We are indebted to Pierre Mattar and Freda Miller for their assistance with the in utero electroporation and to Noel Ghanem and Bensun Fong for creating diagrams. We thank Linda Jui and Jason G. MacLaurin for excellent technical assistance. Equipment was supported by the Centre for Stroke Recovery.
This work was supported by a scholarship from HSFO and OGS to D.S.S. and a CIHR grant to R.S.S.

NOT: Tüm deneyler Hayvan Bakımı Kanada Konseyi Rehberi bağlı kalarak, Ottawa Hayvan Bakım etik kurul Üniversitesi tarafından onaylanmıştır. 1. Ön cerrahi hazırlanması ve Malzeme <ol><li> Plazmid hazırlanması: <ol><li> Üreticinin protokolüne göre Qiagen MegaPrep kullanılarak plazmidler hazırlayın. Yüksek DNA verimi sağlar ve bir gece boyunca inkübe etmek, transforme edilen bakterilerden LB et suyu, en az 400 ml aşılanmasında. </li><li> En az 5 ug / ml&#39;lik bir son konsantrasyona kadar, 100-200 ul TE içinde yeniden süspanse plazmid. -20 ° C&#39;de kısım, DNA ve saklayın. Konsantrasyonu çok düşük ise, çökmez ve bu kirlilikleri ekleyebilirsiniz olarak tekrar süspansiyon. Yeni bir hazırlık üzerine başlayın. </li><li> Ameliyat öncesi% 0.1 seyreltilmiş plazma çözeltisi 10 ya da 20 ul başına (ağırlık / hacim) hızlı yeşil boya 1 ul steril su içinde 2 ug / ml tek bir plazmid (örneğin Kre-GFP) seyreltin. GFP EğerGFP ile işaretlenmiş tüm hücreler hem de-elektroporasyona CO ihtimalini en üst düzeye çıkarmak için 4: ve ilgi konusu genin, GFP plazmidi ve 1 arasında bir oranda ilgilenilen geni taşıyan plazmid enjekte ayrı plasmidler üzerindeki birlikte elektropore edilecekse plazmidler. </li></ol></li><li> 62 ° C &#39;de bir tek aşamalı çekme kullanılarak, standart bir iğne çektirmenin - Cam Mikro kapiller tüpler (bundan sonra iğneler olarak adlandırılır 1 mm dış çapında) çekin. </li><li> Aşağıdaki içeren bir cerrahi paketi Otoklav: iğne sahipleri, yara retraktör, forseps, cerrahi makas, iğne için makas, zımba, zımba, gazlı bez, yara örtüleri, cerrahi örtüler (Şekil 1A, B; Malzeme Tablosu). </li><li> Diğer gerekli araçları toplayın: Electroporator, FemtoJet Microinjecter, Tweezertrodes (5 veya 7 mm), tuzlu, dikiş, 5 ml şırınga (Malzeme Tablo). </li></ol> 2. Cerrahi Fare hazırlayın <ol><li> Ağrı yönetimi için, bir zamanlanmış-p enjektebir saat ameliyat öncesi Buprenorfin (0.05 mg / kg vücut ağırlığı) ile regnant fare. E17.5 embriyonik gün 12.5 (E12.5) yerden zamanlanmış farelere bu protokolü uygulayın. </li><li> Arka yük için Microloader pipet ipuçlarını kullanın plazmid çözümü ile iğne çekti. Bu iğne tıkalı hale gelmesine neden olacaktır henüz iğne ucu içine tüm yol doldurmayın. </li><li> 1 L / dk O 2 ile% 2-5 izofluran gaz kullanarak fare uyuşturan. Bir kez hareketsiz, yüz maskesi yerleştirmek ve kurumasını önlemek için gözlere merhem uygulayın. 1 ml tuzlu suda arka (% 0.9 NaCl), deri altından enjekte edilir. NOT: izofluran gaz ile çalışırken, aşırı gaz hatırlamak ve odasına kaçmasını önlemek için gaz süpürücü kullanın. </li><li> Karın Tıraş. , Chlorohexadine fırçalayın ile karın yıkayın su ile durulayın ve chlorohexadine ve% 70 etanol son hazırlık çözümü uygulayın. Steril örtü veya gazlı bez ile kapak fare kürk kapsayacak, ancak karın maruz (Şekil 1C, D) terk etmek. </li></ol>utero Elektroporasyon olarak <ol><li> Alt meme arasındaki karın üzerinde deride bir kesi yapmak. Bağ dokusu yırtılarak alttaki kas tabakasından cilt çekin, sonra linea alba boyunca karın boşluğuna periton kesti. Ayrı yara kenarlarını çekmek için yara ekartörünü yerleştirin. </li><li> Karın boşluğundan rahim çıkarın ve gösterilen (Şekil 1E) olarak ortaya koymak. <ol><li> Farklı yavrular (örneğin, ilgi konusu gen ile GFP genel GFP gibi), farklı plazmitler ile enjekte edilecek ise, her iki tarafta yavrular sayısı ve her bir plasmid ile kaç enjekte hangilerinin ve karar verirler. Geri sıcak ve yağlanmış embriyolar tutmak için karın içine rahim bir tarafını değiştirin. Hemen serum fizyolojik ile maruz rahim (tercihen vücut sıcaklığına ısıtılmış) nemlendirin. </li><li> Aynı plazmid tüm yavrular enjekte Eğer haftaları (örneğin transgenik embriyolar içine Kre-GFP enjekte olduğu gibi)Y aynı anda rahim bir boynuz çıkarın. </li></ol></li><li> Küçük cerrahi makas veya ince forseps bir çift ile ucu kesilmiş dikkatle enjeksiyon değnek kavrama kafasına dolu bir iğne koyun ve. Henüz akışkan bir (Şekil 1Ji-iv) geçmesine izin verirken, iğne sürece mümkün kalması ucu kesilir. <ol><li> Ayak kürek basıldığında 0.1-0.2 ul küçük ama kolayca görülebilir damlacık iğne ucu görünür şekilde mikropüskürtücü (saniye cinsinden) enjeksiyonu uzunluğunu ve enjeksiyon basıncını ayarlayın. Farklı iğneler arasında mümkün olduğunca tutarlı Bu damlacık hacmini tutun. NOT: Bu adım, embriyo hayatta (tartışma) için önemlidir. </li><li> FemtoJet için aşağıdaki enjeksiyon parametrelerini kullanın: basınç enjekte (pi) = 250-300 hPa; Enjeksiyon süresi (ti) = 0.8-1.2 sn; tazminat basıncı = 10-50 hPa. Gerekirse, iğneler arasında bir farklılaşmanın düzeltileceği bu parametreleri ayarlamak. </li></ol></li><li> SBir embriyo seçme ve yavaşça arka, yukarı eğimli kafa (Şekil 1F) üzerine yerleştirin. NOT: Bu dönüm veya embriyo değişen gerektirebilir; Çok fazla baskı uygulamak ve embriyo manipüle ederken amniyon kesesinin yırtılması önlemek için dikkatli olun. </li><li> Embriyo yerleştirildikten sonra, sagital sinüs bir hilal şeklinde gölge paralel olarak sunuyor ventrikül bulun. Hafif bir açıyla ventrikül üzerindeki rahim iğne ucu dokunun ve rahim duvarından iğne rehberlik. Gerekli basmç miktarı iğne netlik bağlıdır. Ventrikül içine korteks ile iğne itin. NOT: nadiren herhangi bir ek saptanabilir direnç olmasına rağmen, embriyonun kafası genellikle biraz uzağa itti ve sonra iğne ventrikül içine korteks geçerken geri teper. </li><li> Plazmid çözüm enjekte etmek için ayak raket Pompa. Yeşil alan görünen ve uygun kadar pompalamaya devamventrikül (Şekil 1G) ark şekli. Genç embriyoların (E14.5 E12.5 için) boya genellikle karşı ventrikül içine nüfuz edecek. Not: pompa sayısı iğne ucu çapına bağlıdır, embriyo (ve karıncığın elde edilen boyutu) ve istenilen enjeksiyon hacmi yaşı. Hacmi tutun embriyolar arasında mümkün olduğunca tutarlı enjekte. </li><li> Üç ya da dört embriyolar enjekte edilir. Prosedür boyunca tuzlu su ile nemli tüm maruz embriyolar tutun. </li><li> Geri enjekte ilk embriyoların gidin ve pozitif elektrot beynin (Şekil 1 H) istenen bölgeye plazmid çekecektir böyle kürekler yerleştirin. Açı negatif elektrot için elektrotlar arasındaki en kısa yol elektroporasyon için hedef yapı (Şekil 2) içinden geçer. </li><li> Kürekler yerdesiniz kez, şok uygulanır. Embriyoların yaş ve boyutuna bağlı olarak, 35-50 V 5 bakliyat kullanın (T bkzmümkün 1). Aşağıdaki elektroporasyon parametreleri kullanın: 5 msn darbe uzunluğu 950 msn darbe aralığı ile. Tüm enjekte embriyolar ile tekrarlayın. </li><li> Geçen iğne olasılıkla kurutulmuş ve adım 3.9 sırasında tıkanmış gibi adım 3.3 gibi yeni ön-yüklenmiş iğne ucu kesin. Aynı plasmid ya da farklı bir plazmid ya da üç ya da dört embriyo sonraki resim enjekte edilir ve daha sonra adım 3.9 olarak darbe uygulanır. Rahim bir tarafında tüm yavrular enjekte edildiğinde, dişinin karın rahim ikinci tarafını kaldırın ve daha sonra sıcak tutmak için tamamlanmış tarafı değiştirin. </li><li> Amnion kesesi travma önlemek için çok fazla baskı uygulamak için tuzlu su ile değil, nemli yüzey tutmak için dikkatli olmak kadın rahim her iki tarafını dön. Basit bir sürekli dikiş kullanılarak kapatıldı kas tabakası dikin. Zımba cilt kapattı. Zımba mevcut değilse, basit bir kesintiye dikiş ile cilt sütür. (Şekil 1 H) </li><li> Suturi dang, giderek hamile dişinin anestezi hafifletmek için izin izofluran aşağı çevirin. NOT: Ameliyat tamamlandıktan sonra bu barajın uyarılma hızlandırmak olacaktır; embriyo yaşam artıracak mümkün olduğunca kısa bir süre için anestezi kadın tutmak. Toplam ameliyat süresi, kadın ilk kez anestezi ne zaman başlayarak, yaklaşık 30 dakika olmalıdır. </li></ol> 4. Sonrası Bakım <ol><li> Bir inkübatör veya anestezi kurtarmak için bir ısıtma pedi üzerinde dikildi kadın yerleştirin. Temiz yatak onu yatak ve kafes içinde standart bir toparlanma jel hidrasyon korumak yerleştirin. Cerrahi sonrası ağrı tedavisi için, daha sonra ameliyat sonrası 8 saat aralıklarla, 12 saat aralıklarla dört ek dozlar en Buprenorfin (0.05 mg / kg vücut ağırlığı) bir kadın üç doz vermek. </li></ol> 5. Hasat Brain NOT: Brain yetişkinliğe herhangi bir yaş kadar hasat edilebilir. şu geçerlidirdoğumdan önce beyinleri toplamak için. Yavrular dünyaya bir kez, rahim içinde sipariş çöp her yavru ya aynı plazmid ile enjekte edilmelidir, böylece kaybolur, ya da kontrol ve deney hayvanlar arasındaki farklılaşma başka bir yöntem icat gerektiğini unutmayın. <ol><li> Soğuk (4 ° C) Hazırlama kadın ötenazi önce% 4 paraformaldehid (PFA) filtre edilmiştir. </li><li> Sodyum pentobarbital (100 mg / kg vücut ağırlığı) öldürücü bir dozda intraperitonal enjeksiyonu ile dişi öldürülür ve açık karın kesti. Rahim iki tarafını çekin ve yavrular başlangıçta enjekte edildiğinde olarak yatıyordu. <ol><li> Tüm yavrular gibi bir transgenik çöp içine Cre enjekte ederken aynı plazmid, alınan durumunda, embriyo sırası nedenle PBS içinde kadın ve yersiz rahim kesmek, önemli değil. </li><li> Yavrular farklı plazmid ile enjekte olsaydı, herhangi bir ölü yavrular belirterek, her yavru bulmak için emin olun, kontrol ve experimen enjekte edildi hangilerinin belirlemekkadın rahim çıkarmadan önce tal plazmidler. Yavru uterus PBS içinde hangi grup daha sonra ait olduğu izlemek için dikkatli olun. </li></ol></li><li> Her biri tek tek yavru çıkarın keskin bir makas veya bir tıraş bıçağı ile başını kesmek ve PBS içinde kafa yerleştirin. Transgenik farelerin durumunda, genotipleme için doku numuneleri toplamak ve bir 12-çukurlu plaka içindeki gibi, ayrı ayrı her bir kafayı tutun. </li><li> Diseksiyon kapsamında, değil nick dikkatli olmak, kafatası korteks beyin kaldırmak veya orta hat gözyaşı. Bir floresan mikroskop kullanarak, başarılı elektropore belirten, beyinleri bir GFP + yama olup olmadığını görmek için kontrol edin. </li><li> Gece boyunca 4 ° C&#39;de PFA içine daldırma ile beyin sabitleyin. NOT: PFA sık sık sinyal doyurur çünkü önce tespite GFP ifade beyinlerini kontrol edin. </li><li> Dondurulmadan önce en az üç gün boyunca% 20 sukroz içinde daldırma yoluyla cryoprotect beyinleri. T sükroz% 10 gradyan,% 15 ve% 20 (her bir çözelti içinde, 24 saat) kullanarakO ozmotik basınca bağlı doku hasarı önlemek. -80 ° C&#39;de dondurulmuş beyinleri saklayın ve 10-14 mikron kalınlığında kesitler bir kriyostat üzerinde kesti. </li></ol> Anti-GFP ile 6. Ortak boyama Not: antijen geri alma ko-leke anti-GFP ile gerekli ise, GFP sinyali ödün vermeyen hafif bir antijen geri alma protokolü kullanılması önemlidir. Bazı antijen alma protokolleri (hatta GFP karşı bir antikor ile) GFP boyama ile birlikte kullanmak için çok sert. Önce birincil antikor uygulaması temel sitrik asit ön-işlem, en epitoplar 24 için başarılı olur. <ol><li> GKD 2 O. sitrik asit 0.01 M bir solüsyon yapmak bir kalibre edilmiş pH metre kullanılarak 6 pH değerine sahiptir. </li><li> Sitrik asit kadar mikrodalga ya da su banyosu içinde boyanması odasındaki ısı sitrik asit, 75-80 ° C&#39;ye ulaştı. 10-15 dakika boyama odasında slaytlar koyun. Mümkün olduğunca istikrarlı olarak sıcaklığını muhafaza. </li><li> PBS slaytlar yıkayın3x, oda sıcaklığında 3 dakika daha karıştırıldı. Primer antikor uygulayın ve bir standart protokole göre immün devam edin. </li><li> (Şekil 3A) yıkmak onaylamak için GFP eş-etiketleme ve ilgi gen tarafından Cre aracılı eksizyon doğrulayın. </li></ol>

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Cortex. 19, 120-125 (2009).</li><li>Bouabe, H., Gene Okkenhaug, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=targeting+in+mice:+a+review.">targeting in mice: a review.</a> Methods Mol. Biol. 1064, 315-336 (2013).</li><li>Ferguson, K. 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=A+cell-autonomous+requirement+for+the+cell+cycle+regulatory+protein,+Rb,+in+neuronal+migration.">A cell-autonomous requirement for the cell cycle regulatory protein, Rb, in neuronal migration.</a> EMBO J. 24 (24), 4381-4391 (2005).</li><li>Lagace, D. C., 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=Dynamic+contribution+of+nestin-expressing+stem+cells+to+adult+neurogenesis.">Dynamic contribution of nestin-expressing stem cells to adult neurogenesis.</a> J. Neurosci. 27 (46), 12623-12629 (2007).</li><li>Andrusiak, M. G., Vandenbosch, R., Park, D. S., Slack, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+retinoblastoma+protein+is+essential+for+survival+of+postmitotic+neurons.">The retinoblastoma protein is essential for survival of postmitotic neurons.</a> J. Neurosci. 32 (42), 14809-14814 (2012).</li><li>Shan, B., Chang, C. Y., Jones, D., Lee, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcription+factor+E2F-1+mediates+the+autoregulation+of+RB+gene+expression.">The transcription factor E2F-1 mediates the autoregulation of RB gene expression.</a> Mol. Cell. Biol. 14 (1), 299-309 .</li><li>Ferland, R. J., Cherry, T. J., Preware, P. O., Morrisey, E. E., Walsh, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Characterization of Foxp2 and Foxp1 mRNA and protein in the developing and mature brain. 460 (2), 266-279 (2003).</li></ol>

The authors have nothing to disclose

Utero electroporations performans birincil sorunlar iki embriyo öldürücü ve 2) electroporations arasındaki değişkenliği azaltarak engelliyor) 1 bulunmaktadır. Künt iğne embriyo daha fazla hasar gibi öldürücü önlemede en önemli faktörlerden biri, iğne kalitesidir. İğneyi keserken hala ayak kürek (Şekil 1J) bir pompa aşağıdaki görünmesini sıvı 0.1-0.2 ul görünür damlacık izin verirken, mümkün olduğunca uzun ucu tutun. İğne çok sıkıcı ya da şaft çok kısa ise, ortaya çıkan doku hasarı embriyo öldürebilir. Iğne ucu kadar ince ise tersine, yeterli plazmid hacmini enjekte çok uzun sürer; nedeniyle araştırmacının elinde kaçınılmaz titreşim ve embriyo hafif dönüşleri çok uzun nedenler hasar için beyinde iğne bırakarak. Ayrıca, genç içine enjeksiyonlar izin keskin, eğimli ipuçları iğne yapmak için bir microbeveler kullanınDaha kırılgan embriyolar, bu embriyolar E13.5 ve yaşlı için gerekli olmamalıdır rağmen. Hamile kadın stres düzeyi embriyo sağkalımı etkileyen bir başka önemli faktördür. yüksek Baraj stres düzeyi, daha büyük olasılıkla o çöp iptal edecektir. Bu nedenle bu anestezi süresini en aza indirmek ve ameliyat öncesi ve sonrası mümkün olduğunca az konut değiştirmek önemlidir. arka plan suşu da stres düzeyini etkileyebilir ve bu soruşturma kapsamında fenotip farklı arka plan suşları arasında muhafaza olduğunu varsayarak, örneğin CD1 gibi bir zorlanma üzerine anksiyete eğilimli outcross koloniler için gerekli olabilir. İEÜ tek büyük sınırlama electroporations arasındaki doğal değişkenlik olduğunu. Her Electroporated beyin dolayı enjeksiyon hacmi, kürek yerleştirme, kürek temas ve embriyo yaş değişikliklere farklıdır. Iğneler değişken olduğu için, sabit bir hacim enjekte etmek mümkün değildir - yerine investigator tüm embriyoların benzer miktarlarda sunmak için yeşil yama boyutunu tahmin gerekir. Embriyo sıvı dolu bir kese içinde yüzen çünkü stereotaktik enjeksiyon için yapıldığı gibi benzer şekilde, bu, sistematik olarak embriyonun kafasına kanatların konumu ve yönlenişinin ölçmek mümkün değildir. Son olarak, embriyonik yaş küçük farklılıklar deneysel sonuçlar üzerinde önemli etkileri olabilir. Bu nedenle, littermates karşılaştırılması önerilmektedir.

Radyal göç erken kortikal gelişim merkezi bir süreçtir. Bu tür doğru mitotik çıkış ve nöronal farklılaşması, hücre polarite, sitoskeletal dinamikleri ve zar-ötesi reseptörlerin ekspresyonunun düzenlenmesini, hem de bu tür bir radyal glial iskele oluşumu olmayan hücre otonom faktörleri ve hücre otonom faktörler, çeşitli bağlıdır göçmen rehberlik salgılanması 1-3 molekülleri. Bu mekanizmaların herhangi birinin bozulması beri karmaşık ve zor bir süreç olabilir göç bir kusur yatan nedenini belirlemek, bir transgenik fare modelinde 4-8 nöronal göç bozabilir. Utero elektroporasyon (İEÜ) olarak tamamlayacak ve basitleştirmek için kullanılabilir böylece genetik knock-out model fenotip ve yorumlanması radyal göç 7,9-11 için gerekli önemli mekanizmaları aydınlatmak. Utero elektroporasyon (İEÜ) süreci olan bir plazmid carryi tarafından<html> İlgili bir geni ve bir raportör hem ng bir fare veya sıçan embriyo beyninde ventrikül içine enjekte edilir ve daha sonra bir elektrik akımı 12-14 kullanımı ile ventrikül astar hücreleri içine çekilir. Bu araştırmacı yukarı veya geliştirme ya da Electroporated nöronların fonksiyonu ilgi bir gen düzenlenmesi aşağı etkisini analiz etmeyi sağlar. İEÜ sonra, beyinleri immunohistokimyasal 7,9-11, elektrofizyoloji 15 ya da hücre kültürü 16,17 için işlenebilir. İEÜ büyük avantajı, bu gen ekspresyonunun yüksek oranda spesifik müdahale edilebilmesini sağlar olmasıdır. Ayrıca, İUE yönlendirilmiş bir akım (Şekil 2) boyunca gelişen beynin belirli bir bölgeyi hedef için kullanılabilir. İUE çeşitli promotörler ya da plazmid tahrik sistemleri kontrolü altındaki genleri taşıyan plazmid enjeksiyonu yoluyla, belirli bir hücre türü hedef için kullanılabilir 10,18-21 (tetrasiklin ile uyarılan gen ifadesi bir örnektir). &gt; İUE Cre Lox aracılığıyla gen eksizyon sistemi 7-9,11,22 ile bağlantılı olarak kullanılabilir jove_content &quot;. Cre proteini için mesaj kodlayan bir gen içeren bir plazmid floxed alel için bir embriyo homozigot (elektroporasyona edilebilir olup, burada gen veya belirli DNA bölgeleri Cre aracılı DNA rekombinasyon, iki loxP sitelerinin) tarafından kuşatılmıştır. Cre daha sonra, spesifik elektroporasyona sinir ön ilgi konusu genin rekombinasyona neden olacaktır, ve floxed allele.The etkisinin bir knock-out üretilmesi sinir göç ve bireysel nöronlar içinde gelişimine demonte protein daha sonra incelenebilir. Cre Elektroporasyon sağlam destek ortamı terk etkilenen hücrelerin sadece küçük bir popülasyonda rekombinasyonu neden olur. Bunun tersine, belirli bir hücre tipinin kontrol altında Cre dokuya özgü sentezleme promotör, tüm dokusu boyunca meydana hem göç Nöroblastlar ve çevresini etkilenmiş olabileceğini. Böylece, Jux böyleceBu iki yaklaşımın taposition verilen göç kusur özerk ya da hücre dışı özerk mekanizmaları hücre bağlı olup olmadığını belirleyebilir. Her iki deneysel sistemlerde Arızalı göç anlaşılacağı bir hücre özerk mekanizması gözlemlenen fenotip sonuçları olduğunu; bir doku-spesifik Cre modelinde kusurlu göç Cre elektroporasyonu, aşağıdaki normal bir göç ilgi konusu gen, bir hücre olmayan bağımsız bir mekanizma yoluyla etki olduğunu gösterir. İEÜ da nakavt hayvanlar 7,9,10 içine potansiyel etkileşim genleri electroporating kurtarma deneyleri gerçekleştirmek için kullanılabilir. Örneğin, bir araştırmacı bir alt hedef electroporating ve göç kusur Electroporated nöronlar giderilmiştir ise belirleyerek bir transgenik modelinde bir göç fenotip kurtarmak için çalışabilir. Bu başarılı bir kurtarma normal bir göç SPE protein ifadesini manipüle tarafından restore edilebileceğini belirtir ek yararı vardır, spesifik nöron, çevredeki ortam hala hedeflenen gen için eksik olsa. Yine, bu yaklaşım daha fazla zaman ve kusurlu mekanizma hücre özerk olup olmadığını belirlemek yararı, transgenik çizgiler kapısı veya oluştururken daha maliyetli olduğunu. İUE nakavt embriyolar (Şekil 4C) bir haberci plazmid elektroporasyonu yoluyla nöronlar göç izlemek için kullanılabilir. Ameliyat sırasında ventrikül astar sadece nöronlar 17 elektropore olarak, İEÜ in vivo morfolojisi görselleştirme avantajı ile belirli bir zamanda doğmuş nöronlar takip etmek için kullanılabilir. Son olarak, bu tür orta veya dorsal telencephalon, hipokamp veya ganglionik eminensinden (Şekil 2) gibi beyin bölgeleri hedef IUE kullanmak mümkündür. Bu araştırmacılara komplikasyon sonuç belirli bir alanda bağımsız gen fonksiyonunu araştırmak için güç verirkomşu yapılarda demonte proteinden ing. Retinoblastoma proteini (pRb), p107 ve p130 cep protein ailesini oluşturan ve iyi hücre döngüsü çıkış regülatörleri kurulmuştur. Bununla birlikte, bu proteinler, aynı zamanda, nöral gelişimi, hücre döngüsü bağımsız yönlerini düzenleyen dair artan kanıtlar vardır. Daha önce görüldüğü gibi, pRb ve p107 hem teğet 4,23 ve radyal göç 6 önemli bir rol oynamaktadır. Burada, biz bir transgenik modeli Cre utero elektroporasyon kullanımını örneklendirmek için sinir göç 6 cep protein aile üyeleri pRb ve P107 rolünü göstermektedir. Özetle, İEÜ gen silme (veya aşırı ekspresyonu) hücre özerk etkilerini analiz güçlü bir yol sağlar. Modelleri nakavt özel dokusu ile kombine edildiğinde, İEÜ nöral göçü kontrol mekanizmaları ile ilgili ek bilgi sağlayabilir.

<table><tbody><tr><td>ECM 830 Square Wave Electroporator</td><td>BTX Harvard Apparatus</td><td>45-0052</td><td>Electroporator only &amp;ndash; buy cables separately (45-0204)</td></tr><tr><td>1250FS Footswitch for ECM 830</td><td>BTX Harvard Apparatus</td><td>45-0211</td><td>Foot paddle is necessary for hands-free electroporation</td></tr><tr><td>Tweezertrodes</td><td>BTX Harvard Apparatus</td><td>45-0489 (5mm)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 45-0488 (7mm)</td><td>Platinum forcep style electrodes</td></tr><tr><td>Femtojet</td><td>Eppendorf</td><td>920010504</td><td>Microinjector</td></tr><tr><td>Griphead 0</td><td>Eppendorf</td><td>920007414</td><td>Holds the pulled needle</td></tr><tr><td>Universal Capillary Holder</td><td>Eppendorf</td><td>920007392</td><td>Connects griphead to tube</td></tr><tr><td>Injection Tube</td><td>Eppendorf</td><td>920007431</td><td>Connects capillary holder to Femtojet</td></tr><tr><td>Foot Control</td><td>Eppendorf</td><td>920005098</td><td>Foot paddle for hands-free injection</td></tr><tr><td>Microloader Tips</td><td>Eppendorf</td><td>930001007</td><td>Tips for loading the pulled capillary tubes</td></tr><tr><td>Plasmid Mega Kit</td><td>Qiagen</td><td>12181</td><td>5 QIAGEN-tip 2500, Reagents, Buffers</td></tr><tr><td>Fast Green FCF</td><td>Sigma-Aldrich</td><td>F7252-5G</td><td>Non-toxic dye</td></tr><tr><td>Extra Fine Bonn Scissors</td><td>Fine Science Tools</td><td>14084-08</td><td>For trimming the needles</td></tr><tr><td>Bonn Strabismus Scissors</td><td>Fine Science Tools</td><td>14084-09</td><td></td></tr><tr><td>Graefe Forceps</td><td>Fine Science Tools</td><td>11050-10</td><td>Straight</td></tr><tr><td>Colibri Retractors</td><td>Fine Science Tools</td><td>17000-03</td><td></td></tr><tr><td>Stapler</td><td>Fine Science Tools</td><td>12031-07</td><td>For 7mm clips</td></tr><tr><td>Castroviejo Needle Holder</td><td>Medical Tools</td><td>SNH-6737</td><td>Straight, with lock</td></tr><tr><td>Needle Puller</td><td>Narishige</td><td>PC-10</td><td>For pulling microcappillaries</td></tr><tr><td>Microcapillaries</td><td>Drummond</td><td>1-000-800</td><td>0.4 mm inner diameter glass capillary tubes</td></tr></tbody></table>

induction of protein deletion through in utero electroporation to define deficits in neuronal migration in transgenic models

Transgenik fare hatları Cre-Lox sistemini kullanarak Genetik silme protein fonksiyonunu incelemek için kullanılan güçlü bir araçtır. Bununla birlikte, çok özel Cre modellerinde dışında, bir doku veya hücre popülasyonu içinde bir proteinin silme genellikle birden fazla etkileşim mekanizmalardan kaynaklanan karmaşık fenotiplere yol açar. Söz konusu, ya da hücrenin çevre düşüklüğü kaynaklanabilecek olmayan bir hücre özerk bir mekanizma, gelen hücreye özgü bir hücre özerk mekanizmasının bozulması bir fenotip sonuçları, ayırt etmek zor olabilir olup olmadığını belirleme. Utero elektroporasyon, bir in vivo bağlamda protein işlevi anlamak için (İUE), gelişmekte olan korteks ya da seçilen diğer beyin bölgesi içindeki hücrelerin küçük bir alt grubu içerisinde, gen silme sağlar. İUE sırt telencephalon medial telencephalon, hipokampus veya ganglionik eminensinden da dahil olmak üzere spesifik beyin alanları, hedef için kullanılabilir. Bu gözlem o kolaylaştırırsağlıklı bir çevrede bağlamında hücre özerk gen silme sonuçları f. Bu protokolün amacı, İEÜ protein silme hücre özerk olmayan hücre özerk etkileri ayırt üzerinde durularak, floxed transgenik fare doğrultusunda radyal göç bir kusur analiz etmek nasıl kullanılabileceğini göstermektir. Beyin gelişiminde proteinin işlevi sınırlı hücre popülasyonunda İUE aracılı gen silme karşı tüm korteks içinde gen silme kaynaklanan fenotip, daha iyi bir fikir karşılaştırarak izolasyonu ya tekniği kullanılarak daha yüksek verim elde edilebilir.

Different regions of the brain can be targeted for electroporation by varying the orientation of the paddle electrodes over the embryo&#8217;s head (Figure 2). For all parts of the forebrain, the plasmid is injected into the lateral ventricle (the hind brain can be targeted by injecting into the fourth ventricle). In general, place the electrodes so that the straight line between them passes through the region of interest, with DNA being drawn toward the positive electrode. To target the lateral cortex (Figure 2A), place the positive electrode on top of the green crescent, with a slight rostral angle. For the medial cortex (Figure 2B), switch the orientation so that the positive electrode is over the contralateral non-injected ventricle, this time with an even larger forward angle. To target the hippocampus (Figure 2C), place the positive electrode over the contralateral side, but instead angle the positive electrode caudally (electroporations must be done at an age when the neural precursors are still part of the cortical hem, prior to migration to the final location of the hippocampus). To target the ganglionic eminence (Figure 2D) place the paddles on the embryo in line with the ears, with the positive electrode slightly lower than the negative electrode. Representative images of these electroporated regions are shown in Figure 2.
It is important to validate Cre-mediated excision in electroporated brains. The ideal approach is to perform in situ hybridization or immunohistochemistry against the transcript or protein in question. Unfortunately, as is the case for pRb, reliable probes or antibodies are not always available against the deleted gene. In these cases, alternative approaches, such as staining for a downstream target regulated by the deleted gene (Figure 3A) or confirming excision of the targeted allele in electroporated sections by PCR (Figure 3B), can be used. For example, Figure 3A confirms Cre mediated excision by reproducing a well-documented effect of pRb/p107/p130 gene deletion in electroporated neurons. Following electroporation of pRb f/f; p107 -/-; p130 f/f neurons with the pCig2-Cre plasmid, these triple knock out GFP+ cells continue to express the cell cycle progression marker Ki67, which is a known effect following excision of pocket proteins25. Control GFP+ neurons electroporated with the empty pCig2 plasmid do not express Ki67. The Cre plasmid should also be tested in vitro through co-transfection with a plasmid carrying a reporter gene behind a floxed stop codon, such as the NL-dsRed plasmid (Figure 3C) in a cell line, but this should only be used in combination with other in vivo techniques.
Due to the variation between electroporated brains, analysis of electroporated neurons requires calculating the percentage of total GFP+ neurons that portray a specific characteristic, such as expression of a protein marker or location within a given cortical layer. It is crucial to compare electroporated sections from similar anatomical locations within the brain relative to known landmarks. For example, images for Figure 4 were taken at the level of the anterior commissure. Furthermore, due to horizontal migration, images taken near the edge of the electroporated regions have a higher proportion of mature neurons that have reached the cortex, while images taken near the middle of the electroporated region have both mature and immature GFP+ cells. To insure consistency, it is important to quantify images selected from similar places within the electroporated patch.
Following Emx1-Cre mediated deletion of pRb and p107 in pyramidal neurons in the dorsal telencephalon, a population of neurons expressing the upper cortical layer marker FoxP1 were mislocalized to the intermediate zone (IZ) (Figure 4A). There are significantly more FoxP1+ cells in the IZ of double knock out (DKO) brains compared to control brains with a corresponding decrease in the number of FoxP1+ cells in the upper cortical layers (above layer IV) of DKO brains than found in the upper layers of control brains (Figure 4B). There was no corresponding defect in the radial glial scaffold (Figure S2 of Svoboda et al., 2012) 6. Here we compare expression of cortical layer markers Tbr1, FoxP1 and Cux1 in pRb f/+; p017 +/-; Emx1-Cre + (wild type (WT)) brains to expression of these same markers pRb f/f; p107 -/-; Emx1-Cre + (DKO) brains. Heterozygote embryos can be used as controls since pocket proteins exhibit autoregulation at the protein level and heterozygote animals are aphenotypic 26.
It was unclear whether these FoxP1+ cells were found in the IZ because these cells were immature neurons inappropriately expressing FoxP1, or whether they were earlier born neurons that had failed to migrate. To distinguish between these possibilities, we electroporated GFP into the wild type (WT) and double knock out (DKO) embryos at E14.5 and tracked the progress of these neurons over 4 days. Although this technique does not conclusively identify the final cell division, as with BrdU birthdating, it does allow the tracking of newborn neurons since it marks the discrete set of neurons derived from cells lining the ventricle at the time of electroporation 17. As shown in Figure 4C, all of the GFP+ neurons reach the uppermost cortical layer in the WT brains, but only a subset of these neurons reach the top of the cortex in DKO brains, indicating that there is in fact a defect in the migration of upper layer neurons, rather than improper expression of neuronal markers (similar results were obtained through traditional BrdU birthdating 6).
Neuronal migration is a highly complex process that is regulated by a multitude of interconnected mechanisms 1-3. As it is very difficult to identify the mechanism underlying a migration defect in an uncharacterized transgenic model, distinguishing cell autonomous versus non-cell autonomous defects can facilitate phenotype characterization. If following electroporation of Cre-GFP, GFP+ neurons fail to migrate in an otherwise wild type brain, this indicates that the mechanism disrupted by gene deletion is specific to the electroporated cell. Alternatively, if the electroporated neurons migrate normally in the wild type brain, this suggests that the cause of aberrant migration in the transgenic embryo is due to an environmental defect. Following electroporation of Cre-GFP in control pRb f/+; p107 +/- and double knock out pRb f/f; p107 -/- embryos, GFP+ neurons were found to migrate into the cortex in pRb f/f; p107 -/- brains, but many of these neurons failed reach the upper cortical layers as they did in control brains (Figure 4D) 6. There were significantly lower percentage of GFP+ neurons within the layer III/IV, marked by expression of FoxP127, in pRbf/f; p107 -/- electroporated brains than in control brains (Figure 4E). A Cre-GFP fusion protein was used to limit GFP fluorescence to the cell body and facilitate localization of the neuron to a specific cortical layer. Since the phenotype following electroporation of Cre is less severe than the phenotype following gene deletion throughout the cortex, this suggests that the original phenotype shown in Figure 4A is a combination of multiple mechanisms, both cell autonomous and not cell autonomous. Alternatively, it is possible that the time delay following electroporation, during which Cre is expressed, DNA recombination is performed, pRb is downregulated and the effects are manifested, is long enough to allow the electroporated neurons to reach the cortex before a migration defect becomes apparent.
It should be noted that the distinction between cell autonomous and non-cell autonomous mechanisms should be interpreted with caution in brains with high electroporation efficiencies, where 100-200 or more neurons are electroporated in a single 14 &#181;m thick section.If a high enough proportion of neurons are electroporated, GFP+ neurons may influence each other and create a small microenvironment. To be truly cell autonomous, migration defects must be detected even when electroporation efficiency is very low (10-30 GFP+ neurons per section). If necessary, the number of transfected cells can be lowered by decreasing voltage, pulse number or DNA concentration. All of these factors must be taken into consideration when analyzing the results following electroporation of Cre in a transgenic model.
<img alt="Figure 1" fo:content-width="6in" src="/files/ftp_upload/51983/51983fig1highres.jpg" width="475" /> 
Figure 1: Performing In utero electroporation. (A) Sterile surgical set up. (B) Surgical tools from left: fine scissors for cutting needle tips; stapler; forceps; wound retractor; surgical scissors; needle holder. (C) Prepped pregnant mouse with shaved and disinfected abdomen. (D) Mouse covered with sterile drape prior to incision. (E) Uterus with E13.5 embryos removed from abdominal cavity. (F) Injecting pups in the ventricle. (G) Close up of injected pup; note the blue crescent on the embryo&#8217;s head indicating that the ventricle was successfully injected with the plasmid solution (arrow). (H) Paddles are placed to electroporate the lateral cortex. (I) Mouse post-surgery. (J) Preparing sharp needles is a crucial part of in utero electroporation. Examples of uncut needles (i, iv), good needles (ii, v) and poor needles (iii, vi) are shown at low (i-iii) and high (iv-vi) magnification. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51983/51983fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="6in" src="/files/ftp_upload/51983/51983fig2highres.jpg" width="600" /> 
Figure 2: Targeting different brain regions. Diagrams A-D show paddle placement for targeting different regions of the forebrains. The dark blue circle represents the paddle in front of the embryo while the light blue circle represents the paddle behind the embryo. Positive and negative electrodes are indicated with the appropriate sign. (A) Electrodes angled to target the lateral cortex. (A&#8217;, A&#8221;) Example of electroporation of the lateral cortex, three days after surgery. (B) Electrodes angled to target the medial cortex. (B&#8217;, B&#8221;) Example of electroporation of the medial cortex. (C) Electrodes angled to target the hippocampus. (C&#8217;, C&#8221;) Example of electroporation of the hippocampus. (D) Electrodes angled to target the ganglionic eminence (GE). (D&#8217;, D&#8221;) Example of electroporation of the GE. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51983/51983fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" fo:content-width="6in" src="/files/ftp_upload/51983/51983fig3highres.jpg" width="600" /> 
Figure 3: Confirming Cre-mediated gene excision in electroporated neurons. (A) Cre mediated excision can be confirmed by co-staining for a downstream target of the deleted gene. pRb f/f; p107 -/-; p130 f/f embryos were electroporated with either the empty pCig2 plasmid or the pCig2-Cre plasmid and stained for GFP and Ki67, a marker of cell cycle entry. Double stained cells were seen in neurons electroporated with Cre (arrows in inset) but not in neurons electroporated with pCig2 alone (arrowheads). (B) Excision of pRb was also confirmed by performing PCR with genotyping primers on DNA extracted from the tissue sections removed from the slides. Sections were divided down the midline and the electroporated side of the section was pooled. The upper band represents the floxed allele and the lower band represents the wild type allele. The arrow indicates the excised allele in the electroporated side of the brain only. Because pRb f/+; p107 +/- heterozygotes were used as controls, an excised allele can be seen in the electroporated side of the brain in both the control and knockout brains. (C) Cre-mediated excision can also be confirmed in vitro using plasmids with floxed stop codons upstream of RFP, such as NL-dsRed. RFP is only expressed in HEK cells when co-transfected with Cre-GFP in Hek293 cells (bottom row), indicating that excision was successful. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51983/51983fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" fo:content-width="6in" src="/files/ftp_upload/51983/51983fig4highres.jpg" width="600" /> 
Figure 4: Using IUE to study migration in transgenic mouse models. (A) Expression of FoxP1, a marker for layer III/IV cortical neurons, was examined in pRb f/+; p107 +/-; Emx1-Cre + (WT), and pRb f/f; p107 -/-; Emx1-Cre + (DKO) brains at E17.5. Overlay includes Cux1 (blue - marker for layers II-IV) and Tbr1 (green - marker for layers V/VI). A population of cells expressing FoxP1 was observed in the intermediate zone of DKO embryos (arrow), but not WT embryos, with a concurrent decrease in the number of FoxP1+ neurons in layers II-IV of the cortex in DKO brains relative to WT brains (B). The white line indicates the delineation between the cortical plate and intermediate zone, which was determined using DAPI staining (not shown). (C) To trace the migration of a discrete population of late born neurons, WT and DKO embryos were electroporated with GFP at E14.5 and collected the brains at E18.5. Brains were co-stained with FoxP1 (red) and Tbr1 (blue) to show that GFP+ neurons are found almost exclusively in layer II in the WT brain but are delayed in the intermediate zone (IZ) and lower cortical layers in the DKO. (D) To determine if pRb and p107 regulate radial migration cell autonomously, pRb f/f; p107-/- and pRb f/+; p107-/+ embryos were electroporated with Cre-GFP at E13.5 and brains were collected at E17.5. The location of GFP+ neurons was quantified according to cortical layer. Layer II was defined as being above FoxP1 staining (red), layer III/IV was defined as within FoxP1 staining, above Tbr1 (blue) and layer V/VI was defined as within the Tbr1 positive layer (E). We show that a portion pRb and p107 deficient neurons fail to reach layer II but instead remain in layer III/IV. The figure has been modified from Svoboda et al., 2012. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51983/51983fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Embryo Age</td>
			<td>Voltage</td>
		</tr>
		<tr>
			<td>E12.5 &#8211; E13.5</td>
			<td>35V</td>
		</tr>
		<tr>
			<td>E14.5 &#8211; E15.5</td>
			<td>40V</td>
		</tr>
		<tr>
			<td>E16.5</td>
			<td>45V</td>
		</tr>
		<tr>
			<td>E17.5</td>
			<td>50V</td>
		</tr>
	</tbody>
</table>
Table 1: Electroporation voltage by embryo age.

Watch this Scientific Journal Video about Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models at JoVE.com

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

Neuron migration is regulated by numerous cell autonomous and non-cell autonomous factors. This protocol shows how in utero electroporation can be used to determine whether a phenotype in a transgenic mouse is due to disruption of a cell intrinsic mechanism or impairment of interaction between the neuron and its environment.

Protein silinmesi indüksiyonu yoluyla<em&gt; In Utero</em&gt; Elektroporasyon Transgenik Modelleri Nöronal Göç Açıkları tanımlayın

Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific ...

induction-protein-deletion-through-utero-electroporation-to-define

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

10.2K Görüntüleme.  University of Ottawa. Neuron migration is regulated by numerous cell autonomous and non-cell autonomous factors. This protocol shows how in utero electroporation can be used to determine whether a phenotype in a transgenic mouse is due to disruption of a cell intrinsic mechanism or impairment of interaction between the neuron and its environment. 

Video: Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

In utero Elektroporasyon Kortikal nöronlar Altküme Gen Fonksiyonu Öğrenmenin İlköğretim Nöronal Kültür takip

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

Nörobilim

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.
To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

Verimli kullanarak birden çok CNS Topraklar&#39;da Gen Teslimat In Utero Elektroporasyon

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

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 ...

Murine Neural Plate Targeting by In Utero Nano-Injection (NEPTUNE) at Embryonic Day 7.5

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously largely been restricted to the manipulation of embryonic stages post-neurulation. A protocol was developed to inject the amniotic cavity at embryonic day (E)7.5 and deliver lentivirus, encoding cDNA or shRNA, targeting &gt;95% of the neural plate and neural crest cells, contributing to the future brain, spinal cord, and peripheral nervous system. This protocol describes the steps necessary to achieve successful transduction, including grinding of the glass capillary needles, pregnancy verification, developmental staging using ultrasound imaging, and optimal injection volumes matched to embryonic stages. 
Following this protocol, it is possible to achieve transduction of &gt;95% of the developing brain with high-titer lentivirus and thus perform whole-brain genetic manipulation. In contrast, it is possible to achieve mosaic transduction using lower viral titers, allowing for genetic screening or lineage tracing. Injection at E7.5 also targets ectoderm and neural crest contributing to distinct compartments of the eye, tongue, and peripheral nervous system. This technique thus offers the possibility to manipulate gene expression in mouse neural-plate- and ectoderm-derived tissues from preneurulation stages, with the benefit of reducing the number of mice used in experiments.

Murine Neural Plate Targeting by In Utero Nano-Injection NEPTUNE at Embryonic Day 7.5

In this protocol, we describe how to inject the mouse amniotic cavity at E7.5 with lentivirus, leading to uniform transduction of the entire neural plate, with minimal detrimental effects on survival or embryonic development.

Embriyonik Gün 7.5'te In Utero Nano-Enjeksiyon (NEPTÜN) ile Murin Nöral Plaka Hedefleme

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously ...

Gelişim Biyolojisi

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous system. Overexpression of desired genes is just as possible as shRNA mediated loss-of-function studies. Therefore it offers a wide range of applications. The feasibility to target particular cells in a distinct area further increases the range of potential applications of this very useful method. For efficiently targeting specific regions knowledge about the subtleties, such as the embryonic stage, the voltage to apply and most importantly the position of the electrodes, is indispensable.
Here, we provide a detailed protocol that allows for specific and efficient in utero electroporation of several regions of the C57BL/6 mouse central nervous system. In particular it is shown how to transfect regions the develop into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. For this information about the appropriate embryonic stage, the appropriate voltage for the corresponding embryonic stage is provided. Most importantly an angle-map, which indicates the appropriate position of the positive pole, is depicted. This standardized protocol helps to facilitate efficient in utero electroporation, which might also lead to a reduced number of animals.

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

This protocol describes in detail how to specifically transfect different regions in the C57BL/6 central nervous system via in utero electroporation. Included in this protocol are detailed instructions for transfections of regions that develop into the cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus and striatum.

Korteks, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- ve Striatum özgü<em&gt; In Utero</em&gt; C57BL / 6 Fare Elektroporasyon

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous ...

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of intellectual disability and account for 20-40% of drug-resistant epilepsy in childhood. High-resolution brain imaging has facilitated in vivo identification of a large group of MCD phenotypes. Despite the advances in brain imaging, genomic analysis and generation of animal models, a straightforward workflow to systematically prioritize candidate genes and to test functional effects of putative mutations is missing. To overcome this problem, an experimental strategy enabling the identification of novel causative genes for MCD was developed and validated. This strategy is based on identifying candidate genomic regions or genes via array-CGH or whole-exome sequencing and characterizing the effects of their inactivation or of overexpression of specific mutations in developing rodent brains via in utero electroporation. This approach led to the identification of the C6orf70 gene, encoding for a putative vesicular protein, to the pathogenesis of periventricular nodular heterotopia, a MCD caused by defective neuronal migration.

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Periventricular nodular heterotopia (PNH) is the most common form of malformation of cortical development (MCD) in adulthood but its genetic basis remains unknown in most sporadic cases. We have recently developed a strategy to identify novel candidate genes for MCDs and to directly confirm their causative role in vivo.

Dizi-CGH, Bütün exome sıralama ve rahim içinde elektroporasyon Rodents beyin malformasyonlar için sorumlu genlerin tanımlamak için birleştirerek yeni bir strateji

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

Rahimde Elektroporasyon Nöronal alt popülasyonlar heyecanlanma ve Tek hücreli Bağlantı Eğitim Yaklaşımları

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

Embriyonik Fare Beyni Organotipik Dilim Kültüründe Nöronların Geçiş Sürekli Konfokal Görüntüleme Kullanımı<em&gt; Utero&#39;da</em&gt; Elektroporasyon

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To do so requires generating a mosaic in which neurons with loss or gain of function of a gene are surrounded by genetically unperturbed tissue. Here, we combine the Cre-lox recombination system with in utero electroporation in order to generate mosaic brain tissue that can be used to study the cell-autonomous function of genes in neurons. DNA constructs (available through repositories), coding for a fluorescent label and Cre recombinase, are introduced into developing cortical neurons containing genes flanked with loxP sites in the brains of mouse embryos using in utero electroporation. Additionally, we describe various adaptations to the in utero electroporation method that increase survivability and reproducibility. This method also involves establishing a titer for Cre-mediated recombination in a sparse or dense population of neurons. Histological preparations of labeled brain tissue do not require (but can be adapted to) immunohistochemistry. The constructs used guarantee that fluorescently labeled neurons carry the gene for Cre recombinase. Histological preparations allow morphological analysis of neurons through confocal imaging of dendritic and axonal arbors and dendritic spines. Because loss or gain of function is achieved in sparse mosaic tissue, this method permits the study of cell-autonomous necessity and sufficiency of gene products in vivo.

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous functions of genes in the brain can be studied by inducing loss or gain of function in sparse populations of cells. Here, we describe in utero electroporation to deliver Cre recombinase into sparse populations of developing cortical neurons with floxed genes to cause loss of function in vivo.

CRE-lox rekombinasyon fare serebral korteks rahim içinde elektroporasyon ile inducing

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

Eski Utero Elektroporasyon ve embriyonik fare beyni canlı görüntüleme, GABAergic Interneurons geçiş için Organotypic dilim kültürleri

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

Protein silinmesi indüksiyonu yoluyla

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