この作品は、ドイツ研究協会（ドイツ学術振興）によって賄われていた。

1。注射用DNAとガラスマイクロピペットの作製<ol><li>良質のガラスマイクロピペットは、羊水の損失のために初期高い流産率を減少させるために不可欠です。ガラスマイクロピペットを引っ張るための手順は、よく13,18,25を文書化されています。ヒート= 300;; 1.2ミリメートル径の毛細管を使用し、設定P = 500と従来のサターでP-97のデバイスを引っ張っ= 40を引いて、速度= 50;時間= 50。 3ミリメートル &quot;トラフ&quot;フィラメント（サッタートゥルFT330B）でプーラーを取り付けます。 2ミリメートル径フィラメントは私たちのために少なく満足のいく結果が得られている。一方、マイクロピペットの先端を面取りすると、私たちのために結果を改善していないようです。 </li><li> 1から2μgの/μlに最終濃度0.1％ファストグリーンを含むPBSで精製し、エンドトキシンフリーのプラスミドDNA（細胞培養グレード）に溶解する。ファストグリーンは、胚の脳室に注入された目に​​見える解決策を作ります。 </li><li>ガラスマイクロピペットへのDNA溶液10μlをロードします。 </lI&gt; <li>噴射システム（ピコポンプ）や口のピペットにガラスマイクロピペットを接続します。 </li></ol> 2。麻酔加熱パッドや手術器具で手術台を準備します。胚の可視化を容易にするために、冷光源をオンにします。例えばガラスビーズ滅菌器を使用した手術器具を消毒する。 三つの異なる麻酔手順は、（説明を参照）は、このプロトコルのために可能です。ここで我々は麻酔導入（流量0.5 L /分）と同様のメンテナンス（流量1リットル/分）のためにイソフルラン吸入を使用して、最善を考えるものを説明します。 <ol><li>チューブの短い長さによってKomesaroffマーク5麻酔デバイスに接続されている（そのため、マウスが表示されたまま）、透明の小さな容器に妊娠しているマウスをご紹介します。 </li><li>イソフルランを気化器を満たし、その後、デバイスに接続されている酸素ボンベを開きます。 isoflの1つまたは2つのパルスを吹くurane /麻酔が入って設定するためのマウスと時計を保持する容器への酸素（0.5L /分） </li><li>マウスを取り出し、乾燥からそれらを防ぐために、すぐに頭に流れ麻酔マスクに適合するように軟膏との目をカバーしています。 </li></ol> 3。開腹<ol><li>加熱パッド（そうでなければ、その体温が回復の可能性を減少させる、非常に高速に下がるだろう）上に腹マウスを置き、テープで側面に4つの足を固定することにより、所定の場所に、その本体を固定します。反射刺激（しっかり圧例えばつま先またはテールピンチ）への応答の損失のためにチェックすることによって、麻酔の深さを評価する。 </li><li>腹部の表面を剃るとiodophorpovidone-ヨウ素（Braunoderm）でそれを消毒。 </li><li>腹部皮膚上の縦切開（1〜1.5センチメートル長い）を作る。その後、腹膜を切った。切開の周り綿ガーゼを置きます。 1子宮角が見えるようにし、PBS-すすぎgauzに鈍ピンセットで慎重にそれを引き出し電子。非常に多くの場合、それは常に（ 図2Aおよび2B）湿ら保つためにPBSで子宮を洗浄します。全体の手順の間に子宮内部の高い圧力が中絶になり注射により流体の損失の可能性を増加胚に送信しますので、タイトな子宮間膜や子宮を引っ張って避けてください。 </li></ol> 4。脳室へのDNAインジェクション<ol><li>脳室を可視化することができるような方法で子宮を保持する。広範囲に不利な立場にある胚の位置を変えてはいけない - これが唯一の中絶の可能性が高くなります。 </li><li>上部から胚の頭を見て、視覚的に左右の皮質半球間のギャップや亀裂をローカライズ。半球は区別することが容易であり、それらの内側側脳室は（対象としないように）通常はやや暗めの形状として認識することができます。胚（FIGUR DNA注射のために完全に配向させることが判明した場合ES 2C及び2D）、それが貫通する子宮壁可能であり、一度に第三脳室に入る。約1mmのために貫通、皮質半球間のギャップの吻側終わりに子宮壁と穿刺それに45°のガラスマイクロピペットを保持します。このようにして、ガラスマイクロピペットの先端は、脳（ではない側脳室）（ 図2Cおよび2D）の第三脳室に入ります。それは胚の頭部の近傍で最初貫く子宮壁（常に45°）に便利です不利指向の胚では、皮質半球の間に正しい位置にマイクロピペットの先端を配置し、だけにして脳を穿孔。第三脳室（良い注射が緑流体で心室を埋める）にDNA溶液を約1μLを注入します。 1子宮ホーンのすべての胚と同じ手順を繰り返します。これは、DNA溶液を、心室流体で均等に混ぜるとentirに到達するためのいくつかの時間を可能にします電子神経上皮。 </li></ol> 5。エレクトロポレーションのための視床下部を標的<ol><li>にエレクトロを切り替えて胚年齢（E12.5のために我々は5方形波パルスを使用し、50 V、50ミリ秒ON/950ミリ秒OFF）に応じて設定を調整します。正極として、ステンレス製の針電極（CUY550-10）を使用し、負極ラウンドフラット電極（CUY700P4L）として。 （電流だけでなく、それを介して胚の周りを流れそうでないので、それは、電極胚よりも小さいことが重要である。） </li><li>エレクトロポレーションのために、その背側（それらのほとんどがあるように）（ つまり、実験者の方を向いて）アップしているそれらの胚を選択して、その方向性に有利で ​​はないものを捨てる。これは、エタノール消毒指で、または手袋を子宮に触れても安全です。鉗子で子宮を持って、しかし、エレクトロポレーション時の電流の流れを妨げる。 </li><li>ピアースAMNの間に胚の頭で子宮壁針電極の先端とそれを介して下方へ突き出すことによりiotic嚢と胎盤。スラストブロックとしてもう一方の手の人差し指を使用してください。電極先端の5mm程度は中脳（ 数字は2Eと2F）の程度のレベルで、羊膜嚢と子宮壁との間で今でなければなりません。これは &quot;ターゲット電極&quot;であることを忘れないので、その位置は、トランスフェクトされ得る可能性が最も高い視床下部の神経上皮のどの部分を決定します。胚は非常に優しく、電極間に &quot;圧迫&quot;されなければならない。 </li><li>一方、位置と胚の頭（ 図2Eと2G）の反対側の子宮壁の外側ラウンドフラット電極。 </li><li>電圧（50 V、50ミリ秒ON、950ミリ秒OFF、5方形波パルス）を適用するためにペダルスイッチを使用します。 </li><li>もう一方の手の人差し指で子宮をバック保持しながらゆっくりと子宮の針電極を引き出します - もしamnioチック液がパンクした壁を通して失われ、通常胚は妊娠中絶を受けることになる。すべての胚との手順を繰り返します。 </li><li>腹部に子宮ホーンを返し、残りの角の注射およびエレクトロポレーションを繰り返します。 </li></ol> 6。手術を終えて<ol><li>すべての胚の注入とエレクトロポレーションした後、彼らはそれを閉じる前に生理食塩水で腹腔前と湿っていたとおりに正確に配置され、非常に慎重に腹部に子宮を後ろに置きます。 </li><li>手術腸線（VICRYLポリグラクチン910、5-0、エチコンV4914）で腹膜を縫合。肌用の閉鎖methodwithより耐性縫合（Supramidナイロン、6-0、Serag Wiessner 07171L TO） &quot;ステッチが中断&quot;。 </li><li>ポビドンヨード（Braunoderm）と腹部の表面を消毒し、非ステロイド皮下抗炎症（ 例えば 、0.9％NaCl中Rimadyl 1:10の溶液100μl）、または、より良い、BUのようなオピオイドを注入prenorphine（Temgesic、0.9％NaCl中0.05〜mg / kg体重）痛みを緩和する。 </li><li>麻酔マシンから母親を外し、加熱パッドにより加熱されるケージに入れてください。それは完全に麻酔から回復されるまで、継続的にマウスを監視します。その後、感染症や痛みの兆候なしで、彼らはプロシージャから回復して保証するために、毎日動物を確認してください。 </li></ol> 7。結果の分析<ol><li>分析の希望日に応じて胚（またはpostnatals）収穫。出生後のマウス（1から2日齢）を断頭により殺される。 </li><li>以前エレクトロ注釈によると、すべての胚を分離。実体顕微鏡下で脳を解剖して、適切な地域の緑色蛍光シグナル（GFPレポーターから）のために顕微鏡下で確認してください。 </li><li>選択された脳は、 &quot;新鮮&quot;を分析することができます：4％パラホルムアルデヒド中で短い時間のために組織を固定し、アガロース4％またはゼラチンで埋め込むlbumin、その後ビブラトーム型デバイス上のセクション（ 図3）。より詳細な免疫組織化学的分析のために（ 図4）、10月マウンティング培地（ティッシュテック）に埋め込 ​​む30％ショ糖溶液を、脳を凍結保護はその後クライオスタットで20μmのセクションをカット。 </li></ol>

<ol>
	<li>Itasaki, N., Bel-Vialar, S., Krumlauf, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shocking'+developments+in+chick+embryology:+electroporation+and+in+ovo+gene+expression.">Shocking' developments in chick embryology: electroporation and in ovo gene expression.</a> Nat. Cell Biol. 1, E203-E207 (1999).</li><li>Miyasaka, N., Arimatsu, Y., Takiguchihayashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foreign+gene+expression+in+an+organotypic+culture+of+cortical+anlage+after+in+vivo+electroporation.">Foreign gene expression in an organotypic culture of cortical anlage after in vivo electroporation.</a> Neuroreport. 10, 2319-2323 (1999).</li><li>Saito, T., Nakatsuji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+into+the+embryonic+mouse+brain+using+in+vivo+electroporation.">Efficient gene transfer into the embryonic mouse brain using in vivo electroporation.</a> Dev. Biol. 240, 237-246 (2001).</li><li>Tabata, H., Nakajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+in+utero+gene+transfer+system+to+the+developing+mouse+brain+using+electroporation:+visualization+of+neuronal+migration+in+the+developing+cortex.">Efficient in utero gene transfer system to the developing mouse brain using electroporation: visualization of neuronal migration in the developing cortex.</a> Neuroscience. 103, 865-872 (2001).</li><li>Fukuchi-Shimogori, T., Grove, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neocortex+patterning+by+the+secreted+signaling+molecule+FGF8.">Neocortex patterning by the secreted signaling molecule FGF8.</a> Science. 294, 1071-1074 (2001).</li><li>Tabata, H., Nakajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurons+tend+to+stop+migration+and+differentiate+along+the+cortical+internal+plexiform+zones+in+the+Reelin+signal-deficient+mice.">Neurons tend to stop migration and differentiate along the cortical internal plexiform zones in the Reelin signal-deficient mice.</a> J. Neurosci. Res. 69, 723-730 (2002).</li><li>Fukuchi-Shimogori, T., Grove, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emx2+patterns+the+neocortex+by+regulating+FGF+positional+signaling.">Emx2 patterns the neocortex by regulating FGF positional signaling.</a> Nat. Neurosci. 6, 825-831 (2003).</li><li>Shimogori, T., Banuchi, V., Ng, H. Y., Strauss, J. B., Grove, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+signaling+centers+expressing+BMP,+WNT+and+FGF+proteins+interact+to+pattern+the+cerebral+cortex.">Embryonic signaling centers expressing BMP, WNT and FGF proteins interact to pattern the cerebral cortex.</a> Development. 131, 5639-5647 (2004).</li><li>Shimogori, T., Grove, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+growth+factor+8+regulates+neocortical+guidance+of+area-specific+thalamic+innervation.">Fibroblast growth factor 8 regulates neocortical guidance of area-specific thalamic innervation.</a> J. Neurosci. 25, 6550-6560 (2005).</li><li>Saito, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+electroporation+in+the+embryonic+mouse+central+nervous+system.">In vivo electroporation in the embryonic mouse central nervous system.</a> Nat. Protoc. 1, 1552-1558 (2006).</li><li>Dixit, R., 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=Efficient+Gene+Delivery+into+Multiple+CNS+Territories+Using+In+Utero+Electroporation.">Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation.</a> J. Vis. Exp. (52), e2957 (2011).</li><li>Rice, H., Suth, S., Cavanaugh, W., Bai, J., Young-Pearse, T. L. <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+Electroporation+followed+by+Primary+Neuronal+Culture+for+Studying+Gene+Function+in+Subset+of+Cortical+Neurons.">In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons.</a> J. Vis. Exp. (44), e2103 (2010).</li><li>Walantus, W., Castaneda, D., Elias, L., Kriegstein, 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+Intraventricular+Injection+and+Electroporation+of+E15+Mouse+Embryos.">In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos.</a> J. Vis. Exp. (6), e239 (2007).</li><li>Walantus, W., Elias, L., Kriegstein, 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+Intraventricular+Injection+and+Electroporation+of+E16+Rat+Embryos.">In Utero Intraventricular Injection and Electroporation of E16 Rat Embryos.</a> J. Vis. Exp. (6), e236 (2007).</li><li>Borrell, V., Yoshimura, Y., Callaway, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+delivery+to+telencephalic+inhibitory+neurons+by+directional+in+utero+electroporation.">Targeted gene delivery to telencephalic inhibitory neurons by directional in utero electroporation.</a> J. Neurosci. Methods. 143, 151-158 (2005).</li><li>Haddad-Tovolli, R., Heide, M., Zhou, X., Blaess, S., Alvarez-Bolado, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+thalamic+differentiation:+gli-dependent+pattern+and+gli-independent+prepattern.">Mouse thalamic differentiation: gli-dependent pattern and gli-independent prepattern.</a> Front Neurosci. 6, 27 (2012).</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>Matsui, A., Yoshida, A. C., Kubota, M., Ogawa, M., Shimogori, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+in+Utero+Electroporation:+Controlled+Spatiotemporal+Gene+Transfection.">Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection.</a> J. Vis. Exp. (54), e3024 (2011).</li><li>Vue, T. Y., 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=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>Tanaka, S., 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=Transcriptional+regulation+of+the+hypocretin/orexin+gene+by+NR6A1.">Transcriptional regulation of the hypocretin/orexin gene by NR6A1.</a> Biochem. Biophys. Res. Commun. 403, 178-183 (2010).</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>Canteras, N. S., Watson, C., Paxinos, G., Puelles, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Nervous System. , 539-562 (2011).</li><li>Caqueret, A., Yang, C., Duplan, S., Boucher, F., Michaud, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Looking+for+trouble:+a+search+for+developmental+defects+of+the+hypothalamus.">Looking for trouble: a search for developmental defects of the hypothalamus.</a> Horm. Res. 64, 222-230 (2005).</li><li>Petros, T. J., Rebsam, A., Mason, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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. (31), e1333 (2009).</li><li>Nijagal, A., Le, T., Wegorzewska, M., Mackenzie, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Mouse+Model+of+in+Utero+Transplantation.">A Mouse Model of in Utero Transplantation.</a> J. Vis. Exp. (47), e2303 (2011).</li><li>Altman, J., Bayer, S. 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+Development+of+the+Rat+Hypothalamus.">The Development of the Rat Hypothalamus.</a> Adv. Anat. Embryol. Cell Biol. 100, 1-178 (1986).</li><li>Clancy, B., Darlington, R. B., Finlay, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+developmental+time+across+mammalian+species.">Translating developmental time across mammalian species.</a> Neuroscience. 105, 7-17 (2001).</li><li>Clancy, B., 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=Web-based+method+for+translating+neurodevelopment+from+laboratory+species+to+humans.">Web-based method for translating neurodevelopment from laboratory species to humans.</a> Neuroinformatics. 5, 79-94 (2007).</li><li>Ishii, Y., Bouret, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+birthdate+of+hypothalamic+leptin-activated+neurons+in+mice.">Embryonic birthdate of hypothalamic leptin-activated neurons in mice.</a> Endocrinology. 153, 3657-3667 (2012).</li><li>Karim, M. A., Sloper, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histogenesis+of+the+supraoptic+and+paraventricular+neurosecretory+cells+of+the+mouse+hypothalamus.">Histogenesis of the supraoptic and paraventricular neurosecretory cells of the mouse hypothalamus.</a> J. Anat. 130, 341-347 (1980).</li><li>Shimada, M., Nakamura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+of+neuron+origin+in+mouse+hypothalamic+nuclei.">Time of neuron origin in mouse hypothalamic nuclei.</a> Exp. Neurol. 41, 163-173 (1973).</li><li>Alvarez-Bolado, G., Paul, F. A., Blaess, S. <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+lineage+in+the+mouse+hypothalamus:+from+progenitor+domains+to+hypothalamic+regions.">Sonic hedgehog lineage in the mouse hypothalamus: from progenitor domains to hypothalamic regions.</a> Neural. Dev. 7, 4 (2012).</li><li>Vann, S. D., Aggleton, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mammillary+bodies:+two+memory+systems+in+one.">The mammillary bodies: two memory systems in one.</a> Nat. Rev. Neurosci. 5, 35-44 (2004).</li><li>Gratsch, T. E., De Boer, L. S., O'Shea, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+inhibition+of+BMP-4+gene+expression+in+postimplantation+mouse+embryos.">RNA inhibition of BMP-4 gene expression in postimplantation mouse embryos.</a> Genesis. 37, 12-17 (2003).</li><li>Wu, N., Yu, A. B., Zhu, H. B., Lin, X. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+silencing+of+Sry+gene+with+RNA+interference+in+developing+mouse+embryos+resulted+in+feminization+of+XY+gonad.">Effective silencing of Sry gene with RNA interference in developing mouse embryos resulted in feminization of XY gonad.</a> J. Biomed. Biotechnol. 2012, 343891 (2012).</li><li>Kikuchi, N., Nakamura, S., Ohtsuka, M., Kimura, M., 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=Possible+mechanism+of+gene+transfer+into+early+to+mid-gestational+mouse+fetuses+by+tail+vein+injection.">Possible mechanism of gene transfer into early to mid-gestational mouse fetuses by tail vein injection.</a> Gene Ther. 9, 1529-1541 (2002).</li><li>Foust, K. D., 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=Intravascular+AAV9+preferentially+targets+neonatal+neurons+and+adult+astrocytes.">Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes.</a> Nat. Biotechnol. 27, 59-65 (2009).</li><li>Xin, H., 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=The+brain+targeting+mechanism+of+Angiopep-conjugated+poly(ethylene+glycol)-co-poly(epsilon-caprolactone)+nanoparticles.">The brain targeting mechanism of Angiopep-conjugated poly(ethylene glycol)-co-poly(epsilon-caprolactone) nanoparticles.</a> Biomaterials. 33, 1673-1681 (2012).</li><li>Niwa, H., Yamamura, K., Miyazaki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+selection+for+high-expression+transfectants+with+a+novel+eukaryotic+vector.">Efficient selection for high-expression transfectants with a novel eukaryotic vector.</a> Gene. 108, 193-199 (1991).</li><li>Kaufman, R. J., Davies, M. V., Wasley, L. C., Michnick, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+vectors+for+stable+expression+of+foreign+genes+in+mammalian+cells+by+use+of+the+untranslated+leader+sequence+from+EMC+virus.">Improved vectors for stable expression of foreign genes in mammalian cells by use of the untranslated leader sequence from EMC virus.</a> Nucleic Acids Res. 19, 4485-4490 (1991).</li><li>Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+green+fluorescent+protein.">The green fluorescent protein.</a> Annu. Rev. Biochem. 67, 509-544 (1998).</li><li>Londrigan, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+promoters+for+driving+efficient+transgene+expression+in+neonatal+porcine+islets.">Evaluation of promoters for driving efficient transgene expression in neonatal porcine islets.</a> Xenotransplantation. 14, 119-125 (2007).</li></ol>

著者は、彼らが競合する経済的利益を持っていないことを宣言します。

麻酔について：視床下部に子宮内エレクトロポレーションでは技術的に困難であると長い麻酔時間を必要とすることができるので、我々は、酸素とイソフルランの混合物を投与することにより麻酔を誘導し、維持することを好む。我々の経験では、動物は少なくとも1時間までの期間について、このように適切に麻酔残ることができ、母親の回復は非常に高速であり、胚の生存率...

マウス胎児の脳の遺伝子操作が発達規制について学ぶために好ましいアプローチである。変異マウス線の生成は、しかし遅く、高価である。哺乳類の脳の神経細胞の開発に特定の遺伝的変化を導入する一つの有力な方法は、 子宮内エレクトロポレーションである。本質的に、この技術は、胚は、一定時間生き残る脳を収集し、可能な新規な、有益な表現型のためにそれらを調べることができ、その後、電気的パルスによって胚脳の神経上皮へのDNAのトランスフェクションから構成される。この方法では、実験者は、マウス変異体の生産に必要な長い待機期間なしでほとんど即座仮説をテストすることができます。 発展途上胚へのDNAのトランスフェクションは、ニワトリ胚1上のOVOエレクトロポレーションで始まりました。マウスに不可欠な概念実証は、文化の中で行われた<s2&gt;まで。これは、すぐに子宮内 3,4 でマウスの技術の最初の説明が続いた。 主な問題は、それらまたは母を殺すことなく、 子宮内での開発の胚の脳をトランスフェクトすることです。必要な手術（開腹、インジェクション、エレクトロポレーション）を実行するために学ぶことは、長い訓練期間を必要とします。手術が胚の生存率が許容できるポイントに習得された後、次の重要な問題は、次のとおりです。脳構造がアクセス可能である？驚くことではないが、 子宮内エレクトロポレーションで得られた結果を示した最初の論文は、皮質開発5-9に焦点を当てた。外科的処置に最もアクセス現像マウス脳の領域が皮質（ 図1）であるので、これは、この技術を用いて出版物のほとんどは依然として当てはまる。皮質に子宮内エレクトロポレーションでの手順が記載されているプリント10とビデオ11〜14インチ技術の変更が終脳の腹側部分、大脳基底核15を標的するために使用することができる。 終脳を超えて、間脳は（古典的視床と視床下部に分けて）に到達することがより困難脳の領域である。その背と最もアクセス部分、視床16-19の標的論文·レポートの数が少ない。 視床下部は、脳の中で最も腹側部分であるので、1背面（皮質）（ 図1）から最も深くローカライズ。この領域は、遺伝的に子宮内でマウスの脳を操作することを約束し研究者のための困難な課題である。我々の知る限りでは、ごく少数の記事では、マウスの視床下部20,21に子宮内トランスフェクションの結果を報告する。しかし、視床下部cannoの機能的重要性それは食べたり飲んだり、交配、繁殖と子育て22のような行動を規制するため、T、誇張すること。また、視床下部の開発の変化は、肥満、高血圧、糖尿病、思春期早発症23のような生活条件の後半発信に貢献しています。開発中に遺伝的に視床下部を変更することができることは、それを理解するための非常に強力なツールを提供するでしょう。 我々はここで使用している妊娠マウスの開腹するための基本的な外科的プロトコルは、他のプロトコル11,13,14,24で使用されているものと似ています。私たちは、完全を期すためにここで簡単にそれらを記述します。我々の手順の鍵と、他方では、麻酔のタイプ、注入の代わりに、電極の種類や挿入および胚の頭部に対する正電極の位置である。前者は可能にするため、我々は、単純な腹腔内麻酔上ガス吸入による麻酔を誘導し、維持することを好む難しい手術に必要な麻酔のやや長い期間。麻酔からの迅速な復旧にイソフルラン吸入結果、通常、母親は既に手術後の分を通常の動作を示しているので。ガラスマイクロピペットでDNA溶液を注入するための最も簡単な点は、しかし、視床下部のエレクトロポレーションのために完全には不向きである側脳室、です。直接第三脳室に注入は確かに深い間脳構造を標的とすることが重要です。標準の、既製の電極にE12.0またはE12.5から視床下部をトランスフェクトすることができる。私たちは、特にこの目的に適してNEPAジーン（千葉県、日本）によって製造された電極の一部を発見した。 私たちの手順では、我々は全体の視床下部神経上皮や電極の向きに応じて部分的な、地域のトランスフェクションのトランスフェクションを得る。ここで我々は間違いなく、乳頭体をトランスフェクトすることによって技術を実証深いとすべての視床核の最も凹んだ。さらに、我々は、解像度の細胞レベルにトランスフェクションされた細胞の詳細な組織学的分析を下に示す。 子宮内でマウスの脳の発達をトランスに他のアプローチと子宮内エレクトロポレーションでの比較は考察の項に記載されています。

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>Acepromazine</td>
			<td>Sanofi GmbH</td>
			<td>&nbsp;</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter</td>
			<td>HDG9623</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>Ketamin</td>
			<td>Pharma GmbH</td>
			<td>&nbsp;</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>Fast Green</td>
			<td>Fluka</td>
			<td>44715</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rimadyl</td>
			<td>Pfizer</td>
			<td>&nbsp;</td>
			<td>non-steroidal anti-inflammatory</td>
		</tr>
		<tr>
			<td>Braunoderm</td>
			<td>Braun</td>
			<td>3887138</td>
			<td>povidone-iodine</td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline PBS</td>
			<td>Gibco</td>
			<td>14190</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Temgesic (buprenorphine)</td>
			<td>Essex Pharma</td>
			<td>&nbsp;</td>
			<td>opioid analgesic</td>
		</tr>
		<tr>
			<td>Eye Ointment</td>
			<td>Pan-Ophtal</td>
			<td>7136926</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>Anaesthetic Device Komesaroff Mark-5</td>
			<td>Medical Developments Australia</td>
			<td>ACN 004 903 682</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Capillary puller P-97</td>
			<td>Sutter Instrument Co.</td>
			<td>P-97</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Compresstome</td>
			<td>Precisionary Instr.</td>
			<td>VF-300</td>
			<td>Vibratome-type device</td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Zeiss</td>
			<td>LSM700</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM3050S</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Electroporator</td>
			<td>Nepa Gene Co. Ltd.</td>
			<td>CUY21EDIT</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Electrode 1</td>
			<td>Nepa Gene Co. Ltd.</td>
			<td>CUY550-10</td>
			<td>Stainless Steel Needle Electrode, 10 mm-Tip, 0. 5 mm diam.</td>
		</tr>
		<tr>
			<td>Electrode 2</td>
			<td>Nepa Gene Co. Ltd.</td>
			<td>CUY700P4L</td>
			<td>Cover Round Platinum Plate 4 mm diameter</td>
		</tr>
		<tr>
			<td>Fiberoptic cold light source</td>
			<td>Leica</td>
			<td>KL2500 LCD</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass capillaries</td>
			<td>Harvard Apparatus</td>
			<td>GC120T-15</td>
			<td>1. 2 mm O.D. x 0. 94 mm I.D.</td>
		</tr>
		<tr>
			<td>Glass bead sterilizer</td>
			<td>Fine Science Tools</td>
			<td>FST250</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Harvard Apparatus</td>
			<td>py872-5272</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Injection device</td>
			<td>World Precision Instruments</td>
			<td>Pneumatic Pico Pump PV820</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Suture Thread Coated Vicryl</td>
			<td>Ethicon</td>
			<td>V4914</td>
			<td>Peritoneal Suture</td>
		</tr>
		<tr>
			<td>Suture Thr. Supramid</td>
			<td>Serag Wiessner</td>
			<td>TO07171L</td>
			<td>Skin Suture</td>
		</tr>
	</tbody>
</table>

genetic manipulation of the mouse developing hypothalamus through in utero electroporation

開発哺乳類の脳の特定の領域の遺伝子組み換えは非常に強力な実験的なアプローチです。しかし、小説のマウス変異体を生成することはしばしばいらいら遅いです。これは、合理的なポスト手術用生存率と子宮内現像マウスの脳へのアクセスが可能であることが示されている。それでも、この手順と結果は脳の発達の最も表面的かつ簡単にアクセスできる部分はほぼ全面的に報告されており、皮質、すなわち 。視床、狭く、より内側の領​​域は、ターゲットへのより困難であることが判明しました。特に深い核、視床下部のものへのトランスフェクションは、最も困難なので、非常にいくつかの結果が報告されているかもしれないです。ここでは、全体の視床下部の神経上皮またはエレクトロを通じてトランスフェクションのためにその一部を（視床下部の領域）が対象とする手順を示しています。我々のアプローチの鍵は、長い昏睡回、T中の注射ですhird心室、適切な種類と電極の位置。さらに、我々はターゲティング最も凹ん視床核、乳頭体のその後の組織学的分析の結果を示す。

ほとんどの視床下部のニューロンはやや短いマウス開発27,28に翻訳ラット26で出生デートの分析によると、E11.5 E15.2の間に生まれています。視床下部の神経発生のピークはE12.5 29-31に到達する。そこで、本研究（E12.5）のために選択されたトランスフェクションの年齢で、視床下部ニューロンの大部分は、任意の与えられた吻側 - 尾レベルでラベルを付けることができま...

Watch this Scientific Journal Video about Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation at JoVE.com

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

視床下部の機能や医療の重要性にもかかわらず、<em&gt;子宮内</emその開発の&gt;遺伝子操作はほとんど試みられていない。私たちは、の詳細な手順を示す<em&gt;子宮内</em&gt;マウスの視床下部へのエレクトロポレーションと合計と部分（地域）視床下部のトランスフェクションの代表的な結果を示しています。

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 ...

genetic-manipulation-mouse-developing-hypothalamus-through-utero

Research

JoVE Journal

Neuroscience

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

15.7K ビュー.  University of Heidelberg. 視床下部の機能や医療の重要性にもかかわらず、子宮内遺伝子操作はほとんど試みられていない。私たちは、の詳細な手順を示す子宮内マウスの視床下部へのエレクトロポレーションと合計と部分（地域）視床下部のトランスフェクションの代表的な結果を示しています。

ビデオ: Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

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 ...

神経科学

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 ...

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6.
The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. 
We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

開発マウス内耳への遺伝子導入による<emインビボで&gt;</em&gt;エレクトロポレーション

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

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 ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

小脳顆粒ニューロンの遺伝子操作<em&gt;体外</em&gt;と<em&gt;インビボ</em&gt;ニューロンの形態と移行を研究するために、

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

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

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

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

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

Cortex-、Hippocampus-、Thalamus-、Hypothalamus-、横中隔Nucleus-と線条体固有<em&gt;子宮内</em&gt; C57BL / 6マウスでのエレクトロポレーション

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

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.

子宮内エレクトロポレーションは、神経部分集団の興奮とシングルセルの接続を研究するためのアプローチ

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

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

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

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

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

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 ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

新生児マウス脳の線条体細胞の遺伝的操作の定位手術

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...