著者らは、CS研究室で子宮内エレクトロポレーション技術の確立に彼らの初期の仕事のためにエヴァHadzimova、ピエールMattarとクリストファーコバックに感謝します。この作品は、カナダ保健研究所（CIHR）助成金（MOP 44094）とDMKにCSとアルバータ州の小児病院研究財団助成するチームグラント（00933から000を）新興CIHR /財団ファイティング失明（FFB）によって賄われていた。 RDがCIHRカナダホープ奨学金によってサポートされていた、RCは、FFBの学生の身分によってサポートされており、LMLは、遺伝学のCIHRトレーニンググラントと子どもの発達によってサポートされていました。

<ol>
	<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, 237-246 (2001).</li><li>Takahashi, M., Sato, K., Nomura, T., Osumi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulating+gene+expressions+by+electroporation+in+the+developing+brain+of+mammalian+embryos.">Manipulating gene expressions by electroporation in the developing brain of mammalian embryos.</a> Differentiation. 70, 155-162 (2002).</li><li>Langevin, L. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+and+ex+vivo+electroporation+for+gene+expression+in+mouse+retinal+ganglion+cells.">In utero and ex vivo electroporation for gene expression in mouse retinal ganglion cells.</a> J Vis Exp. , (2009).</li><li>Garcia-Frigola, C., Carreres, M. I., Vegar, C., Herrera, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+delivery+into+mouse+retinal+ganglion+cells+by+in+utero+electroporation.">Gene delivery into mouse retinal ganglion cells by in utero electroporation.</a> BMC Dev Biol. 7, 103-103 (2007).</li><li>Kataoka, A., 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=Fgf8+controls+regional+identity+in+the+developing+thalamus.">Fgf8 controls regional identity in the developing thalamus.</a> Development. 135, 2873-2881 (2008).</li><li>Vue, T. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+hedgehog+signaling+controls+thalamic+progenitor+identity+and+nuclei+specification+in+mice.">Sonic hedgehog signaling controls thalamic progenitor identity and nuclei specification in mice.</a> J Neurosci. 29, 4484-4497 (2009).</li><li>Tsuchiya, R., Takahashi, K., Liu, F. C., Takahashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrant+axonal+projections+from+mammillary+bodies+in+Pax6+mutant+mice:+possible+roles+of+Netrin-1+and+Slit+2+in+mammillary+projections.">Aberrant axonal projections from mammillary bodies in Pax6 mutant mice: possible roles of Netrin-1 and Slit 2 in mammillary projections.</a> J Neurosci Res. 87, 1620-1633 (2009).</li><li>Buffo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+pattern+of+the+transcription+factor+Olig2+in+response+to+brain+injuries:+implications+for+neuronal+repair.">Expression pattern of the transcription factor Olig2 in response to brain injuries: implications for neuronal repair.</a> Proc Natl Acad Sci U S A. 102, 18183-18188 (2005).</li></ol>

利害の衝突は宣言されません。

子宮内エレクトロポレーションは、発生過程の様々な分析に使用することができます。例えば、このようなGFP、mCherryまたはアルカリホスファターゼなどのレポーター遺伝子のトランスフェクションは、系統のトレースとニューロン移動の実験を行うために使用することができます。また、Creリコンビナーゼは、一時的に選択的に空間的および/または時間的に制御された方法でfloxed対?...

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>カテゴリ</th></tr><tr><td>ファインはさみ</td><td>ファインサイエンスツール株式会社</td><td> 14078〜10 </td><td> 外科ツール </td></tr><tr><td>湾曲したアイリスはさみ、 </td><td>ファインサイエンスツール株式会社</td><td> 14061〜10 </td><td> 外科ツール </td></tr><tr><td>オルセン-ヘーガル元繊細なニードルホルダー</td><td>ファインサイエンスツール株式会社</td><td> 12002〜12 </td><td> 外科ツール </td></tr><tr><td>リング鉗子、9mmの</td><td>ファインサイエンスツール株式会社</td><td> 11103〜09 </td><td> 外科ツール </td></tr><tr><td>アイドレッシングForcep </td><td>ファインサイエンスツール株式会社</td><td> 11051〜10 </td><td> 外科ツール </td></tr><tr><td>デュモン＃7 DMX Forcep </td><td>ファインサイエンスツール株式会社</td><td> 11271〜30 </td><td> 外科ツール </td></tr><tr><td>デュモン＃5 DMX Forcep </td><td>ファインサイエンスツール株式会社</td><td> 11251〜30 </td><td> 外科ツール </td></tr><tr><td>組織forcep -アドソン</td><td>ファインサイエンスツール株式会社</td><td> 11027〜12 </td><td> 外科ツール </td></tr><tr><td>反射のクリップア</td><td>世界の精密機器</td><td> 500343 </td><td> 外科ツール </td></tr><tr><td>穴あきスプーン、直径15mm </td><td>ファインサイエンスツール株式会社</td><td> 10370〜18 </td><td> 外科ツール </td></tr><tr><td> Autoclipリムーバー</td><td>ミクロン</td><td> 427637 </td><td> 外科ツール </td></tr><tr><td>シルクブラック編組縫合糸</td><td>エチコン社</td><td> K871 </td><td> 外科ツール </td></tr><tr><td>反射スキンクロージャーステンレス鋼創傷クリップ</td><td>世界の精密機器</td><td> 500346 </td><td> 外科ツール </td></tr><tr><td> ECM 830方形波のエレクトロポレーションシステム</td><td> VWR - CanLab </td><td> 58018-004 </td><td> 楽器 </td></tr><tr><td>ピンセットのw /可変ギャップ2ラウンド、5mm、プラチナプレート電極</td><td>プロテックインターナショナル（株） </td><td> CUY650P5 </td><td> 楽器 </td></tr><tr><td>ピンセットのw /可変ギャップ2ラウンド7ミリメートルプラチナプレート電極</td><td>プロテックインターナショナル（株） </td><td> CUY650P7 </td><td> 楽器 </td></tr><tr><td>エッペンドルフFemtojetマイクロインジェクター</td><td> VWR CanLab </td><td> CA62111 - 488 </td><td> 楽器 </td></tr><tr><td>エッペンドルフFemtojetマイクロインジェクター用フットコントロール</td><td> VWR CanLab </td><td> CAACCESS（MISC.） </td><td> 楽器 </td></tr><tr><td> Bransonic超音波洗浄機モデル1510R - DTH </td><td> VWR CanLab </td><td> CA33995 - 534 CPN - 952〜118 </td><td> 楽器 </td></tr><tr><td>サターP97マイクロピペットプラー</td><td>サターインストゥルメント、Carsenグループ本社</td><td> P - 97 </td><td> 楽器 </td></tr><tr><td>マイクロピペット - フィラメント径を持つホウケイさ：1mm、ID：0.78ミリメートル、10 cmの長さ</td><td>サターのインストゥルメント</td><td> BF100 - 78から10 </td><td> 楽器 </td></tr><tr><td> 3軸粗動マニピュレーター</td><td>カールツァイスカナダ株式会社</td><td> M - 152 </td><td> 楽器 </td></tr><tr><td>マイクロマニピュレータのための磁気保持装置</td><td>世界の精密機器</td><td> M1 </td><td> 楽器 </td></tr><tr><td>マイクロマニピュレータ用スチールベースプレート</td><td>世界の精密機器</td><td> 5052 </td><td> 楽器 </td></tr><tr><td>マイクロピペットホルダー</td><td>世界Prのecisionインスツルメンツ</td><td> MPH3 </td><td> 楽器 </td></tr><tr><td>マイクロピペットハンドル</td><td>世界の精密機器</td><td> 5444 </td><td> 楽器 </td></tr><tr><td>実体顕微鏡</td><td>ライカ</td><td> MZ6 </td><td> 楽器 </td></tr><tr><td>イソフルラン麻酔用気化器</td><td>ポーターインストルメンツカンパニー</td><td> MODEL 100 - F </td><td>楽器</td></tr><tr><td>手術器具を超音波処理するためのMetriclean2低起泡性ソリューション</td><td> Metrexリサーチ株式会社</td><td> 10から8100 </td><td> 外科的試薬 </td></tr><tr><td>縫合後、0.2グラムのメチレンブルー抗生物質噴霧のゲンタマイシン40mg/ml </td><td>シグマアルドリッチ</td><td> G1264 </td><td> 外科的試薬 </td></tr><tr><td>手術器具を滅菌するためのGermex </td><td> Vétoquinol </td><td> DIN＃00141569 </td><td> 外科的試薬 </td></tr><tr><td> BNPの眼軟膏</td><td> Vétoquinol </td><td> DIN＃00516414 </td><td> 外科的試薬 </td></tr><tr><td>ナイル® </td><td>教会＆ドワイト株式会社によって配布</td><td>市販</td><td> 外科的試薬 </td></tr><tr><td> Stanhexidine 4パーセントw / vの皮膚洗浄剤</td><td>オメガラボラトリーズ株式会社</td><td> 01938983 </td><td> 外科的試薬 </td></tr><tr><td>ブプレノルフィン（Temgesic）鎮痛剤</td><td>シェリングプラウ</td><td> 531から535 </td><td> 外科的試薬 </td></tr><tr><td> Sulpha&quot;25&quot;sulphamethazine経口抗生物質</td><td>専門の獣医研究所</td><td> DIN＃00308218 </td><td> 外科的試薬 </td></tr><tr><td>乳酸リンゲル液</td><td>バクスター株式会社</td><td> DIN＃0061085 </td><td> 外科的試薬 </td></tr><tr><td>生理食塩水 - 0.9％の塩化ナトリウム</td><td> B -ブラウンメディカル株式会社</td><td> DIN＃01924303 </td><td> 外科的試薬 </td></tr><tr><td>吸入麻酔 - イソフルランUSP </td><td>カナダ社の製薬パートナー</td><td> DIN＃02237518 </td><td> 外科的試薬 </td></tr><tr><td>ファストグリーンFCF </td><td>シグマアルドリッチ</td><td> F7252 </td><td> 外科的試薬 </td></tr></tbody></table>

efficient gene delivery into multiple cns territories using in utero electroporation

遺伝子発現を操作する機能は、複数の発達経路の解明につながる、現代の実験発生学の基礎となるものです。いくつかの強力で定評のトランスジェニック技術の喪失と機能獲得型モデルの両方の生成を可能にする、マウスの遺伝子発現レベルを操作するために用意されています。しかし、マウス遺伝子組換えの世代は、コストと時間がかかるものです。遺伝子操作の代替の方法では、広く求められている。 子宮内エレクトロポレーションで、我々は正常に3,4を適応しているライブのマウス胚の1,2への遺伝子送達の方法です。それは、主に一般的にニワトリ5で使用されているOVOエレクトロポレーション技術での成功に基づいています。簡単に言えば、DNAは細胞へのDNAの取り込みを可能にする、脳の発達と電流の原因は細胞膜に一時的な細孔の形成のアプリケーションのオープン脳室に注入されます。若い胚のターゲティングは、超音波ガイド下マイクロインジェクションのプロトコル、前述の6に説明を必要としながら私たちの手では、胚を効率的に、胎生（E）11.5早くも電気穿孔することができます。逆に、E15.5は、脳へのマイクロインジェクションを妨げる頭頂と前頭骨分化の開始、のために我々が簡単にエレクトロできる最新の舞台です。対照的に、網膜は胚発生の終わりを介してアクセス可能です。胚は、胚や初期の産後の全期間を通して、いつの時点で収集することができます。レポーターコンストラクトの注入は、トランスフェクトされた細胞の同定を容易にする。 日付に、子宮内エレクトロポレーションで最も広く新皮質の発達1,2,3,4の分析に使用されています。最近の研究では、胚性網膜7,8,9と視床10,11,12を標的にしている。ここで、我々は簡単胚中枢神経系の別のドメインをターゲットに適応させることができる子宮内エレクトロポレーションプロトコルに変更を提示。我々は、このテクニックを使用することによって、我々は胚の終脳、間脳や網膜をターゲットにできるという証拠を提供する。代表的な結果は、私たちは胚の終脳の前駆細胞の成熟、分化と移行を監視することが、最初の側脳室へのDNAの発現コンストラクトを導入するためにこのテクニックの使用を示す、表示されます。我々はまた、この技術は間脳核に神経細胞を分化回遊経路をモニタすることで、第3脳室周囲の間脳の領域にDNAをターゲットとするために使用することができることを示している。最後に、我々は、マイクロマニピュレータの使用は私たちが網膜の開発に遺伝子発現を操作するの効果を分析できるように、網膜下腔を含め、私たちは正確に小さなターゲットの領域にDNA構築物を導入することができることを示している。

Watch this Scientific Journal Video about Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation at JoVE.com

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

子宮内エレクトロポレーションでは発達中の中枢神経系（CNS）における空間的および時間的に制御された方法で迅速な遺伝子デリバリーを可能にする。ここでは、終脳、間脳や網膜など、複数の胚中枢神経系領域、中に発現構築物を提供するために使用することができる子宮内エレクトロポレーションプロトコルにおける適応性の高いを説明します。

使用した複数のCNS準州への効率的な遺伝子送達子宮内でエレクトロポレーション

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

efficient-gene-delivery-into-multiple-cns-territories-using-utero

Research

JoVE Journal

Neuroscience

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

19.7K ビュー.  University of Calgary. 子宮内エレクトロポレーションでは発達中の中枢神経系（CNS）における空間的および時間的に制御された方法で迅速な遺伝子デリバリーを可能にする。ここでは、終脳、間脳や網膜など、複数の胚中枢神経系領域、中に発現構築物を提供するために使用することができる子宮内エレクトロポレーションプロトコルにおける適応性の高いを説?...

ビデオ: Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. However, because the uterine wall is opaque during early embryogenesis, the ability to target specific parts of the embryo for microinjection is greatly limited. Fortunately, high-frequency ultrasound imaging permits the generation of images that can be used in real time to guide a microinjection needle into the embryonic region of interest. Here we describe the use of such imaging to guide the injection of retroviral vectors into the ventricular system of the mouse forebrain at embryonic day (E) 9.5. This method uses a laparotomy to permit access to the uterine horns, and a specially designed plate that permits host embryos to be bathed in saline while they are imaged and injected. Successful surgeries often result in most or all of the injected embryos surviving to any subsequent time point of interest (embryonically or postnatally). The principles described here can be used with slight modifications to perform injections into the amnionic fluid of E8.5 embryos (thereby permitting infection along the anterior posterior extent of the neural tube, which has not yet closed), or into the ventricular system of the brain at E10.5/11.5. Furthermore, at mid-neurogenic ages (~E13.5), ultrasound imaging can be used direct injection into specific brain regions for viral infection or cell transplantation. The use of ultrasound imaging to guide in utero injections in mice is a very powerful technique that permits the molecular and cellular manipulation of mouse embryos in ways that would otherwise be exceptionally difficult if not impossible.

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. Here we describe the use of high-frequency ultrasound imaging to guide the injection of retroviral vectors into the mouse brain at embryonic day (E) 9.5.

超音波ガイド下のマウスの前脳へのマイクロインジェクション子宮内で E9.5で

In utero survival surgery in mice permits the molecular manipulation of gene expression during development.  However, because the uterine wall is opaque ...

神経科学

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.

子宮内エレクトロポレーションは、皮質ニューロンのサブセットで、遺伝子機能の研究のために初代神経培養した

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

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

非心室プラスミド注入し、エレクトロポレーションにより産後のラット脳への遺伝子デリバリー

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

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.

マウス子宮内でエレクトロポレーション：制御時空間遺伝子導入

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

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.

使用して、マウス大脳皮質および海馬における可視化と樹状突起と棘の遺伝子操作<em&gt;子宮内で</em&gt;エレクトロポレーション

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 ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

局所トランスジェニックラットの発生により<em&gt;子宮内</em&gt;エレクトロポレーションおよび<em&gt;インビボ</em&gt;生物発光スクリーニング

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

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.

マウスの遺伝子操作は、視床下部を介して開発<em&gt;子宮内</em&gt;エレクトロポレーション

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

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous substances into the brain. These exogenous substances may induce injurious impact or constitute therapeutic strategy. The most common delivery methods of exogenous substances into the brain are based on systemic deliveries, such as subcutaneous or intravenous routes. Although commonly used, these approaches have several limitations, including low delivery efficacy into the brain. In contrast, surgical methods that locally deliver substances into the CNS are more specific and prevent the uptake of the exogenous substances by other organs. Several surgical methods for CNS delivery are available; however, they tend to be very traumatic. Here, we describe a mouse infusion microsurgery technique, which effectively delivers substances into the brain via the internal carotid artery, with minimal trauma and no interference with normal CNS functionality.

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

The present protocol describes a mouse microsurgery infusion technique, which effectively delivers substances directly into the brain via the internal carotid artery.

CNSへの標的化物質送達のためのマウス顕微注入法<i&gt;経由で</i&gt;内頸動脈

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous ...

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

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

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

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

胚性マウス脳の器官型スライス培養における移行ニューロンの経時的共焦点イメージング<em&gt;ウテロの</em&gt;エレクトロポレーション

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

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

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

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

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

子宮内エレクトロポレーション マウス大脳皮質における Cre lox 組換えを誘導します。

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