Name: cortex-, hippocampus-, thalamus-, hypothalamus-, lateral septal nucleus- and striatum-specific in utero electroporation in the c57bl/6 mouse
Uploaded: 2016-01-18T14:00:00.000+00:00
Duration: 6 min
Description: Watch this Scientific Journal Video about Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse at JoVE.com

Technical supported by Melanie Pfeifer and Nikolai Schmarowski (Institute for Microscopic Anatomy and Neurobiology, University Medical Center Mainz).

Etik Beyanı: Farelerde ve deneysel prosedürlerin işleme hayvan bakımı için, Avrupa, ulusal ve kurumsal kurallara uygun olarak yapılmıştır. 1. Giriş Utero Elektroporasyon Not: Daha önce 12,14 yayınlanan utero elektroporasyon gerçekleştirildi. Bu nedenle, yöntem aşağıdaki (Şekil 1) kısaca tarif okunur. <ol><li> Hazırlıklar <ol><li> Daha önce açıklandığı gibi 12 (1,5 pCAGGS içeren) Hızlı Yeşil renkli endotoksin içermeyen gelişmiş transfeksiyon dereceli plazmid çözüm hazırlayın. </li><li> Enjeksiyonlar için (35 ° açı) borosilikat cam kılcal (0,8-0,9 çap) çekin ve eziyet. </li><li> Aletleri sterilize edin. </li></ol></li><li> Cerrahlık <ol><li> Ameliyat öncesi analjezikler (karprofen, 4 mg / kg vücut ağırlığı, sc, 24 saat depo) en az 30 dakika uygulayın. </li><li> Uzunluğunu tutun(- cerrahi anestezi aşaması guedel 15&#39;e göre - Evre III maksimal 25-30 dakika) 35-45 dakika arasında en fazla anestezi. </li><li> (: Maske ile% 2.8, ameliyat:% 2.5 indüksiyon bir odasında) izofluran ile zamanlanmış hamile fareler anestezi. Ayak tutam yanıtsızlık ile anestezi onaylayın. </li><li> Anestezi sırasında göz kuruluğu önlemek için göz merhemi sürün. </li><li> % 7.5 providon-İyot çözeltisi ile cerrahi alan sterilize ve maruz sadece cerrahi alana sahip (% 0.9 benzil alkol tuzlu su çözeltisi ile nemli) steril gazlı bez ile fare kaplayın. </li><li> (: 1.5-2 cm, kas kesi: 1-1.5 cm cilt kesisi) bir neşter ile karın boşluğuna açın kesin. </li><li> Veteriner personel veya komite tarafından yönlendirilen ısındı% 0.9 benzil alkol tuzlu su çözeltisi ya da diğer steril izotonik solüsyon ile nemlendirerek kurumasını açılan karın boşluğuna koruyun. </li><li> Dikkatle parmağınızla rahim dokunmayın, halka forseps kullanarak rahim boynuzları (ayıklamaks). </li></ol></li><li> DNA ve elektroporasyon enjeksiyonu. Not: Belirli bölgeleri hedefleyen için kesin ayrıntılar 2. bölümünde belirtilmiştir. <ol><li> Hazırlanan borosilikat camdan aracılığıyla Şekil 1 ve 2. bölümünde belirtildiği gibi yavaşça ventrikül içine renkli DNA çözüm enjekte edilir. </li><li> İlgili embriyonik aşama için uygun gerilim uygulayın (örn. E14 için, 36-38 V) özel forseps tipi platin elektrot (aralık döngüsü uzunluğu 50 ms, aralık duraklama 950 msn) aracılığıyla. Rahim duvarının hasarı en aza indirmek için voltajın uygulanması sırasında elektroporasyon kürekler Pulse. </li></ol></li><li> Mesaj elektroporasyon / post-operatif bakım <ol><li> Dikkatle karın boşluğunda rahim boynuzları değiştirin. </li><li> Kas ve ayrı ayrı cilt kesisi dikin. </li><li> Kurtarma için bir termal destek cihazı fare dinlendirin. </li><li> Kurtarma sırasında fare (davranış, yem denetlemek veSu tüketimi), 4 saat, 24 saat ve 48 saat sonra ameliyat. Gerekirse, 2 gün daha fazla analjezik (karprofen, 4 mg / kg vücut ağırlığı, 24 saat depo) uygulanır. </li></ol></li></ol> Belirli Serebral Alanlarının 2. Transfeksiyon Not: pozitif kutup pozisyonu DNA ana madde çözeltisi (sağ ya da üçüncü ventrikül) ve pozitif kutup konumu ile dolu ventrikül ile dikey merkez çizgisi arasındaki açı olarak gösterilir. Sol ventrikül doldururken pozisyonları ayna ters bulunmaktadır. Şekil 4&#39;te kulak Primordial pozisyonları önemli referans noktaları sağlayan gösterilir. <ol><li> Kortikal alanlara transfeksiyonu (Şekil 2A, Tablo 1). <ol><li> Bölüm 1.2&#39;de anlatıldığı gibi ameliyat. Kortikal alanların in utero elektroporasyon için E14 embriyolar kullanın. </li><li> Sağ lateral ventrikül 4 ug DNA içeren DNA çözeltisi ul 1.5-2 enjekteHazırlanan borosilikat kılcal damarların üzerinden. <ol><li> Dikkatle Ponksiyon üzerinde hiçbir görünür gemiler vardır, böylece halka forseps kullanarak embriyo yerleştirin. </li><li> Yavaş yavaş lambdoidal sütür yaklaşık 0.75 mm koronal ve sagital sütür (Şekil 3) 0,5 mm lateral DNA çözüm enjekte. Ekleme derinliği 1,2 mm (rahim duvarından ölçülen E14) olduğundan emin olun. Keskin bir sınır ile bir mavi-yeşil renk olup olmadığını kontrol edin. </li></ol></li><li> Özel forseps tipi platin elektrotlar yoluyla gerilim (aralık döngüsü uzunluğu 50 msn&#39;den, aralık duraklama 950 msn) uygulayın. <ol><li> 3 ya da 5 mm elektrot raket kullanın. </li><li> 36-38 V arasında değişen bir gerilim kullanın </li><li> Elektrotların kulak primordia sadece ön merkezini yerleştirin. </li><li> , Motor korteks 0-40 ° transfect transfect, somatosensoriyel korteks 40-95 ° transfect, 330-0 ° den retrosplenial korteks kullanım açıları transfectpiriform korteks 95-110 ° (Şekil 2A). </li></ol></li><li> Bölüm 1.4 de anlatıldığı gibi sonrası elektroporasyon / post-operatif bakım gerçekleştirin. Fareler kurban ve bölüm 3&#39;te tarif edildiği gibi elektroporasyon (E18) sonra, embriyolar 4 gün analiz eder. </li></ol></li><li> Hipokamp (Şekil 2B, Tablo 1) transfeksiyonu. <ol><li> Bölüm 1.2&#39;de anlatıldığı gibi ameliyat. Hipokampal formasyonun utero elektroporasyon için E15 embriyoların kullanın. </li><li> Hazırlanan borosilikat kılcal damarların üzerinden sağ lateral ventrikül içinde 4 ug DNA içeren DNA çözeltisi ul 2 enjekte edilir. </li><li> Özel forseps tipi platin elektrotlar yoluyla gerilim (aralık döngüsü uzunluğu 50 msn&#39;den, aralık duraklama 950 msn) uygulayın. <ol><li> 3 ya da 5 mm elektrot raket kullanın. </li><li> 38-40 arasında değişen bir gerilim kullanın. </li><li> Negatif kutup kulak primordiumunun sadece ön merkezi ve merkezi o koyunKulak primordiumunun ortasında pozitif kutup f. </li><li> Dentat girus CA1 250-275 ° (Şekil 2B) transfekte etmek üzere, Ca + 2 230-250 ° transfekte etmek, cornu ammonis (CA) 3 210-230 ° transfekte etmek, 190-210 ° &#39;den açılarıyla transfekte etmek. </li></ol></li><li> Bölüm 1.4 de anlatıldığı gibi sonrası elektroporasyon / post-operatif bakım gerçekleştirin. 3. bölümde açıklandığı gibi (doğum, p21 sonra) tamamlandıktan hipokampal oluşumundan sonra analiz gerçekleştirin. </li></ol></li><li> Lateral septal çekirdeği ve striatum transfeksiyonu (Şekil 2C, Tablo 1) Not: açıları hipokampus transfecting için olanlar ile örtüşüyor. Etkin bir yanal septal çekirdeğe ve istenmeyen hipokampus transfeksiyon erken embriyonik aşamalarda (E12) olmadan striatum transfect için kullanılmalıdır. <ol><li> Bölüm 1.2&#39;de anlatıldığı gibi ameliyat. Lateral septal çekirdek ve striat in utero elektroporasyon içinum E12 embriyoları kullanılır. </li><li> Hazırlanan borosilikat kılcal damarların üzerinden sağ lateral ventrikül içinde 4 ug DNA içeren DNA çözeltisi ul 1 enjekte edilir. </li><li> Özel forseps tipi platin elektrotlar yoluyla gerilim (aralık döngüsü uzunluğu 50 msn&#39;den, aralık duraklama 950 msn) uygulayın. <ol><li> 0.5 mm&#39;lik bir elektrot kullanın. </li><li> 33 V kullanın </li><li> Kulak primordia düzeyinde elektrotların merkezini yerleştirin. </li><li> Striatum 170-240 ° (Şekil 2C) transfekte edilmesi için, 100-170 ° yanal septal çekirdeği kullanımı açıları transfekte etmek. </li></ol></li><li> Bölüm 1.4 de anlatıldığı gibi sonrası elektroporasyon / post-operatif bakım gerçekleştirin. Fareler kurban ve bölüm 3&#39;te tarif edildiği gibi elektroporasyon (E18) sonra, embriyolar 6 gün analiz eder. </li></ol></li><li> Talamus ve hipotalamus Transfeksiyonu (Şekil 2B, Tablo 1) <ol><li> Bölüm 1.2&#39;de anlatıldığı gibi ameliyat. U içintalamus ve hipotalamus kullanımı E12-E13 embriyoların tero elektroporasyon. </li><li> Hazırlanan borosilikat camdan aracılığıyla lateral ventrikül (sağ veya sol) 4 ug DNA içeren 1-1.5 ul DNA çözümü enjekte edilir. Çözüm, üçüncü ventrikül içine yayılır kadar, 2-3 dakika bekleyin. Alternatif doğrudan üçüncü ventrikül içine enjekte edilir. Not: Bu özgüllüğünü arttırır fakat azalmış sağkalım oranı (hasar riski yüksek) neden olabilir. </li><li> Özel forseps tipi platin elektrotlar yoluyla gerilim (aralık döngüsü uzunluğu 50 msn&#39;den, aralık duraklama 950 msn) uygulayın. <ol><li> 0.5 veya 3 mm elektrot kullanın. </li><li> 33-35 V arasında değişen bir gerilim kullanın </li><li> Kulaktan Primordia posterior elektrot merkezi yerleştirin. </li><li> Hipotalamus 90-180 ° (Şekil 2D) transfect, 25-40 ° ile talamus kullanım açıları transfect. </li></ol></li><li> Des gibi post elektroporasyon / post-operatif bakım gerçekleştirinbölüm 1.4 açıklanan şekilde. Fareler kurban ve bölüm 3&#39;te tarif edildiği gibi elektroporasyon (E18) sonra, embriyolar 6 gün analiz eder. </li></ol></li></ol> 3. Perfüzyon Fixation <ol><li> Hazırlıklar <ol><li> Sıcak% 4 paraformaldehid (PFA) 37 ° C arasındadır. </li><li> PBS ile 50 ml şırınga (4 ° C) ve ısıtılmış PFA ile ikinci bir şırınga doldurun. Kabarcıkları kaçının. </li><li> Üç yollu için şırınga bağlayın. Kanatlı infüzyon seti (kelebek) üçüncü ucunu. </li><li> PBS ile kanatlı infüzyon seti yıkayın. </li></ol></li><li> Serpilme <ol><li> Derin subkutan ketamin hidroklorür uygulama (60 mg / kg) ve ksilazin (7.5 mg / kg) ile (gebe veya doğum sonrası transfekte edilmiş) fareler anestetize. </li><li> Ayak tutam yanıtsızlık cerrahi tolerans sahne onaylayın. </li><li> Sadece göğüs kafesine altında karın boşluğuna açın. </li><li> Dikkatle diyaframı kesti. </li><li> Dikkatle e bir kesim üzerinden göğüs kafesini açmakköprücük kemiği kadar ach sitesi. </li><li> (Kalbin apeks başlayarak) sol kalp odasına kelebeğin ucu takın. </li><li> Sol atriyal apendiks kesin. </li><li> Yavaş yavaş PBS (yaklaşık 10 mi) ile fare serpmek. </li><li> Üç yönlü stopcock geçin. </li><li> 5-10 ml% 4 PFA ile fareyi serpmek. </li><li> Makas kullanılarak fare başını kesmek ve foramen magnum başlayan beyin teşrih. Daha önce 16 yayınlanan diseksiyon. </li><li> % 4 PFA 2 gün beyin Postfixate. Karanlıkta 4 ° C&#39;de tutun beyinleri. </li></ol></li></ol> 4. İmmünhistokimya <ol><li> (Bir vibratome ile örneğin) 70 mikron bölümleri oluşturun. </li><li> İsteğe bağlı: antikor boyama ile floresan geliştirin. <ol><li> Oda sıcaklığında 1 saat için bloke bölümleri (% 0.1-0.2 Triton X-100,% 5 normal keçi serumu) </li><li> Uygun bir antikor (: 1000 anti-GFP, 1) ile O / N inkübe </li><li> Yıkama 30.1 M fosfat tamponu ile -5 kez. </li><li> Uygun floresan-konjuge sekonder antikor (: 1000 Alexa Fluor 488 konjuge edilmiş anti-tavşan IgG, 1), 3-5 saat inkübe edin. </li><li> İsteğe bağlı: 1 dakika süre ile (0,1 M fosfat tampon maddesi içinde DAPI, 0.2 ug / ml), 4-6-diaminodino-2-fenilindol ile Counterstain. </li><li> Uygun bir floresan mikroskop ile floresan analiz. </li></ol></li></ol>

<ol>
	<li>Fukuchi-Shimogori, T., Grove, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neocortex+patterning+by+the+secreted+signaling+molecule+FGF8.">Neocortex patterning by the secreted signaling molecule FGF8.</a> Science. 294 (5544), 1071-1074 (2001).</li><li>Saito, T., Nakatsuji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+into+the+embryonic+mouse+brain+using+in+vivo+electroporation.">Efficient gene transfer into the embryonic mouse brain using in vivo electroporation.</a> Dev Biol. 240 (1), 237-246 (2001).</li><li>Tabata, H., Nakajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+in+utero+gene+transfer+system+to+the+developing+mouse+brain+using+electroporation:+Visualization+of+neuronal+migration+in+the+developing+cortex.">Efficient in utero gene transfer system to the developing mouse brain using electroporation: Visualization of neuronal migration in the developing cortex.</a> Neuroscience. 103 (4), 865-872 (2001).</li><li>LoTurco, J., Manent, J. B., Sidiqi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+and+improved+tools+for+in+utero+electroporation+studies+of+developing+cerebral+cortex.">New and improved tools for in utero electroporation studies of developing cerebral cortex.</a> Cereb Cortex. 19 (July), i120-i125 (1991).</li><li>Nakahira, E., Yuasa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+generation,+migration,+and+differentiation+in+the+mouse+hippocampal+primoridium+as+revealed+by+enhanced+green+fluorescent+protein+gene+transfer+by+means+of+in+utero+electroporation.">Neuronal generation, migration, and differentiation in the mouse hippocampal primoridium as revealed by enhanced green fluorescent protein gene transfer by means of in utero electroporation.</a> J Comp Neurol. 483 (3), 329-340 (2005).</li><li>Navarro-Quiroga, I., Chittajallu, R., Gallo, V., Haydar, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term,+selective+gene+expression+in+developing+and+adult+hippocampal+pyramidal+neurons+using+focal+in+utero+electroporation.">Long-term, selective gene expression in developing and adult hippocampal pyramidal neurons using focal in utero electroporation.</a> J Neurosci. 27 (19), 5007-5011 (2007).</li><li>Borrell, V., Yoshimura, Y., Callaway, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+delivery+to+telencephalic+inhibitory+neurons+by+directional+in+utero+electroporation.">Targeted gene delivery to telencephalic inhibitory neurons by directional in utero electroporation.</a> J Neurosci Methods. 143 (2), 151-158 (2005).</li><li>Kolk, S. M., de Mooij-Malsen, A. J., Martens, G. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+Molecular+Approach+of+in+utero+Electroporation+to+Functionally+Decipher+Endophenotypes+in+Neurodevelopmental+Disorders.">Spatiotemporal Molecular Approach of in utero Electroporation to Functionally Decipher Endophenotypes in Neurodevelopmental Disorders.</a> FNMOL. 4 (November), 37 (2011).</li><li>Tomita, K., Kubo, K. I., Ishii, K., Nakajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disrupted-in-schizophrenia-1+(Disc1)+is+necessary+for+migration+of+the+pyramidal+neurons+during+mouse+hippocampal+development.">Disrupted-in-schizophrenia-1 (Disc1) is necessary for migration of the pyramidal neurons during mouse hippocampal development.</a> Hu Mol Gen. 20 (14), 2834-2845 (2011).</li><li>Niwa, M., Kamiya, A., 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=Knockdown+of+DISC1+by+In+Utero+Gene+Transfer+Disturbs+Postnatal+Dopaminergic+Maturation+in+the+Frontal+Cortex+and+Leads+to+Adult+Behavioral+Deficits.">Knockdown of DISC1 by In Utero Gene Transfer Disturbs Postnatal Dopaminergic Maturation in the Frontal Cortex and Leads to Adult Behavioral Deficits.</a> Neuron. 65 (4), 480-489 (2010).</li><li>Nakahira, E., Kagawa, T., Shimizu, T., Goulding, M. D., Ikenaka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+evidence+that+ventral+forebrain+cells+migrate+to+the+cortex+and+contribute+to+the+generation+of+cortical+myelinating+oligodendrocytes.">Direct evidence that ventral forebrain cells migrate to the cortex and contribute to the generation of cortical myelinating oligodendrocytes.</a> Dev Biol. 291 (1), 123-131 (2006).</li><li>Baumgart, J., Grebe, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C57BL/6-specific+conditions+for+efficient+in+utero+electroporation+of+the+central+nervous+system.">C57BL/6-specific conditions for efficient in utero electroporation of the central nervous system.</a> J Neurosci Methods. 240, 116-124 (2015).</li><li>Saito, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+electroporation+in+the+embryonic+mouse+central+nervous+system.">In vivo electroporation in the embryonic mouse central nervous system.</a> Nat Protc. 1 (3), 1552-1558 (2006).</li><li>Matsui, A., Yoshida, A. C., Kubota, M., Ogawa, M., Shimogori, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+in+utero+electroporation:+controlled+spatiotemporal+gene+transfection.">Mouse in utero electroporation: controlled spatiotemporal gene transfection.</a> JoVE. (54), (2011).</li><li>Guedel, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Inhalation anesthesia: a fundamental guide. , (1937).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+Animal+Perfusion+Fixation+for+Rodents.">Whole Animal Perfusion Fixation for Rodents.</a> JoVE. (65), e3564 (2012).</li><li>Mizutani, K., Saito, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progenitors+resume+generating+neurons+after+temporary+inhibition+of+neurogenesis+by+Notch+activation+in+the+mammalian+cerebral+cortex.">Progenitors resume generating neurons after temporary inhibition of neurogenesis by Notch activation in the mammalian cerebral cortex.</a> Development. 132 (6), 1295-1304 (2005).</li><li>Tabata, H., Nakajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labeling+embryonic+mouse+central+nervous+system+cells+by+in+utero+electroporation.">Labeling embryonic mouse central nervous system cells by in utero electroporation.</a> Dev, Growth &amp; Differ. 50 (6), 507-511 (2008).</li></ol>

The authors have nothing to disclose.

This protocol describes in detail how to transfect 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 of C75BL/6 mice. With all the provided information this is the first protocol, which supplies all necessary information to easily recreate transfections of these cerebral regions in the C57BL/6 mouse. Previous publications are mostly focused only on a few specific regions and therefore the detailed information needed for specific transfections is only scarcely standardized. 
The position and the size of the electrode as well as the applied voltage for the corresponding embryonic stage, which are the critical steps for effective and specific transfections, are precisely described. The provided angle-map helps to orientate the electrodes for specific electroporation. The locations of the electrodes are additionally shown on an embryo in the uterus, an embryo in the amniotic sac and also on a pure, dissected embryo. All of this is intended to help in identifying the correct positions of the electrodes. 
Usage of the appropriate voltage is a central aspect in the provided protocol. High voltages decrease the survival rate, whereas low voltages reduce the transfection efficiency 2,3,12. Voltage recommendations given in this protocol are specific for C57BL/6 mice and slightly differ from these for CD12,3,12. CD1 mice tolerate higher voltages than C57BL/6 mice and therefore higher voltages might be used for transfecting CD1 mice12. No data exist for other mouse strains, but the standards might need some modifications. 
A further central point is the size of the electrode. The usage of a smaller electrode, rather than a bigger one, is always recommended for experiments where specificity is important. Smaller electrodes enhance specificity4,12. If the specificity of the transfection is not the main point, especially for inexperienced experimenters, bigger electrodes enhance the chance of hitting the desired region. Thereby one has to keep in mind that electrodes covering too much of the embryo, might affect the heart rhythm and lead to reduced survival. For example a 10 mm electrode covers the whole E12 embryo and therefore is not recommended for transfecting E12 embryos, even if specificity is not the primary goal. 
The stages used in this protocol were chosen exemplary. Considering the fact that progenitors that are transfected on a certain time point give rise to a particular type of neuron the embryonic stages for transfection can be adapted to the specific aim of the study. For instance during cortical development, neurons are born at different time points, forming the six cortical layers in a sequential manner. Hence, depending of the time point of transfection the transfected progenitors give rise to neurons of a specific layer2,17.
For a successful transfection the following points also should be observed. First, the careful preparation and injection of the glass capillaries play a crucial role for the survival of the embryos and therefore for effective transfection. Second, the selection of an appropriate DNA concentration can also be very important and might have to be specifically adapted for different vectors18. Third, application of high voltages affects neighboring embryos, and therefore transfection of neighboring embryos might reduce the survival of these embryos. Further it has also been recommended to avoid the embryos closest to the vaginal duct, because the damage of these embryos might lead to abortion18.
Besides great advantages the technique of in utero electroporation has its drawbacks, which should be taken into consideration while designing experiment. First the regions, which can be efficiently transfected, are manly restricted to areas surrounding the ventricles. This is due to the fact that the plasmid-solution has to be injected into a ventricle. Further, even with all the details provided in this protocol it is not possible to transfect all cells out of a specific region. Even the number of transfected cells is hardly controllable. The analysis of the gene function is not restricted to embryonic stages. Adult mice can also be analyzed because the transfection of postmitotic neurons lasts at least up to 4 months13, but is not passed to the next generation. 
Further specifications, such as specifically electroporating thalamic and hypothalamic nuclei or temporally addressing cortical layers can additionally broaden the scope. The combination of genetically engineered mice, or inducible systems in with the in utero electroporation also additionally enhances possible applications. 
With all the given facts, this protocol facilitates specific transfections and therefore reduces the number of animal used. Standardization of the protocol additionally enhances the comparability of the obtained results.

Üç bağımsız gruplar 1-3 2001 yılında ilk açıklama beri i n utero elektroporasyon kemirgen, merkezi sinir sisteminde gen ifadesini analiz etmek için yaygın olarak kullanılan standart bir araç haline gelmiştir. Sürekli gelişen teknikler, hala zaman ve para kolaylığından dolayı, rahimde elektroporasyon temyiz tüketen rağmen bir nakavt farelerin nesil ile karşılaştırıldığında. Yani, in utero elektroporasyon hızlı ve verimli gain- ve zarar-fonksiyon çalışmalarını 4 sağlar. Serebral bölgeleri transfekte etmek için, negatif yüklü plasmid ihtiva eden bir çözelti, ventrikül içine enjekte edilir. Elektrik akımının sırasında, negatif yüklü DNA pozitif kutbuna doğru hareket eder ve bu nedenle, transfekte edilmiş bölgenin pozitif kutup konumunu değiştirerek basitçe seçilebilir. Sık sık, merkezi sinir sisteminin pek çok bölge ta olabilir gösterilmiştir3,5-8 rgeted. Örneğin, son çalışmalar hipokampus, striatum piriform kortekste veya 9-11 belirli transfections göstermektedir. Ancak, uygun pozisyonlar hakkında bilgiler genellikle sadece nadiren standardize ve her zaman farklı fare suşları aktarmak kolay değildir. Bazı embriyonik evrelerinin Transfeksiyon önemsiz olmaktan uzaktır. Utero elektroporasyon spesifik için set-up seçerken Birçok etkileyen faktörler dikkate alınmalıdır. İlk olarak, optimum ilgili embriyonik aşamalarında transfect uygun gerilimlerinin hakkında bilgiye ihtiyaç vardır. Düşük gerilimler transfeksiyon verimi 2,3,12 azaltmak ise yüksek gerilim, hayatta kalma oranını düşürmek. Ayrıca elektrot raket boyutu, önemli bir rol oynar düşürülmüş özgüllük çok büyük sonuçlar ya bağlı kalp ritmi 4,12,13 ve sevgi ile ölüme neden olabilir elektrot kürekler kullanımı nedeniyle. Uygulanangerilim ve boyut ve elektrot kürek konumu dikkat edilmesi gereken en önemli özelliklerdir, ancak DNA çözümün uygulanan miktarı gibi, elektroporasyon sonucu etkileyen başka faktörler de vardır. Biz C57BL / 6 fare 12 farklı beyin bölgelerinin hızlı ve etkin transfeksiyon sağlayan ayrıntılı bir protokol geliştirdik. Bu protokolde gerilimlerde hakkında ayrıntılı bilgiler kullanılmak üzere geliştirilmiş ve özgünlük için elektrot raket boyutu sağlanır. Bundan başka, ventrikül ilgili bilgiler plazmid solüsyonu miktarı ve elektrodun pozisyonu için öneriler sağlanır birlikte doldurulacak. Bir harita ve bu pozisyonlarda daha görselleştirme ayrıntılı konum bilgisi göstergesi retrosplenial korteks, motor korteks, somatosensoriyel korteks, piriform korteks, t utero elektroporasyon basit özgü ve verimli sağlarO ammonis 1-3, dentat girus, striatum, yan septal çekirdek, talamus ve hipotalamus Cornu.

<table><tbody><tr><td>EndoFree Plasmid Maxi Kit</td><td>QIAGEN</td><td>12362</td><td></td></tr><tr><td>Fast Green</td><td>Roth</td><td>0301.1</td><td></td></tr><tr><td>pCAGGS</td><td>Addgene</td><td></td><td></td></tr><tr><td>borosilicate glass capillaries (0.8-0.9 mm diameter)</td><td>Wold Precision Instrument Inc.</td><td>1B100F-4</td><td></td></tr><tr><td>Isoflurane (Forene)</td><td>Abbott</td><td>PZN 4831850</td><td></td></tr><tr><td>Carprofen (Rimadyl)</td><td>Pfizer GmbH</td><td>approval number: 400684.00.00</td><td></td></tr><tr><td>eye ointment (Bepanthen Augen und Nasensalbe)</td><td>Bayer&amp;nbsp;</td><td>PZN 01578681</td><td></td></tr><tr><td>0.9% benzyl alcohol 0.9% saline solution</td><td>Pharmacy&lt;br/&gt; of the University Medical Center Mainz</td><td></td><td></td></tr><tr><td>gauze (ES-Kompressen)</td><td>Hartmann</td><td>407835</td><td></td></tr><tr><td>sterile 5-0 Perma-Hand Silk Suture</td><td>Ethicon Johnson &amp;amp; Johnson</td><td>K890H</td><td></td></tr><tr><td>ring forceps 1/ 1.5 mm</td><td>Fine Science Tools</td><td>11101-09</td><td></td></tr><tr><td>ring forceps 4.8/ 6 mm</td><td>Fine Science Tools</td><td>11106-09</td><td></td></tr><tr><td>ring forceps 2.2/ 3 mm</td><td>Fine Science Tools</td><td>11103-09</td><td></td></tr><tr><td>Adson Forceps-Serrated Straight 12 cm</td><td>Fine Science Tools</td><td>1106-12</td><td></td></tr><tr><td>IrisScissors-Delicate Straight-Sharp/Blunt 10 cm</td><td>Fine Science Tools</td><td>14028-10</td><td></td></tr><tr><td>Mayo-Stille Scissors-Straight 15 cm</td><td>Fine Science Tools</td><td>14012-15</td><td></td></tr><tr><td>Dumont #5 Forceps-Inox</td><td>Fine Science Tools</td><td>11251-20</td><td></td></tr><tr><td>Castroviejo NeedleHolder-with Lock-Tungsten Carbide 14 cm</td><td>Fine Science Tools</td><td>12565-14</td><td></td></tr><tr><td>Elektroporator CUY21 SC&amp;nbsp;</td><td>Nepa Gene Co.</td><td></td><td></td></tr><tr><td>FST 250 Hot Bead Sterilizer</td><td>Fine Science Tools</td><td>18000-45</td><td></td></tr><tr><td>Microgrinder EG-44</td><td>Narishige</td><td></td><td></td></tr><tr><td>P-97 Micropette Puller</td><td>Sutter Instrument Company</td><td>P-97</td><td></td></tr><tr><td>Platinum electrodes 650P 0.5 mm</td><td>Nepagene</td><td>CUY650P0.5</td><td></td></tr><tr><td>Platinum electrodes 650P 3 mm</td><td>Nepagene</td><td>CUY650P3</td><td></td></tr><tr><td>Platinum electrodes 650P 5 mm</td><td>Nepagene</td><td>CUY650P5</td><td></td></tr><tr><td>Platinum electrodes 650P 10 mm</td><td>Nepagene</td><td>CUY650P10</td><td></td></tr><tr><td>Anesthesia system</td><td>Rothacher-Medical GmbH</td><td>CV-30511-3 Vapor 19.3</td><td></td></tr><tr><td>Heating plate</td><td>Rothacher-Medical GmbH</td><td>HP-1M</td><td></td></tr><tr><td>Temperature Controller 220V AC</td><td>Rothacher-Medical GmbH</td><td>TCAT-2LV</td><td></td></tr></tbody></table>

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.

Şekil 2, retrosplenial korteks içine gelişen bölgelerin utero elektroporasyon belirli için örnekler gösterir, motor korteks, somatosensorial korteks, piriform korteks, cornu 1-3 ammonis, dentat girus, striatum, yan septal çekirdek , talamus ve hipotalamus. Transfeksiyonlarının sonuçları tavsiye açı (Şekil 2) yanında gösterilmiştir. In vivo açıları daha iyi görüntülenmesi için elektrot (0,5 mm) konumu da (amniotik kese içinde Şekil 5&#39;te, rahimde embriyo, embriyo, disseke embriyo) bir embriyo üzerinde gösterilir. Sağlamak için genel bir bakış Tablo 1 spesifik transfeksiyon için ilgili gerçekleri özetlenmiştir. Bundan başka, bu elektrodun boyutu özgüllük (Şekil 6) çok önemli bir rol oynadığı gösterilmiştir. Elektrot kürekler büyük boyutlu la yol açarörnek teşkil eden, Şekil 6&#39;da gösterildiği gibi, rger alanları, transfekte edildi. Bundan başka, embriyo çok çok büyük örten elektrodlar, ve böylece embriyolar kalp ritmini (Şekil 7) etki riski artar. Örneğin 10 mm elektrot kürek bütün E12 embriyo (Şekil 7) kapsar. <img alt="figür 1" src="/files/ftp_upload/53303/53303fig1.jpg" /> Utero elektroporasyon Şekil 1.. Utero elektroporasyon performans temel adımlar gösterilmektedir. Bir ocak yerleştirilmiştir fare zaten izofluran ile anestezi ile (A), cerrahinin tüm kurulum gösterir. (B), cerrahi alanın dezenfeksiyonu farenin karın boşluğuna (C) açmadan önce çok önemli bir adımdır. (D) DNA içeren çözelti, hızlı G ile renklendirilirBaşarılı enjeksiyon görselleştirme sağlar reen, (E, F). Lateral ventrikül içine Başarılı enjeksiyon hilal şeklinde spot içine sonuçlanır. Uygun voltaj forseps türü platin elektrotları ile uygulanır Enjeksiyondan sonra (G). (H) dikkatlice cerrahi yara dikilir karın boşluğuna rahim değiştirdikten sonra. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53303/53303fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 2," src="/files/ftp_upload/53303/53303fig2.jpg" /> Murin korteks, hipokampüs, yanal septal çekirdek, striatum, talamus belirli alanlara elektroporasyonu ve hipotalamusta Şekil 2. Açılı-haritası. Pozitif ve negatif kutup için uygun pozisyonlar, şematik çizimde gösterilmiştir. (AC için </stro<html> ng&gt;) konumları sağ ventrikül içine bir DNA-çözeltisi enjekte etmek için endikedir. Sol ventrikül içine enjeksiyonlar için, açılar ayna ters bulunmaktadır. Belirli transfeksiyonlar histolojik teyit edildi. (A) kortikal alanlar transfecting için pozitif ve negatif kutup pozisyonları gösterir (retrosplenial korteks (RSC), motor korteks (MC), somatosensorial korteks (SSC) ve piriform korteks (PC). (B) gösterir hipokampal oluşumları transfecting için pozitif ve negatif kutup pozisyonları (cornu 1-3 (CA1, CA2, CA3) ve dentat girus (DG). (C) striatum transfecting için pozitif ve negatif kutup pozisyonları gösterir ammonis (STR) ve Baumgart J. &amp; batağan N., 2015, 12 uyarlanmıştır yanal septal çekirdeği (LS). (D) talamus (TH) ve hipotalamus (HY) transfecting için elektrotların uygun pozisyonları gösterir..<html> ps: //www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53303/53303fig2large.jpg &quot;target =&quot; _ blank &quot;&gt; bu rakamın büyük halini görmek için lütfen buraya tıklayınız. <img alt="Şekil 3," src="/files/ftp_upload/53303/53303fig3.jpg" /> Şekil 3. Delinme sitesi. DNA çözüm enjeksiyon için en uygun pozisyon Yaklaşık lambdoidal sütür ve 0.5 mm koronal 0.75 mm sagital sütür gelen yanal. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53303/53303fig3large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 4," src="/files/ftp_upload/53303/53303fig4.jpg" /> Kulak primordiumunun Şekil 4. Pozisyonu. Kulak primordiumunun pozisyonu açıkça (ok) tanımlanır.&quot;nk&gt; bu rakamın büyük halini görmek için lütfen buraya tıklayınız. <img alt="Şekil 5A," src="/files/ftp_upload/53303/53303fig5A.jpg" /><img alt="Şekil 5B," src="/files/ftp_upload/53303/53303fig5B.jpg" /><img alt="Şekil 5C" src="/files/ftp_upload/53303/53303fig5C.jpg" /><img alt="Şekil 5D" src="/files/ftp_upload/53303/53303fig5D.jpg" /> Bir embriyo üzerinde açıları Şekil 5. Görselleştirme. Daha iyi görselleştirme için kortikal alanların belirli transfeksiyonları (retrosplenial korteks, motor korteks, somatosensoriyel korteks, piriform pozitif (mavi artı işareti ile gösterilir) ve negatif kutup için uygun pozisyonları korteks), hipokampal alanlar (cornu ammonis (CA) 1-3, dentat girus (DG)),Yanal septal çekirdek, striatum, talamus ve hipotalamus utero bir embriyo gösterilir, amniyon kesesinde ve disseke embriyo (bütün E15,5) bir embriyo. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53303/53303fig5large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 6," src="/files/ftp_upload/53303/53303fig6.jpg" /> Elektrot boyutuna bağlı olarak transfeksiyonunun Şekil 6. Özgünlük. Elektrot raketin Artırıldı boyutu transfeksiyonunun özgünlüğünü azaltır. Bu geç embriyonik aşamalarda (E17, alt) özellikle genç embriyonik aşamalarında (E12, üst) bariz, ama aynı zamanda görülebilir. Baumgart J. &amp; batağan N., 2015, 12 uyarlanmıştır. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53303/53303fig6large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a><img alt="Şekil 7," src="/files/ftp_upload/53303/53303fig7.jpg" /> Bir E12 embriyo büyüklüğüne göre elektrot raket Şekil 7. Boyut. Embriyonun çok çok büyük kapak ve bu nedenle kalp ritminin sevgi riskini artırabilir elektrotlar. Örneğin, (sağda) 10 mm elektrot özgünlüğü temel amacı olmasa bile, bütün E12 embriyo kapsar ve bu nedenle tavsiye edilmez. Baumgart J. &amp; batağan N., 2015, 12 uyarlanmıştır. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53303/53303fig7large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <table fo:keep-together.within-page="1"><tbody><tr><td> Hedeflenen bölge </td><td> Açı </td><td colspan="2"> Elektrot konumu </td><td> Embriyonun Yaş </td><td> DNA Amount / konsantrasyon </td><td> Voltaj </td><td> Elektrot boyutları </td></tr><tr><td> Retrosplenial kortekste </td><td> 330 ° - 0 ° </td><td colspan="2"> Kulak primordia elektrotların merkezi sadece ön </td><td> E14 (E11-E17) </td><td> 1.5- 2 ul / / Ml 2.7- 2 ug </td><td> 36- 38 </td><td> 3- 5 mm </td></tr><tr><td> Motor korteks </td><td> 0 ° - 40 ° </td><td colspan="2"> Kulak primordia elektrotların merkezi sadece ön </td><td> E14 (E11-E17) </td><td> 1.5- 2 ul / / Ml 2.7- 2 ug </td><td> 36- 38 </td><td> 3- 5 mm </td></tr><tr><td> Somatosensori Cortex </td><td> 40 ° - 95 ° </td><td colspan="2"> Kulak primordia elektrotların merkezi sadece ön </td><td> E14 (E11-E17) </td><td> 1.5- 2 ul / / Ml 2.7- 2 ug </td><td> 36- 38 </td><td> 3- 5 mm </td></tr><tr><td> Piriform Cortex </td><td> 95 ° - 110 ° </td><td colspan="2"> Kulak primordia elektrotların merkezi sadece ön </td><td> E14 (E11-E17) </td><td> 1.5- 2 ul / / Ml 2.7- 2 ug </td><td> 36-3 8 </td><td> 3- 5 mm </td></tr><tr><td> Cornu ammonis (CA) 1 </td><td> 250 ° - 275 ° </td><td colspan="2"> Kulak primordiumunun ortasında pozitif kutup Merkezi </td><td> (E14-) E15 </td><td> 2 ul / / Ml 2 ug </td><td> 38- 40 </td><td> 3- 5 mm </td></tr><tr><td> CA2 </td><td> 230 ° - 250 ° </td><td colspan="2"> Kulak primordiumunun ortasında pozitif kutup Merkezi </td><td> (E14-) E15 </td><td> 2 ul / / Ml 2 ug </td><td> 38- 40 </td><td> 3- 5 mm </td></tr><tr><td> CA3 </td><td> 210 ° - 230 ° </td><td colspan="2"> Kulak primordiumunun ortasında pozitif kutup Merkezi </td><td> (E14-) E15 </td><td> 2 ul / / Ml 2 ug </td><td> 38- 40 </td><td> 3- 5 mm </td></tr><tr><td> Kıvrımlarının </td><td> 190 ° - 210 ° </td><td colspan="2"> Kulak primordiumunun ortasında pozitif kutup Merkezi </td><td> (E14-) E15 </td><td> 2 ul / / Ml 2 ug </td><td> 38- 40 </td><td> 3- 5 mm </td></tr><tr><td> Striatum </td><td> 170 ° - 240 ° </td><td colspan="2"> Kulak primordia seviyesinde elektrot hizmetleri </td><td> E12 </td><td> 1 ul / / Ul 4 ug </td><td> 33 </td><td> 0,5 mm </td></tr><tr><td> Yanal Septal Nucleus </td><td> 100 ° - 170 ° </td><td colspan="2"> Kulak primordia seviyesinde elektrot hizmetleri </td><td> E12 </td><td> 1 ul / / Ul 4 ug </td><td> 33 </td><td> 0,5 mm </td></tr><tr><td> Talamus </td><td> 25 ° - 40 ° </td><td colspan="2"> Electodes merkezi sadece kulak pri posteriormordia </td><td> E12- E13 </td><td> 1-1,5 ul / / Ml 4-2,7 ug </td><td> 33- 35 </td><td> 0.5- 3 mm </td></tr><tr><td> Hipotalamus </td><td> 90 ° - 180 ° </td><td colspan="2"> Electodes merkezi sadece kulak Primordia posterior </td><td> E12- E13 </td><td> 1-1,5 ul / / Ml 4-2,7 ug </td><td> 33- 35 </td><td> 0.5- 3 mm </td></tr></tbody></table> Tablo 1:. Sonuçların Özeti bu tabloda dahil açıklanan kortikal alanların belirli transfeksiyon ilgili tüm bilgiler. Bu hedeflenen bölge, açı, elektrodun pozisyonu, embriyonik aşamada, DNA çözeltisi, gerilim uygulanan miktarı ve konsantrasyonu ve elektrot boyutu hakkında ayrıntılı bilgiler yer almaktadır.

Watch this Scientific Journal Video about Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse at JoVE.com

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 ...

cortex-hippocampus-thalamus-hypothalamus-lateral-septal-nucleus

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

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

27.9K Görüntüleme.  University Medical Center, JGU Mainz. 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.

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

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

Fare Hipokampal Doku Ex Utero Elektroporasyon ve Organotipik Dilim Kültür

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

Gelişim Biyolojisi

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol we outline the experimental methodology for preparing mice for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E13-E16 mice, however, it is most commonly performed at E15, as shown in this video. 

E15 Fare Embriyolar Utero İntraventriküler Enjeksiyon ve Elektroporasyon

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors ...

Biyoloji

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 ...

Nörobilim

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 ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

Tarafından Embriyonik ve Erişkin Nöral Kök Hücre Genişletme<em&gt; In Utero</em&gt; Elektroporasyon veya Viral Stereotaksik Enjeksiyon

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

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 ...

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 ...

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, ...

Double In Utero Electroporation to Target Temporally and Spatially Separated Cell Populations

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian corticogenesis. This procedure takes advantage of the brain ventricles to allow the introduction of DNA of interest and uses a pair of electrodes to direct the entrance of the genetic material into the cells lining the ventricle, the neural stem cells. This method allows researchers to label the desired cells and/or manipulate the expression of genes of interest in those cells. It has multiple applications, including assays targeting neuronal migration, lineage tracing, and axonal pathfinding. An important feature of this method is its temporal and regional control, allowing circumvention of potential problems related with embryonic lethality or the lack of specific CRE driver mice. Another relevant aspect of this technique is that it helps to considerably reduce the economic and temporal limitations that involve the generation of new mouse lines, which become particularly important in the study of interactions between cell types that originate in distant areas of the brain at different developmental ages. Here we describe a double electroporation strategy that enables targeting of cell populations that are spatially and temporally separated. With this approach we can label different subtypes of cells in different locations with selected fluorescent proteins to visualize them, and/or we can manipulate genes of interest expressed by these different cells at the appropriate times. This strategy enhances the potential of in utero electroporation and provides a powerful tool to study the behavior of temporally and spatially separated cell populations that migrate to establish close contacts, as well as long-range interactions through axonal projections, reducing temporal and economic costs.

Double in utero electroporation allows targeting cell populations that are spatially and temporally separated. This technique is useful to visualize interactions between those cell populations using fluorescent proteins in normal conditions but also after functional experiments to perturb genes of interest.

Geçici ve Mekansal Olarak Ayrılmış Hücre Popülasyonlarını Hedef lemek için Utero Elektroporasyonunda Çift

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian ...

In Vivo Targeting of Neural Progenitor Cells in Ferret Neocortex by In Utero Electroporation

Manipulation of gene expression in vivo during embryonic development is the method of choice when analyzing the role of individual genes during mammalian development. In utero electroporation is a key technique for the manipulation of gene expression in the embryonic mammalian brain in vivo. A protocol for in utero electroporation of the embryonic neocortex of ferrets, a small carnivore, is presented here. The ferret is increasingly being used as a model for neocortex development, because its neocortex exhibits a series of anatomical, histological, cellular, and molecular features that are also present in human and nonhuman primates, but absent in rodent models, such as mouse or rat. In utero electroporation was performed at embryonic day (E) 33, a midneurogenesis stage in ferret. In utero electroporation targets neural progenitor cells lining the lateral ventricles of the brain. During neurogenesis, these progenitor cells give rise to all other neural cell types. This work shows representative results and analyses at E37, postnatal day (P) 1, and P16, corresponding to 4, 9, and 24 days after in utero electroporation, respectively. At earlier stages, the progeny of targeted cells consists mainly of various neural progenitor subtypes, whereas at later stages most labeled cells are postmitotic neurons. Thus, in utero electroporation enables the study of the effect of genetic manipulation on the cellular and molecular features of various types of neural cells. Through its effect on various cell populations, in utero electroporation can also be used for the manipulation of histological and anatomical features of the ferret neocortex. Importantly, all these effects are acute and are performed with a spatiotemporal specificity determined by the user.

Presented here is a protocol to perform genetic manipulation in the embryonic ferret brain using in utero electroporation. This method allows for targeting of neural progenitor cells in the neocortex in vivo.

Utero Elektroporasyon in Ferret Neokorteks nöral progenitor hücrelerinin Vivo Hedefleme

Manipulation of gene expression in vivo during embryonic development is the method of choice when analyzing the role of individual genes during mammalian ...

Korteks, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- ve Striatum özgü

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Fare Hipokampal Doku Ex Utero Elektroporasyon ve Organotipik Dilim Kültür

E15 Fare Embriyolar Utero İntraventriküler Enjeksiyon ve Elektroporasyon

Verimli kullanarak birden çok CNS Topraklar'da Gen Teslimat In Utero Elektroporasyon

Fare Utero Elektroporasyon: Kontrollü Spatiotemporal Gen Transfeksiyon

Tarafından Embriyonik ve Erişkin Nöral Kök Hücre Genişletme

Kullanarak Fare Serebral Korteks ve Hippocampus Görselleştirme ve Dendritler ve Spine Genetik Manipülasyon

Fare Genetik Manipülasyon yoluyla Hipotalamus geliştirilmesi

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

Geçici ve Mekansal Olarak Ayrılmış Hücre Popülasyonlarını Hedef lemek için Utero Elektroporasyonunda Çift

Utero Elektroporasyon in Ferret Neokorteks nöral progenitor hücrelerinin Vivo Hedefleme