この作品は、国防総省（アイデア開発賞、W81XWH-10-1-0041、AB）、CTの幹細胞助成金（A-B）、および健康NRSA 10668225ホルムアミド（DMF）の国立研究所からの補助金によって支えられている。本材料は、部分的にコネチカット幹細胞研究助成金プログラムの下コネチカット州によってサポートされた仕事に基づいています。その内容はもっぱら著者の責任であり、必ずしもコネチカット、コネチカットやCTイノベーションの州の公衆衛生学教室の国の公式見解を示すものではない、（株）出資者は、データ収集を研究デザインに何の役割を持っていませんと分析、公表することを決定、または原稿の準備。

この手順では、エール大学動物実験委員会の要求に従ったものである。科学者は、動物実験委員会のガイドラインは、その制度上の要件に応じて承認され、使用されていることを確認する必要があります。 1。第1節。 DNAは、ソリューション、およびガラスピペットの作製<ol><li>高純度（OD 280分の260&gt; 1.80）、高濃度（μg/μlの&gt; 2.5）エンドトキシンフリーのDNAを生成します。 </li><li> 22μmのフィルターを通して0.9％生理食塩水や滅菌フィルターを準備します。 </li><li>カンタル線を使用したモデルのPP-830ナリシゲPC-10ガラスピペットプラー場所に10cmの火災洗練されたホウケイ酸ガラスキャピラリーチューブ（1.10 mm外径：1.5ミリメートル、ID）を指定します。 70.5一歩ずつ加重プル℃にプーラーをセット円形の鉗子とのヒントを破ると、ギザギザの縁のための解剖顕微鏡で検査します。 15分間UVランプ下ベベルのヒントや場所。引っ張らピペットは先端の端から2ミリメートルでマークする必要があります。 </li><li>重量/体積速いグリーンsoluti 0.1パーセントを準備乾いた速いグリーンで滅菌濾過0.9％食塩水を混合することにより、上の。 </li></ol> 2。第2節。動物の準備、ガラスピペットの読み込みおよびプラスミドインジェクション<ol><li>ガラスシャーレ4〜予冷に、ケージ0から1までの子犬生後個別に場所を取り外し℃にした後、濡れた氷の上に置きます。 </li><li>約5分後、足のピンチ応答を利用することによって、麻酔の状態を確認してください。脳室内注射への移動が発生しない場合は、件名子犬。 </li><li>ファストグリーン、DNA、および食塩水の組み合わせは急速な蒸発、結晶化、ガラスピペットの封鎖をもたらすことができることに注意するのは重要です。したがって、原液や注射直前にパラフィルム上にアリコート1から2μlを生成します。 </li><li>引っ張らガラスピペットで分注し全体音量を吸引します。 </li><li>親指とあなたの小さい利き手の人差し指で子犬の頭を保持する。 </li><li>ランプを点灯させdがちょうど光ビームの外に子犬の頭を保持する。必要であれば、子犬によって反射された光の量を減らすために、頭の上に生理食塩水を入れた。心室は現在点灯しなければならない。 </li><li>あなたの利き手を使用すると、（ 図1Aおよび1B）に最も近い側脳室に針を挿入します。注射部位は、ラムダ縫合および目と矢状縫合に横2ミリメートルからほぼ等距離にあるべきである。挿入は、側脳室への浸透性を確保すべき、P0は子犬のために2mmのマークにピペットを引っ張った。偶然にも、あなたは、頭蓋内圧のリリースからピペット内に髄液の充填バック表示される場合があります。プラスミドの注入を支援頭蓋内圧を減少させるために、pup.Step 2.8の頭の上にあなたのグリップを緩めます。エアプレッシャーインジェクター（Picospritzer、パーカー）を使用して、側脳室へのプラスミドの約0.5μLを注入します。 </li></ol> 3。第3節。エレクトロポレーションおよびリカバリ<ol><li> SEトン5平方パルス、950ミリ秒間隔で100ボルトで50ミリ秒/パルス、エレクトロポレーション用の発電機の電圧。プラスミドエレクトロの空間特異性は、電荷転送の方向性によって決定される。細心の注意は、SVZ 8に沿って幹細胞集団の異質性を与えられた配置に与えられるべきである。増殖と細胞の運命のレートの違いは、腹と背吻側-尾位置9,10ために特定されました。 </li><li>プラスミドを注入された後、PBSにピンセット電極を浸す。このステップでは、電荷移動を最大にし、抵抗による熱傷を防止するためである。 </li><li>プラスミド注射部位（ 図1Cおよび1D）と同側の耳の近くに子犬の背側壁上に正極を配置します。子犬の鼻面に対側半球腹側部に負極を配置します。 </li><li>パルスフーツを押すことによって、現在の転送を開始する魔女は、背から約25°の角度間隔を使用して横方向に電極をペダルと掃く。 </li><li> 5分間加熱パッド上にエレクトロポレーションした子犬を置きます。優しく穏やかな刺激で子犬を30秒毎に回転させます。 </li></ol>

<ol>
	<li>Kriegstein, A., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+glial+nature+of+embryonic+and+adult+neural+stem+cells.">The glial nature of embryonic and adult neural stem cells.</a> Annual Review of Neuroscience. 32, 149-184 (2009).</li><li>Imayoshi, I., Sakamoto, M., Kageyama, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+methods+to+identify+and+manipulate+newly+born+neurons+in+the+adult+brain.">Genetic methods to identify and manipulate newly born neurons in the adult brain.</a> Frontiers in Neuroscience. 5, 64 (2011).</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, i120-i125 (2009).</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, 865-872 (2001).</li><li>Boutin, C., Diestel, S., Desoeuvre, A., Tiveron, M. C., Cremer, H. <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+vivo+electroporation+of+the+postnatal+rodent+forebrain.">Efficient in vivo electroporation of the postnatal rodent forebrain.</a> PloS ONE. 3, e1883 (2008).</li><li>Chesler, A. T., 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=Selective+gene+expression+by+postnatal+electroporation+during+olfactory+interneuron+nurogenesis.">Selective gene expression by postnatal electroporation during olfactory interneuron nurogenesis.</a> PloS ONE. 3, e1517 (2008).</li><li>Platel, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMDA+receptors+activated+by+subventricular+zone+astrocytic+glutamate+are+critical+for+neuroblast+survival+prior+to+entering+a+synaptic+network.">NMDA receptors activated by subventricular zone astrocytic glutamate are critical for neuroblast survival prior to entering a synaptic network.</a> Neuron. 65, 859-872 (2010).</li><li>Alvarez-Buylla, A., Kohwi, M., Nguyen, T. M., Merkle, F. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+heterogeneity+of+adult+neural+stem+cells+and+the+emerging+complexity+of+their+niche.">The heterogeneity of adult neural stem cells and the emerging complexity of their niche.</a> Cold Spring Harbor Symposia on Quantitative Biology. 73, 357-365 (2008).</li><li>Fernandez, M. E., Croce, S., Boutin, C., Cremer, H., Raineteau, O. <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+of+defined+lateral+ventricular+walls:+a+novel+and+rapid+method+to+study+fate+specification+during+postnatal+forebrain+neurogenesis.">Targeted electroporation of defined lateral ventricular walls: a novel and rapid method to study fate specification during postnatal forebrain neurogenesis.</a> Neural Development. 6, 13 (2011).</li><li>de Chevigny, 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=miR-7a+regulation+of+Pax6+controls+spatial+origin+of+forebrain+dopaminergic+neurons.">miR-7a regulation of Pax6 controls spatial origin of forebrain dopaminergic neurons.</a> Nature Neuroscience. 15, 1120-1126 (2012).</li><li>Lacar, B., Young, S. Z., Platel, J. C., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+and+recording+subventricular+zone+progenitor+cells+in+live+tissue+of+postnatal+mice.">Imaging and recording subventricular zone progenitor cells in live tissue of postnatal mice.</a> Frontiers in Neuroscience. 4, (2010).</li><li>Feliciano, D. M., Quon, J. L., Su, T., Taylor, M. M., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+neurogenesis+generates+heterotopias,+olfactory+micronodules+and+cortical+infiltration+following+single-cell+Tsc1+deletion.">Postnatal neurogenesis generates heterotopias, olfactory micronodules and cortical infiltration following single-cell Tsc1 deletion.</a> Human Molecular Genetics. 21, 799-810 (2012).</li><li>Iguchi, T., Yagi, H., Wang, C. C., Sato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tightly+controlled+conditional+knockdown+system+using+the+Tol2+transposon-mediated+technique.">A tightly controlled conditional knockdown system using the Tol2 transposon-mediated technique.</a> PloS ONE. 7, e33380 (2012).</li></ol>

著者が作る全く開示していません。

ここでは詳細新生児SVZのエレクトロポレーションの技術は、急速かつ確実にラベルを付けたり、SVZの幹細胞およびその子孫を操作するためのテクニックを。エレクトロポレーションは、他の技術と比較して持っていることはいくつかの利点があります。まず、セ​​ルの焦点ラベリングを考えると、1は細胞自律的かつ非細胞自律的な効果を識別することです。誘導系を用いて、第2の、遺伝?...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td>重いポリッシュホウケイ酸チューブ</td><td>サター·インストゥルメント</td><td> BF150-110から10 </td><td></td></tr><tr><td>デュアルステージガラスマイクロピペットプラー</td><td>ナリシゲ</td><td> PC-10H </td><td></td></tr><tr><td>速いグリーン</td><td>フィッシャー·サイエンティフィック</td><td> 0521192205 </td><td></td></tr><tr><td> ECM 830方形波パルスジェネレータ</td><td>ハーバード装置</td><td> 45から0052 </td><td></td></tr><tr><td> Tweezertrodes </td><td>ハーバード装置</td><td> 45から0488 </td><td></td></tr><tr><td>光ファイバー光源</td><td>フィッシャー·サイエンティフィック</td><td> 12-562-36 </td><td></td></tr><tr><td>タングステンハロゲンランプ</td><td>ウシオアメリカ株式会社</td><td> 1002247 </td><td></td></tr><tr><td> Picospritzer II </td><td>パーカー·インスツルメンツ</td><td> 052-0312-900 </td><td></td></tr></tbody></table>

neonatal subventricular zone electroporation

神経幹細胞（NSC）ライン生後側脳、ニューロン、アストロサイト、上衣細胞1を含む複数の細胞型を生じさせる。 NSCの自己複製、コミットメント、および分化に関わる分子経路を理解することは、活用のための脳を修復し、より良いの中枢神経系障害を理解するための独自の可能性が重要です。哺乳類のシステムを操作するための従来の方法は、全体の動物レベル2で、遺伝子工学の時間がかかり、高価な努力が必要でした。したがって、研究の大半は、in vitroまたは無脊椎動物におけるNSCの分子の機能を検討されている。 ここでは、新生児の脳室下帯（SVZ）エレクトロポレーションと呼ばれている新生児のNPCを操作するための簡単​​かつ迅速な技術を実証した。同様の技術は、胚NSCを研究するために十年前に開発されcorticaに関する研究を支援している3,4リットル開発。さらに最近では、これは5月7日出生後のげっ歯類の前脳を研究するために適用した。この手法は、SVZのNSCおよびその子孫の堅牢なラベリング結果。したがって、生後SVZエレクトロポレーションは、哺乳動物のNSCの遺伝子工学のためのコストと時間の効果的な代替手段を提供します。

背側、背側、そしてピンセット電極の&quot;抜本的な&quot;運動（ 図1E）に続く横下帯と連続してほぼすべての放射状グリアの標識における新生児の脳室下帯エレクトロポレーション結果。しかし、エレクトロポレーションは、それぞれの実験の要件に合わせたが、電極を席巻し、ビデオに記載されている特定の配置と向きを使用していないことができます。例えば、背側に限局放?...

Watch this Scientific Journal Video about Neonatal Subventricular Zone Electroporation at JoVE.com

Neonatal Subventricular Zone Electroporation

我々は、新生児の脳室下帯のエレクトロポレーションと呼ばれる低侵襲技術を実証した。テクニックは、新生児の仔の側脳室にプラスミドDNAを注入し、提供し、遺伝的に神経幹細胞を操作するために電流を印加することで構成されています

新生児の脳室下帯のエレクトロ

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal ...

neonatal-subventricular-zone-electroporation

Research

JoVE Journal

Neuroscience

12.7K ビュー.  Yale University School of Medicine. 我々は、新生児の脳室下帯のエレクトロポレーションと呼ばれる低侵襲技術を実証した。テクニックは、新生児の仔の側脳室にプラスミドDNAを注入し、提供し、遺伝的に神経幹細胞を操作するために電流を印加することで構成されています

ビデオ: Neonatal Subventricular Zone Electroporation

The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an extensively studied model system for understanding the behavior of neural stem cells and the regulation of adult neurogenesis. Traditionally, these studies have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region. Compared to sections, wholemounts preserve the complete cytoarchitecture and cellular relationships within the SVZ. This approach has recently revealed that the adult neural stem cells, or type B1 cells, are part of a mixed neuroepithelium with differentiated ependymal cells lining the lateral ventricles. In addition, this approach has been used to study the planar polarization of ependymal cells and the cerebrospinal fluid flow they generate in the ventricle. With recent evidence that adult neural stem cells are a heterogeneous population that is regionally specified, the wholemount approach will likely be an essential tool for understanding the organization and parcellation of this stem cell niche.

The lateral ventricle walls contain the largest germinal region in the adult mammalian brain. Traditionally, studies on neurogenesis in this region have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region.

脳室下帯恩顔：Wholemount染色と上衣の流れ

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an ...

神経科学

In vivo Electroporation of Developing Mouse Retina

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

In vivo Electroporation of Developing Mouse Retina

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

開発マウス網膜の in vivoエレクトロポレーションで

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

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

カルシウムイメージングの急性脳室下帯スライスの調製

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

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.

新生児軟膜表面エレクトロ

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.

皮質介在ニューロンのサブタイプ選択的エレクトロポレーション

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

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

セル成体マウス脳室下帯から神経幹細胞および前駆細胞の選別とその細胞周期ダイナミクスのライブイメージング

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

マウス梁下ゾーンから成体神経幹細胞の単離および培養

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Cryo-section Dissection of the Adult Subependymal Zone for Accurate and Deep Quantitative Proteome Analysis

The subependymal neurogenic niche consists of a paraventricular ribbon of the lateral ventricular wall of the lateral ventricle. The subependymal zone (SEZ) is a thin and distinct region exposed to the ventricles and cerebrospinal fluid. The isolation of this niche allows the analysis of a neurogenic stem cell microenvironment. However, extraction of small tissues for proteome analysis&#160;is challenging, especially for the maintenance of considerable measurement depth and the achievement of reliable robustness. A new method termed cryo-section-dissection (CSD), combining high precision with minimal tissue perturbation, was developed to address these challenges. The method is compatible with state-of-the-art mass spectrometry (MS) methods that allow the detection of low-abundant niche regulators. This study compared the CSD and its proteome data to the method and data obtained by laser-capture-microdissection (LCM) and a standard wholemount dissection. The CSD method resulted in twice the quantification depth in less than half the preparation time compared to the LCM and simultaneously clearly outperformed the dissection precision of the wholemount dissection. Hence, CSD is a superior method for collecting the SEZ for proteome analysis.

Cryo-section-dissection allows fresh, frozen preparation of the largest neurogenic niche in the murine brain for deep quantitative proteome analysis. The method is precise, efficient, and causes minimal tissue perturbation. Therefore, it is ideally suited for studying the molecular microenvironment of this niche, as well as other organs, regions, and species.

正確で深い定量プロテオーム分析のための成人皮下帯のクライオセクション解剖

The subependymal neurogenic niche consists of a paraventricular ribbon of the lateral ventricular wall of the lateral ventricle. The subependymal zone ...

Delivering Intrathecal Injections to Neonatal Mice

新生仔マウスへの髄腔内注射の送達

<meta charset="utf8"></head><body>マウス脳室内領域から単離されたNSCの効率的なヌクレオフェクション

Isolation, Expansion, and Nucleofection of Neural Stem Cells from Adult Murine Subventricular Zone

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) as mitogens, producing clonal aggregates known as neurospheres. This in vitro system is a valuable tool for studying NSC potential. Transfection of siRNAs or genes carried in plasmids can be used to induce perturbations to gene expression and study NSC biology. However, the exogenous nucleic acid delivery to NSC cultures is challenging due to the low efficiency of central nervous system (CNS) cells transfection. Here, we present an improved nucleofection system that achieves high efficiency of gene delivery in expanded NSCs from adult murine SVZ. We demonstrate that this relatively simple method enhances gene perturbation in adult NSCs, surpassing traditional transfection protocols with survival rates exceeding 80%. Moreover, this method can also be applied in primary isolated NSCs, providing a crucial advancement in gene function studies through gene expression manipulation via knockdown or overexpression in neurosphere cultures.

<meta charset="utf8"></head><body>著者スポットライト:マウス脳室下ゾーン由来神経幹細胞培養における高効率遺伝子導入のためのヌクレオフェクションの改善

Here, we describe a nucleofection system designed to enhance gene delivery efficiency in expanded neural stem cells (NSCs) isolated from the adult murine subventricular zone. The findings demonstrate that this method significantly improves gene perturbation in NSCs, surpassing the effectiveness of traditional transfection protocols and enhancing cell survival rate.

成体マウス脳室下領域からの神経幹細胞の単離、増殖、およびヌクレオフェクション

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented ...