </li><li> İstenen kültür zamanı geldiğinde, (bkz. aşağıda) analizi için embriyo düzeltin. </li></ol> 5.. Analiz için Embriyolar hazırlanması <ol><li> Beş dakika süreyle 4 ° C &#39;de 1 X PBS içinde embriyo çalkalayın. Tekrarlayın. </li><li> 4 ° C&#39;de 2 saat süreyle 1XPBS içinde% 2 paraformaldehit analiz (PFA) sabitlemek için embriyosu </li><li> Beş dakika süreyle 4 ° C &#39;de 1 X PBS içinde embriyo çalkalayın. Tekrarlayın. </li><li> 4 gece 1 X PBS içinde% 30 sükroz embriyoların dengeye ° C </li><li> Bir kuru buz / etanol banyosu kullanılarak Optimum Kesme Sıcaklığı (OCT) bileşik embriyoların Embed. Embriyolar -20 ° C&#39;de saklanabilir </li><li> 30 mikron bölüme kriyostat (Leica) ve (VWR, SuperFrost Plus) kızaklar üzerinde monte bölümü embriyoların. -20 ° C de saklayınız </li></ol> 6. İmmünoreaktifnohistochemistry Analizi <ol><li> 16 içinde açıklandığı gibi İmmünohistokimya yapıldı. Fare α-Mash1 (BD Biosciences) 1:100;; tavşan α-Ngn1 (Jane Johnson) 1:5000; tavşan α-Ptf1a (Jane Johnson bu çalışma için kullanılan primer antikor dilüsyonları tavşan α-GFP (Invitrogen) 1:2500 dahil ) 1:2500; tavşan Math1 (Jane Johnson) 1:100. 4 ° C&#39;da bir boyama tepsi üzerinde slayt düz, örnek tarafı yukarı inkübe edin. </li><li> Slaytlar bir bileşik mikroskobu (Olympus BX51) üzerinde analiz edildi. </li></ol> 7. Temsilcisi Sonuçlar Şekil 1A şematik elektroporasyon deneyi anlatıyor. Şekil 1B manipülasyon önce bir E11.5 embriyonun sagittal bir görünümdür. % 0.01 Fast Green plazmid içeren CAG :: GFP enjeksiyonu takiben aynı embriyo Şekil 1C ve tek taraflı GFP sergileyen bir temsilci sabitlenmemiş embriyo gösterilirdorsal ardbeyin 24 saat kültüre aşağıdaki ifade Şekil 1B &#39;de gösterilmiştir. Başarıyla electroporated edilir LRL alanının ölçüde değişkenlik göstermektedir ve bu elektrot konumlandırma çok bağımlı olarak görünmektedir. Bizim çalışmalarda electroporated embriyoların 65 (% 80) 52 dışında başarıyla GFP ifade bulunmuştur. Doku bu tespit ve immünohistokimyasal analizi (bkz. aşağıda) sonra çeşitli bölümler üzerinde lokalize bölgelerde GFP pozitif ise başarıyla electroporated olarak kabul edildi. Bu kriterlere uymayan Embriyolar elektroporasyon de başarısız girişimler olarak skorlandı. Elektroporasyon verimliliği daha fazla incelenmesine dördüncü ventrikül düzeyinde electroporated embriyoların enine kesitlerinde GFP karşı immünohistokimya yapılarak tespit edilebilir. Şekil 2 tek taraflı gösteren bir embriyodan temsilcisi seri bölümleri gösterirGFP. Şekil 2A embriyonun sol tarafına pozitif elektrot doğru olduğunu gösteren bir idealize şematik gösterir. Enine bölümleri (Şekil 2A, orta çizgi ile gösterilen seviyelerde) arka beyin doku (Şekil 2C ve 2B) sol tarafında münhasıran lokalize GFP tanımı ortaya koymaktadır. Şekil 2C gösterildiği üzere 300 mikron rostral veya daha büyük bölümlerde muayene express GFP (Şekil 2B) olmadığı için LRL ile electroporated alan nöral tüpün tüm ön-arka eksen vermemiştir. Gen ekspresyonunun manipülasyonu için Bu testin yardımcı endojen proteinler için ifadesi etki stabilite bağlıdır. LRL proneural transkripsiyon faktörlerinin diferansiyel ifadesi (1 Yorumlar) ile karakterize benzersiz progenitör etki sahip olarak karakterize edilmiştir.Bu faktörlerin bir alt kümesi (Mash1, Math1, Ngn1 ve Ptf1a) LRL, gelecekteki çalışmalarda 16-18 bir konuda precerebellar nöral alt tiplerinin özellikleri kendi önerilen ve / veya karakterize rolleri nedeniyle analiz için seçilmiştir. Dört proteinler E11.5 16-18 az kaudal ardbeyin oldukça karakteristik ifadeler etki var. Biz kültürler yerleştirildi embriyo büyüklüğü artırmak için başarısız oldu ve aynı zamanda koroid pleksus epitel ve E11.5 ve E12.5 5 arasında meydana LRL ve roofplate, morfolojik olaylar invajinasyon üretimi başlatmak için başarısız olduğu görülmektedir. Bu gözlemlere dayanarak bu embriyoların normal gelişimini durdurdu veya fena halde geciktirdi ve kültürlü embriyolar için karşılaştırılabilir kontrol E11.5 az kültürsüz embriyo olmalıdır tespit edildi. Kültür ve elektroporasyon endojen protein düzeyleri rahatsız etmemelerini sağlamak için, immunohistoch tarafından dört farklı proteinler analizelectroporated ve en az 24 saat sonra kültür alındı ​​34 farklı embriyolarda emistry (İHK). Tablo muhafaza Normal protein düzeyleri. Şekil 3 temsilcisi IHC veriler gösteriyor ki analiz her marker ve embriyoların yüzdesi için analiz embriyo sayısını gösterir proteinler iki analiz gelen, Mash1 (Şekil 3A ve 3B) ve Math1 (Şekil 3C ve 3B). Biz E11.5 (Şekil 3A ve 3C) de kontrol embriyo kıyasla embriyo çoğunluğu ifadesi takiben elektroporasyon ve kültür (Şekil 3B ve 3B) normal seviyede muhafaza gözlemlenmiştir. Önemli olarak, bu proteinlerin karakteristiği etki ifadesi pertürbe edilmemiştir. <img src="/files/ftp_upload/3983/3983fig1.jpg" alt="Şekil 1"/> Şekil 1.. Th E11.5 az Embryolarının Elektroporasyon. (A) Şematike elektroporasyon deney. Bir E11.5 embriyo izole edilir ve% 0.01 Fast Green plazmid ifade dördüncü ventrikül içine enjekte edilir. Embriyo daha sonra elektrot kürekler tarafından yanlı ve önceden kültürü içine enjeksiyon önce bir E11.5 embriyo (B) Sagital görünümü konuyor için 50 V darbe tabi tutulur. (C) aynı E11.5 embriyo% 0.01 Fast Green plazmid enjeksiyonu takiben. (D) GFP tek taraflı arka beyin ifade kültür 24 saat takip E11.5 embriyo gözlenmiştir. mb-orta beyin; 4v-dördüncü ventrikül. <img src="/files/ftp_upload/3983/3983fig2.jpg" alt="Şekil 2"/> Şekil 2. Electroporated Doku GFP ekspresyonu. (A) sol karikatür bir E11.5 embriyonun çevresindeki elektrot yerleştirme gösteriyor. Orta karikatür embriyonun sol tarafında plazmid kodlama GFP alımı ve ifade resmediyor. Sağdaki Karikatür l embriyo üzerinden alınan idealize enine bölümün şemasıevels orta karikatür siyah çizgiler ile gösterilir. Kültür 24 saat sonra bir electroporated E11.5 embriyo ile enine kesitlerinde GFP (B-D) İmmünohistokimya. Oklar roofplate (rp) hapseden sekonder antikor göstermektedir. Görüntüler 10X büyütme alınır. Gösterilen kesitlerin nispi seviyeleri (A) &#39;in, orta çizgi boyunca yatay çizgiler ile tasvir edilmektedir. mb-orta beyin, l 4v-dördüncü ventrikül; rp-roofplate. <img src="/files/ftp_upload/3983/3983fig3.jpg" alt="Şekil 3"/> Şekil 3. Alt Rombik Dudak Endojen Proteinlerin ifadesi Mash1 (A ve B) veya CAG ile electroporated edildi embriyolu (A, C) kültür değillerdi E11.5 embriyo enine bölüm karşılaştırarak Math1 (C ve D) için İmmünohistokimya.: : GFP ve 24 saat (B ve D) için kültür. Görüntüler 10X büyütmede alınmıştır. <table border="1"><tbody><tr><td> Proneural TranscriptiFactor üzerinde Analyzed </td><td> Embriyolar sayısı İncelendi </td><td> Normal İfade Desenler İstinat Yüzde </td></tr><tr><td> Math1 </td><td> 15 </td><td> % 86.7 </td></tr><tr><td> Mash1 </td><td> 12 </td><td> % 83.3 </td></tr><tr><td> Ngn1 </td><td> 7 </td><td> % 71.4 </td></tr><tr><td> Ptf1a </td><td> 6 </td><td> % 100 </td></tr></tbody></table> LRL Normal Proneural Transkripsiyon Faktörü Alanlar İstinat Electroporated ve kültür Embriyoların Tablo I. yüzdesi.

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Çıkar çatışması ilan etti.

Bu çalışmada sunulan vitro elektroporasyon tekniği verimli gebeliğin 12. günden küçük embriyolarda gen ekspresyonu işlemek için kullanılabilir yeni bir yöntemdir. Kültürüne embriyoların Yerleştirme tanıttı gen ekspresyonu izin verir ve electroporated embriyolar in vivo olarak kalmasına izin verildiğinde öldürücü gözlenen circumvents. Bu teknik elektroporasyon tabanlı çalışmalar için daha önce ulaşılamayan embriyonik ataları gen ifadeleri değişiklik yapıla...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> Kriyostat </td><td> Leica </td><td> CM-1850 </td><td></td></tr><tr><td> Biologie ucu Dumoxel DUMONT forseps tedavi </td><td> Güzel Bilimsel Araçları </td><td> 11252-30 </td><td></td></tr><tr><td> 20 mm Moria delikli kaşık </td><td> Güzel Bilimsel Araçları </td><td> 10370-17 </td><td></td></tr><tr><td> ECM 830 Kare Dalga Elektroporasyon Generator </td><td> BTX (VWR) </td><td> 47745-928 </td><td></td></tr><tr><td> Harvard Apparatus 7 mm Tweezertrodes * Elektrotlar </td><td> BTX (Fisher) </td><td> BTX450165 </td><td></td></tr><tr><td> Fisher Isotemp CO 2 İnkübatör </td><td> Balıkçı </td><td> 1325525 </td><td></td></tr><tr><td> NAPCO CO 2 Gaz Regülatörü </td><td> Balıkçı </td><td> 15497020 </td><td></td></tr><tr><td> 12 Peki Doku Kültürü Tabaklar </td><td> BD Falcon (Fisher) </td><td> 877229 </td><td></td></tr><tr><td> HyClone Sıvı Ortamda DMEM/F-12 (1:01); L-glutamin ve HEPES grubu; 500ml </td><td> Thermo Scientific (Fisher) </td><td> SH3002301 </td><td></td></tr><tr><td> HyClone * Donör Atçılık Serum </td><td> Thermo Scientific (Fisher) </td><td> SH3007402 </td><td></td></tr><tr><td> İnaktive Fetal Bovine Serum, Nitelikli, Isı </td><td> Invitrogen </td><td> 16140-063 </td><td></td></tr><tr><td> cellgro * 10.000 IU Penisilin, 10,000 mcg / mL Streptomisin </td><td> Mediatech (Fisher) </td><td> MT-30-002-CI </td><td></td></tr><tr><td> HyClone * L-Glutamin L-Glutamin; 20% 0.85 NaCl 0mm </td><td> Thermo Scientific (Fisher) </td><td> SH3003401 </td><td></td></tr><tr><td> Fast-Yeşil </td><td> Balıkçı </td><td> AC41053-0250 </td><td> % 0.01 </td></tr></tbody></table>

in vitro electroporation of the lower rhombic lip of midgestation mouse embryos

Eşkenar dudak nöral tüp ve dördüncü ventrikül (1 Yorumlar) ile roofplate arasındaki kavşakta arka beyin bulunan bir embriyonik neuroepithelium olduğunu. Eşkenar dudak rhombomere 1 (R1) kapsar ve beyincik nöronlar ve çeşitli nöronal beyin soy 2-4 sebebiyet veren alt eşkenar dudak (LRL) oluşturduğu eşkenar üst dudak (URL) bölünmüştür edilebilir. LRL türevleri işitsel koklear çekirdeklerin nöronlar ve denge ve motor kontrol 5-8 düzenleyen katılan precerebellar çekirdeklerin olanları içerir. LRL gelen nörogenez embriyonik gün (E) 9,5-16,5 5, 9 kapsayan geniş bir zamansal pencere içinde oluşur. Farklı nöronal soy bu nörojenik penceresi sırasında farklı gelişimsel gün boyunca postmitotik hücreleri (veya doğar) gibi LRL çıkarlar. Gen ifadesi yapıları elektroporasyon için kullanılabilirLRL ataları gen ekspresyonu işlemek ve potansiyel olarak bu bölgede 10-12 üretilen nöronların kaderini değiştirebilirsiniz. Utero elektroporasyon yılında aracılığıyla fare LRL progenitörlerin değiştirilmesi gen ekspresyonu embriyonik gün E12.5 veya daha sonra 10, 12-14 doğumlu soy işlenmesi için oldukça başarılı olmuştur. Utero electroporations yılında öncesinde E12.5 öncelikle nedeniyle başarısız olmuştur ölümcül dördüncü ventrikül roofplate, LRL içine electroporated olan eksojen DNA sağlanmasında gerekli bir adım delinme ile ilişkilidir. Ancak, birçok LRL edilen soyların önceki E12.5 9&#39;dan LRL kaynaklanmaktadır. Bunlar daha önce doğan soy lateral retiküler oluşturan nöronlar, dış cuneate ve beyincik 5 omurilik ve korteks girişleri bağlamak için işlev precerebellar sisteminin alt olivary çekirdekleri içerir. LRL ifade işlemek amacıylaE12.5 daha genç embriyo, bu embriyosu elektroporasyon takiben kültürü içine yerleştirilmiş olduğu bir in vitro sistemi geliştirilmiştir. Bu çalışma E11.5 az LRL progenitörlerin gen ifadesini değiştirmek için etkin ve verimli bir yöntem sunar. Genel olarak aktif CAG organizatörü sürüldü yeşil floresan protein (GFP) ile electroporated embriyolar tekrarlanabilir kültür 24 saat sonra GFP dile getirdi. Bu testin kritik bir yönü olduğunu gen ekspresyonu çünkü sadece eksojen geninin ifade değil, çünkü ikincil etkiler değiştirilmiş olmasıdır elektroporasyon ve kültür teknikleri sonucu bu. Bu endojen gen ekspresyonu electroporated ve kültürlü embriyolarda bozulmadan kalır olduğu tespit edilmiştir. Bu test için aşırı plazmidlerin tanıtım yoluyla E12.5 daha genç embriyoların LRL çıkan hücrelerin kaderini değiştirebilir veya (RNAi yoluyla) yıkmak için kullanılabilecek farklı pro-nöral transkripsiyon faktörleri.

Watch this Scientific Journal Video about In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos at JoVE.com

In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos

Bu çalışma, bir gelişmesi açıklar<em&gt; In vitro</emMidgestation embriyoların eşkenar alt dudakta gen manipülasyonu için izin&gt; elektroporasyon tekniği.

Midgestation Fare Embryolarının Alt Rombik Dudak in vitro Elektroporasyon yılında

The rhombic lip is an embryonic neuroepithelium located in the hindbrain at the junction between the neural tube and the roofplate of the fourth ventricle ...

in-vitro-electroporation-lower-rhombic-lip-midgestation-mouse

Research

JoVE Journal

Neuroscience

In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos

10.3K Görüntüleme.  University of Illinois at Springfield. Bu çalışma, bir gelişmesi açıklar In vitro elektroporasyon tekniği.

Video: In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

Fare Retina Ganglion Hücreleri Transfeksiyon tarafından In vivo Elektroporasyon

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Nörobilim

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to cis-regulatory elements (CREs), short (&tilde;300-600 bp) stretches of genomic DNA which can lie upstream, downstream, or within the introns of the genes they control. CREs (i.e., enhancers/promoters) typically consist of multiple clustered binding sites for both transcriptional activators and repressors1-3. They serve as logical integrators of transcriptional input giving a unitary output in the form of spatiotemporally precise and quantitatively exact promoter activity. Most studies of mammalian cis-regulation to date have relied on mouse transgenesis as a means of assaying the enhancer function of CREs4-5. This technique is time-consuming, costly and, on account of insertion site effects, largely non-quantitative. On the other hand, quantitative assays for mammalian CRE function have been developed in tissue culture systems (e.g., dual luciferase assays), but the in vivo relevance of these results is often uncertain. 
Electroporation offers an excellent alternative to traditional mouse transgenesis in that it permits both spatiotemporal and quantitative assessment of cis-regulatory activity in living mammalian tissue. This technique has been particularly useful in the analysis of cis-regulation in the central nervous system, especially in the cerebral cortex and the retina6-8. While mouse retinal electroporation, both in vivo and ex vivo, has been developed and extensively described by Matsuda and Cepko6-7,9, we have recently developed a simple approach to quantify the activity of photoreceptor-specific CREs in electroporated mouse retinas10. Given that the amount of DNA that is introduced into the retina by electroporation can vary from experiment to experiment, it is necessary to include a co-electroporated 'loading control' in all experiments. In this respect, the technique is very similar to the dual luciferase assay used to quantify promoter activity in cultured cells. 
When assaying photoreceptor cis-regulatory activity, electroporation is usually performed in newborn mice (postnatal day 0, P0) which is the time of peak rod production11-12. Once retinal cell types become post-mitotic, electroporation is much less efficient. Given the high rate of rod birth in newborn mice and the fact that rods constitute more than 70% of the cells in the adult mouse retina, the majority of cells that are electroporated at P0 are rods. For this reason, rod photoreceptors are the easiest retinal cell type to study via electroporation. The technique we describe here is primarily useful for quantifying the activity of photoreceptor CREs.

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

This protocol describes a simple and inexpensive way to quantify the activity of cis-regulatory elements (i.e., enhancer/promoters) in living mouse retinas via explant electroporation. DNA preparation, retinal dissection, electroporation, retinal explant culture, and post-fixation analysis and quantification are described.

Aktivitesi miktarının Cis</emEksplant Elektroporasyon Fare Retina> Düzenleme Elemanları

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

Gelişmekte olan Fare Retina in vivo Elektroporasyon olarak

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

Fare Utero Elektroporasyon: Kontrollü Spatiotemporal Gen Transfeksiyon

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

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 ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

Kullanarak Fare Serebral Korteks ve Hippocampus Görselleştirme ve Dendritler ve Spine Genetik Manipülasyon<em&gt; Utero olarak</em&gt; Elektroporasyon

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox&#x2013;plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

Civciv Embriyo aksonal Yörüngeler ve Synaptic Hedefler Trace için Beyin elektroporasyon

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

Fare Genetik Manipülasyon yoluyla Hipotalamus geliştirilmesi<em&gt; Utero olarak</em&gt; Elektroporasyon

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

Bir<em&gt; In-vitro</emYetişkin Mouse myenterik Pleksus izole edilen enterik Nöronlar ve Glia bir&gt; hazırlanması

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.