Yazarlar Sharmin Hossain SCE micromicropipette ipuçları elektron mikroskobu görüntüleri olgunlaşmamış bir nöron ve Derek Dunfield time-lapse yakalama büyüme için teşekkür ederiz.

Ekipman set-up: <ol><li> Bu teknik için gerekli elektrik ekipmanları, bir uyarıcıdır bir osiloskop ve gümüş tel mikropipet eklenecek ile donatılmış bir mikropipet sahibi. Biz genellikle bir Axon aletleri Axoporator 800A cihazın kullanımı, ancak bu Çim aletleri gibi diğer yaygın uyarıcıları SD9 uyarıcıları tek hücreli elektroporasyon için de uygundur. Cihazda dahili bir iletken solüsyon içeren cam mikropipet içine yerleştirilen gümüş tel elektrot ile arayüzleri headstage, bağlanır. Stimülatörü diğer kurşun tetra dış yerden giriş aldığı osiloskop girişi, bağlı. Tetra dış zemin elektroporasyon sırasında tetra electrotonic temas halinde kalan bir gümüş tel. Mikropipet larva ile temas konulduğunda devreyi tamamlamak. </li><li> Bu manevranın oda verir ve yaklaşık 30-45 derecelik bir açıyla mikropipet getirilerek sağlar yılından bu yana tek bir hücre elektroporasyon, iyi bir çalışma mesafesi (en az 5 cm) olan bir mikroskop gerektirir. </li></ol> Mikropipetler Fabrikasyon: Uygun mikropipetler hazırlanması, bu protokol için önemli bir adımdır. Buradaki zorluk, dar bir ucu (çapı 1 mikrona kadar) daha az doku delinmesiyle zaman kolayca kırmayacak bir dengeleme yatıyor. Ayrıca, 10 dereceden daha fazla bir konik açısı ile ipuçları çok tercih edildiğini bulduk. <ol><li> 1 mikrona kadar daha az bir ucu çapı ve 10 dereceden daha fazla bir ucu konik açısı ile çekilmiş bir cam mikropipet hazırlayın. <ol><li> Dış çapı 1.5mm ve 0.75mm iç çapı ile bir iç filament borosilikat cam ve bu amaç için uygundur. <ol><li> Nispeten daha kalın bir cam ucu katılık derecesi sağlar. </li><li> Iç filament mikropipet ucuna doğru geri-sıvı dolu çizim önemlidir. </li></ol></li></ol></li><li> Uygun ipuçları Fabrikasyon sıklıkla gözlenen sonuçların dayalı bazı değişiklikler gerekecektir. Farklı dokular, biraz daha farklı bir şekil veya bahşiş çapı bir ipucu isteyebilir. </li></ol> Tek hücreli elektroporasyon protokolü: Yeni kullanıcılar için, genetik olarak kodlanmış floresan proteinleri aksine, onlar hemen Epifloresans altında görülebilir, çünkü floresan dekstran boyalar ile başlamak tavsiye edilir. Floresan boyaların kullanımı kullanıcının doğrudan doku içinde mikropipet ucunun yerini görmek için sağlar ve üzerine elektroporasyon ekipman düzgün mikropipet ucu uygun olup olmadığını kurmak ve olup olmadığını anında geri bildirim verir. <ol><li> Mikropipet bir yükleme şırınga kullanarak veya arka doldurarak electroporated bileşik solüsyonu ile doldurun. <ol><li> Eğer plazmid DNA&#39;sı kullanarak, suda hazırlanan örnekleri endotoksin ücretsiz olduğunu sağlamak ve 1-2μg/ml arasında bir konsantrasyon. </li><li> Floresan dekstran boyaların konsantrasyonu ampirik olarak tespit edilmelidir. Biz sık sık su yaklaşık 300μM sulandırılmış floresan boya kullanın. </li><li> Tipik olarak, 0.5-1μl hacimleri yeterlidir. </li><li> Yüklemeden önce, kısaca tıkanmaya yol açabilir mikropipet, içine çekilir partikül enkaz miktarını azaltmak için yüksek hızda çözümler santrifüj. </li><li> Ucu içinde hava kabarcıkları genellikle akım etkileyebilir ve şiddetle headstage, montaj öncesinde mikropipet Flicking yerinden olmalıdır. </li></ol></li><li> Tutucunun içine mikropipet yerleştirin veya headstage, 3-eksen mikromanipülatör üzerine monte edilmiş. Mikropipet o gümüş tel elektrot yerleştirilir ve iç çözümü ile electrotonic temas içinde olduğunu emin olun. </li><li> Larva yetiştirme ortamı (Steinberg solüsyonu, pH 7.4) hazırlanan% 0,02 MS-222 (3-aminobenzoik asit etil ester) batırılarak tetarlar anestezisi. <ol><li> Biz normalde deneylerde evre 44-48 albino Xenopus laevis tetarlar kullanın. </li><li> Tetarlar yaklaşık 5 dakika sonra genellikle tamamen anestezi. </li><li> Çeşitli tetarlar bekleme süresini azaltmak için aynı anda narkoz olabilir, ancak 1 saat dışındaki süreler için MS-222 maruz kalmaktan kaçının. </li></ol></li><li> Plastik bir transfer mikropipet kullanarak elektroporasyon odasına tek bir anestezi larva transferi. <ol><li> Elektroporasyon odası, küçük bir Sylgard ® silikon blok tetra-şekilli bir kavite, oyma ve gümüş bir topraklama kablosu takmadan odasında larva ile electrotonic temas halinde olacağı gibi boşluğuna kolayca ev yapılabilir. </li></ol></li><li> Kullanılarak pozisyon larva dorsal tarafına fırça temizleyin. </li><li> Mikropipet, cilt ile temas halinde doğrudan ilgi bölgeye bitişik olduğu kadar düşürün. <ol><li> Yoğun dolu hücre gövdeleri bölgeleri hedeflemek, başarılı elektroporasyon şansını büyük ölçüde artıracaktır.Biz genellikle, tetra optik tectum hedef. </li><li> 30 ila 45 derecelik bir açıyla mikropipet getirin. Dik açıları genellikle bir görüşü engelleyen ise, sığ yörüngeleri, cilt ponksiyon daha zor hale getirir. </li></ol></li><li> Devamı mikropipet düşürücü cilt başlangıçta Köşelerini ve altta yatan doku içine geçmesine olacak. <ol><li> Yüzeysel hücreleri in vivo görüntüleme için tercih edilir ve mikropipet ucu cilt dimpling üzerine yavaş yavaş indirilir sağlamak hedeflenmiş olabilir. </li></ol></li><li> Bir kez dokusu içinde yer mikropipet direnci sürekli izlemeniz gerekir. Eğer bir Axoporator 800A (Axon Instruments), yaklaşık 10-40MΩ dirençler kullanarak en uygun olan. Direnç ucu çapı tek hücreli elektroporasyon için uygun olup olmadığını iyi bir gösterge. <ol><li> Yüksek mikropipet dirençleri (&gt; 100MΩ), çok dar ipuçları ile tıkanmış ip uçlarına ya da mikropipetler göstergesidir. </li><li> Düşük mikropipet dirençleri (10M), genellikle, bir kırık ucu bir sonucu çok büyük bir ucu çapı göstergesidir. </li><li> Doğrudan direncini ölçmek sağlamaz stimülatörü kullanıyorsanız, mikropipet direnç osiloskop (aşağıda) ele tarafından ölçülen gerilim darbeleri genliği gözlemleyerek dolaylı ölçülebilir. </li></ol></li><li> Gerilim tren uygulayın. <ol><li> Plazmid DNA için, darbe, darbe tren süresi 500ms 300Hz&#39;de teslim süresi 1ms negatif kare dalga bakliyat, oluşan en etkili olduğu tespit eğitir. <ol><li> Darbe gerilimi osiloskop ölçülen genlik bakliyat 0.75 ve 1.5μA arasında olduğunu ampirik gibi belirlenir. </li></ol></li><li> Floresan dekstran boyalar için, pozitif kare dalga darbeleri 300μs süresi 10ms darbe tren süresi ile 300Hz frekans teslim edilir. <ol><li> DNA protokol ile, darbe gerilimi osiloskop ölçülen genlik bakliyat 0.75 ve 1.5μA arasında olduğunu ampirik gibi belirlenir. </li><li> Epifloresans altında elektroporasyon sonuçlarını gözlemleyerek dayalı uyaran parametrelerine ek ayarlamalar yapılabilir. <ol><li> Electroporated hücreler loş görünür hücreleri parlak görünene kadar, darbe parametreleri giderek artmış olmalıdır. </li><li> Öte yandan hücre kümeleri etiketli, tek hücre etiketli kadar, darbe parametrelerin azaltılması olmalıdır. </li></ol></li></ol></li></ol></li><li> Mikropipet geri çekin, doku içinde farklı bir site tekrar takın, ve gerilim tren uygulayın. <ol><li> Verimi artırmak için, biz genellikle o etiketli hücrelerin üst üste binmemesi sağlamak için yeterli boşluk ile larva optik tectum her yarımkürede birden fazla sitede electroporate. </li></ol></li><li> Steinberg yetiştirme çözümü ile bir kap içine electroporated larva transferi. <ol><li> Tetarlar anestezi birkaç dakika içinde hızla iyileşir. </li></ol></li><li> Aynı mikropipet sayısız iribaşların kullanılabilir. Osiloskop gözlenen bakliyat genliği sürekli gözlemlemek önemlidir. <ol><li> Darbe genlik darbe yoğunluğu önemli ölçüde artan sonra düşük kalırsa, bu durum, bir oturuşta birçok tetarlar electroporating sık görülen tıkanmış olan ucu, bir sonucudur. <ol><li> Küçük bir ipucu yapışmasına neden çıkarmak için kullanılan bir yöntem, doku içine mikropipet yerleştirdikten sonra tek bir pozitif darbe uygulamaktır. </li><li> Yapışmasına neden yerinden ya da sık sık kendini yinelemektedir tıkanma değil, mikropipet değiştirin. </li></ol></li><li> Darbe genlik nabız parametrelerini önemli ölçüde azalan sonra çok büyük kalırsa, bu, ucu kırık olduğunu ve değiştirilmesi gerekir bir işaretidir. </li></ol></li></ol> Başarıyla electroporated hücreler için Gösterimi: <ol><li> Elektroporasyon sonra, tetarlar Epifloresans dik bir mikroskop kullanılarak etiketli hücreler için taranmaktadır. </li><li> Floresan boyaların durumda, en az 30 dakika sonrası elektroporasyon tetarlar sonra gösterilebilir. <ol><li> Biz daha uzun aralıklarla düşük eşiğe düzeyleri ile ilişkili olduğunu, ancak bulduk. </li></ol></li><li> Genetik olarak kodlanmış floresan proteinleri için, tarama genellikle en az 12 saat sonrası elektroporasyon yapılır. <ol><li> Floresan proteinlerle ifade hücreler zamanla parlak almaya devam edecektir, bu yüzden loş hücreleri 12-24 saat ek bir aradan sonra yeniden gösterildi olabilir. </li></ol></li><li> Sylgard ® odasında yerleştirilen, yukarıda açıklanan ve dakika muamele tetarlar anestezi. Bireysel tetarlar sonra Epifloresans altında incelenir. <ol><li> Epifloresans ışığa aşırı derecede maruz etiketli hücre öldürebilir fototoksisite neden olacağını unutmayın. Bu nedenle, floresan ışık hücrelerinin maruz kaldığı süreyi en aza indirir. </li></ol></li><li> Steinberg yetiştirme çözümü ile konteyner tetarlar dönün. </li></ol>

<ol>
	<li>Bestman, J. E., Ewald, R. C., Chiu, S. L., Cline, H. 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+single-cell+electroporation+for+transfer+of+DNA+and+macromolecules.">In vivo single-cell electroporation for transfer of DNA and macromolecules.</a> Nature Protocols. 1, 1267-1272 (2006).</li><li>Dunfield, D., Haas, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+electroporation.">Single cell electroporation.</a> Encyclopedia of Neuroscience (4th Ed.). , .</li><li>Haas, K., Jensen, K., Sin, W. C., Foa, L., Cline, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+electroporation+in+Xenopus+tadpoles+in+vivo--from+single+cells+to+the+entire+brain.">Targeted electroporation in Xenopus tadpoles in vivo--from single cells to the entire brain.</a> Differentiation. 70, 148-154 (2002).</li><li>Haas, K., Sin, W. C., Javaherian, A., Cline, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+electroporation+for+gene+transfer+in+vivo.">Single-cell electroporation for gene transfer in vivo.</a> Neuron. 29, 583-591 (2001).</li></ol>

Tek hücreli elektroporasyon (SCE), gen fonksiyonu belirleyen ve hedeflenen genetik manipülasyon yapmak için güçlü bir araçtır. Albino larva şeffaflık ve beyin erişilebilirlik Bu model sistem canlı bir organizma içinde nöronal büyüme ve hücre içi olayları görselleştirmek için ideal olun. SCE, tek bir nöronun büyüme görselleştirme izin verir, ve aksi halde değiştirilmemiş beynin içinde hücre özerk manipülasyonlar gerçekleştirebilirsiniz. Bu video makale Xenopus laevis tetarlar tek hücreli elektroporasyon i...

single cell electroporation in vivo within the intact developing brain

Tek hücreli elektroporasyon (SCE), in vivo hazırlıkları da dahil olmak üzere sağlam doku içindeki tek tek hücrelerin içine DNA veya diğer makromoleküller teslim sağlayan özel bir tekniktir. Bu tekniğin en belirgin avantajı, çevresindeki doku değişmeden bırakarak bu deneysel manipülasyonlar ve böylece küresel tedaviler sonucunda hücre özerk etkileri ayırt bireysel hücreleri üzerinde olabilir. Ileri in vivo görüntüleme teknikleri ile kombine edildiğinde, floresan belirteçlerin SCE hücresel morfoloji, hücre büyümesi ve hücre içi olayları saniye gün değişen zaman çizelgesi üzerinde doğrudan görselleştirme izin verir. Bu tekniği, in vivo ve ex vivo hazırlıkları içinde çeşitli olsa da, biz Xenopus laevis tetarlar kullanmak için bu tekniği optimize var. Bu video makalede, detaylı albino Xenopus larva bozulmamış beyin içinde nöronlar bir floresan boya veya plazmid DNA SCE prosedürü. Ayrıca verim optimize yöntemleri tartışmak ve canlı SCE tarafından floresan etiketli nöronlar iki foton floresan görüntüleme örnekler göstereceğiz.

Watch this Scientific Journal Video about Single Cell Electroporation in vivo within the Intact Developing Brain at JoVE.com

Single Cell Electroporation in vivo within the Intact Developing Brain

Tek hücreli elektroporasyon (SCE), in vivo hazırlıkları da dahil olmak üzere sağlam doku içindeki tek tek hücrelerin içine DNA veya diğer makromoleküller teslim sağlayan özel bir tekniktir. Xenopus laevis larva bozulmamış beyin içinde nöronların bir floresan boya veya plazmid DNA SCE için burada ayrıntılarıyla prosedür.

Tek Hücre Elektroporasyon bozulmamış Gelişmekte olan beyin içindeki in vivo

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact ...

single-cell-electroporation-in-vivo-within-the-intact-developing-brain

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

13.2K Görüntüleme.  University of British Columbia - UBC. Tek hücreli elektroporasyon (SCE), in vivo hazırlıkları da dahil olmak üzere sağlam doku içindeki tek tek hücrelerin içine DNA veya diğer makromoleküller teslim sağlayan özel bir tekniktir. Xenopus laevis larva bozulmamış beyin içinde nöronların bir floresan boya veya plazmid DNA SCE için burada ayrıntılarıyla prosedür.

Video: Single Cell Electroporation in vivo within the Intact Developing Brain

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 ...

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

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

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

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

Fare Akut Beyin Dilimleri Neuroblast Göç vivo Doğum Sonrası Elektroporasyonla ve Time-lapse Görüntüleme

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Neonatal Pial Surface Electroporation

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and gliogenesis, including after injury. However, methods for genetically interrogating these progenitor populations and tracking their lineages had been limited owing to a lack of specificity or time consuming production of viruses. Thus, progress in this region has been relatively slow with only a handful of investigations of this location. Electroporation has been used for over a decade to study neural stem cell properties in the embryo, and more recently in the postnatal brain. Here we describe an efficient, rapid, and simple technique for the genetic manipulation of pial surface progenitors based on an adapted electroporation approach. Pial surface electroporation allows for facile genetic labeling and manipulation of these progenitors, thus representing a time-saving and economical approach for studying these cells.

The pial surface is a unique progenitor zone in the CNS that is receiving increasing attention. Herein, we detail a method for rapid genetic manipulation of this progenitor zone using a modified electroporation method. This procedure can be used for cellular and molecular investigations of cell lineages and signaling pathways involved in cell differentiation and to elucidate the fate and properties of daughter cells.

Yenidoğan Pial Yüzey Elektroporasyon

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and ...

Subtype-selective Electroporation of Cortical Interneurons

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression of genes of interest to specific neuronal subtypes has proven remarkably challenging due to the relative scarcity of specific promoter elements. GABAergic interneurons constitute a neuronal population with extensive genetic and morphological diversity. Indeed, more than 11 different subtypes of GABAergic interneurons have been characterized in the mouse cortex1. Here we present an adapted protocol for selective targeting of GABAergic populations. We achieved subtype selective targeting of GABAergic interneurons by using the enhancer element of the homeobox transcription factors Dlx5 and Dlx6, homologues of the Drosophila distal-less (Dll) gene2,3, to drive the expression of specific genes through in utero electroporation.

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

Kortikal internöronlar alt tip-selektif Elektroporasyon

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

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, ...

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 ...

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 ...