Name: culture of embryonic mouse cochlear explants and gene transfer by electroporation
Uploaded: 2015-01-12T14:45:00.000+00:00
Duration: 9 min 3 s
Description: Watch this Scientific Journal Video about Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation at JoVE.com

We would like to acknowledge Dr. Bradley Schulte for comments on this protocol. This work was supported by National Institutes of Health grant R00 (5R00DC010220). This project was performed in a renovated laboratory space supported by Grant C06RR014516.

注：生きた動物を使用するすべてのプロトコルを見直し、施設内動物管理使用委員会（IACUC）によって承認され、実験動物の管理と使用のために公式に承認された方法に従わなければなりませんしなければなりません。すべての解剖清浄な層流ベンチで滅菌技術を使用して行われるべきである。手袋とマスクは、必要に応じて、この手順の間に着用してください。 マウス胎児インナーイヤー1.解剖<ol><li>コルチ植片培養のオルガンのためのセットアップ： <ol><li>約30分間、UV光をオンにすることによって、層流組織培養フードを滅菌し、使用前に70％エタノールで表面を消毒する。また、オートクレーブを介して、または、使用前に少なくとも20分間、70％エタノールに浸漬することによって、胚の解剖のための細かい解剖ツールとシルガードコーティングされた料理など、すべての解剖器具を滅菌する。 70％エタノールをオフに注ぎ、料理や楽器がきれいな層流CLEAで空気乾燥することができますn個のベンチ使用前。 </li><li>準備無菌ハンクス平衡塩溶液（HBSS）/（4-（2-ヒドロキシエチル）-1-ピペラジンエタンスルホン酸）、HEPES 10×HBSSおよび0.5％の10％を添加し、約7.2にpHを調整し、フィルター滅菌することにより（HEPES）混合4℃での最終溶液とストア。より良い作業中は組織を維持するために冷やしたHBSS / HEPES溶液中のすべての解剖を行ってください。 </li><li>基底膜マトリックスで被覆ガラスボトムディッシュの滅菌15mlのポリプロピレンチューブ中の基底膜マトリックスの300μlのアリコートを冷（4℃）滅菌ダルベッコ改変イーグル培地（DMEM）を5ml加えることによって。内容をボルテックスで混合し、それは皿の中央にも存在する全体の文化を覆うように、各培養皿の中心に基底膜マトリックス-DMEM混合物の150μlを添加する。使用前に少なくとも45分間37℃のインキュベーター中で料理や店舗をカバーしています。 </li><li>目にHBSSを冷やし4〜5ミリリットルを注ぐREE無菌ポリスチレンペトリ皿やフード内に閉じたまま。 </li><li> DMEMの9ミリリットル、N2サプリメント100μlの、10グラム/ mlのシプロとFBSの1ミリリットルを追加することにより、滅菌15ミリリットルポリプロピレンチューブに培養液を準備します。 </li></ol></li><li>胚の単離： 注：胚のマウスから蝸牛外植体を培養するこの手順では、収穫内耳日胚（E）から13頭骨E1 9として検討されて受精後の日と。プロトコルは、E12からE18に始まる胚性マウス内耳と一緒に使用することができる。 <ol><li>頸椎脱臼または適切な承認されたプロトコルによってCO 2と犠牲動物と時限妊娠マウスを安楽死させる。組織培養室で、無菌性を維持するために、離れた層流の組織培養フードから安楽死を実施する。 </li><li>上に向けて腹部をペーパータオルに安楽死させたマウスを置き、70％エタノールで腹部を消毒する。 </li><li>皮膚をつかんによって腹腔を開き別の手を使用してハサミで正中線に沿って湾曲した一方の手で鉗子とカット表皮と筋肉と。両側の2つの垂直なカットを作成し、慎重に子宮を引き出す鉗子で二国間の子宮角を持ち上げて、ハサミを使って結合組織の下からそれを切り離す。 </li><li>チルド解剖溶液（HBSS / HEPESの混合物）と層流クリーンベンチへの転送を含むペトリ皿に胚チェーンを配置します。 </li><li>胎盤から慎重に胚を取り出し、解剖液を含む滅菌ペトリ皿の1にそれらを配置します。解が血まみれになった場合、新たなペトリ皿に移動します。 注：この時点で、胚はタイラーステージングガイドを使用して上演することができます。 </li><li>清潔な鉗子やハサミで首領域でつまんで胚を斬首し、チルド解剖溶液を含む新鮮なペトリ皿に頭を置く。 </li></ol></li><li>内耳の解剖： <ol><li>置きO冷たい解剖を含む滅菌シルガード皿の中のNE胚ヘッド</li><li>ソリューション。 〜1.6倍の倍率で解剖顕微鏡の下で働く、Minutienの周りにピンまたは眼の領域（ 図1A）に近くに配置することによって、胚の頭部を固定する。 </li><li>滅菌ピンセット二組を使用すると、慎重に皮膚を除去し、正中線（ 図1B）に沿って背側の頭蓋骨を開く。頭蓋腔から脳を取り出し、側頭骨内にある内側の耳は、この段階で識別することができます（ 図1C、D）。通常、内側の耳の周りに存在する血管のライニングは、この段階（ 図1D）で内耳を識別するための目印として使用することができる。 </li><li>組織の下に鉗子を配置し、冷解剖溶液を含む新しい皿に頭蓋骨転送のベースから内耳を単離することによって側頭骨から内耳を分析（ 図1E、F）。 </li></ol></li></ol>オルガンコルチ植片の培養の2世代注：開発のこの段階では、組織は軟骨であり、容易に鉗子を用いて解剖することができる。 <ol><li>オリエント内耳腹側は、（ 図2A）を上に向け、静かに内耳（ 図2B）の前庭部分を通ってMinutienピンの鋭い端を挿入することにより、内耳の安定化されるように。 </li><li>超微細鉗子を使用すると、楕円形の窓の近くに鉗子の一端を使用して切開を行うことによって、上にある軟骨を開き、カットし、慎重に蝸牛（ 図2C）から軟骨を取り除く。 注：これは、その上の軟骨が、時にはそれが下にこすることによって、基礎となる蝸牛管から軟骨を解放するために、閉じた鉗子を使用すると便利である場合には、蝸牛管と融合されているように鉗子が軟骨にあまりにも深く挿入されていないことを確認することが重要ですS軟骨のurface。開発のこの段階では、蝸牛のらせんの長さは、ターンの唯一の四分の三である。 </li><li>次に、ベースまたは非常に頂点のいずれかで、蝸牛管の好ましい領域で鉗子を配置することによって感覚上皮を公開、そして優しく蝸牛（ 図2D）の屋根を引き出す。 注：それは分析を妨害感覚上皮をマスクすることができるように蝸牛の屋根が完全に除去しなければならない。 注：それは感覚上皮を引き裂くことは容易であるように、この手順を静かに行う必要があります。 </li><li>蝸牛外植片のベースが一様に平らになるように、最後のステップとして、慎重にさらさ感覚上皮から下にある結合組織を除去。 </li><li>ピンセットを用いて、内耳の前庭部分から解剖蝸牛を分離します。これは、（ 図2E）の前またはステップ2.4の後に行うことができる。 </li><li>地下membraに解剖蝸牛感覚上皮を転送よく1.5ミリメートル無菌すくう（ 図2F）を使用して、NEマトリックスでコーティングされた文化。 </li><li>オリエントは、上皮の内腔表面を持つ蝸牛外植片が上に向け、慎重に組織をフラット化し、静かに皿に追加することによって、新鮮な培養培地150μlでそれを置き換えるために基底膜マトリックス-DMEM溶液を吸引。注意が各植片がコーティングされたガラスカバースリップによく付着し、培養液中に浮遊していないことを確認するために注意する必要があります。蝸牛外植片の損傷を防ぐために、ガラスウェルに貼付しながら、鉗子の先端がアップ指摘されていることを確認してください。 </li><li>穏やかに所望の期間、 インビトロでの 、通常3-6日間（DIV）、5％CO 2、37℃で組織培養インキュベーター中で無菌の150mm培養皿、所定の位置に培養皿に移す。 1 DIVの後、蝸牛外植片を培養皿によく付着していることを確認するために解剖顕微鏡下での文化を調べます。 </li><li>イン後時間の所望の長さのために、in vitroで cubationは、培養物を免疫組織化学のために処理される。 注：植片培養は通常の発達段階、P0と同等である6 DIVインキュベートする。 インビトロでのインキュベーション後、培養物を、in situハイブリダイゼーション 、および/ ​​またはウェスタンブロットにおいて 、免疫組織化学（ 図3B）、RT-PCRのような更なる下流の適用のために処理することができる。 </li></ol> 3.エレクトロ媒介遺伝子導入<ol><li>エレクトロポレーションのためのセットアップ： <ol><li>約20分間、70％エタノール中でオートクレーブ処理または浸漬ずつ100ミリメートルのSylgard被覆ガラス皿を滅菌し、使用前にクリーンベンチ内で空気乾燥させ。 </li><li>適切な最大またはミディプレップキットを使用して、選択した発現ベクターからDNAを準備します。このプロトコルで使用される発現ベクターは、たpIRES2-Atoh1.EGFPとpCLIG-NeuroD1.EGFPです。 DNAの最終濃度は、無菌のDNA中に少なくともを1μg/μlのあるべき/RNaseフリー水。 </li></ol></li><li>胚性蝸牛外植片にDNAをエレクトロポレーション： <ol><li>新鮮な100ミリメートルのシルガードコートディッシュにプラスミドDNA溶液10μlを加え、DNA溶液に（ステップ2.5から得た）完全に解剖蝸牛外植片を移す。 </li><li>それは皿の面に垂直だように、上向きに上皮の管腔表面では、わずかに蝸牛を傾ける。 </li><li>感覚上皮と蝸牛のベースに隣接して配置された正パドル（アノード）に向かって負のパドル（カソード）との蝸牛の両側に電極パドルを置きます。 注：DNAが負に帯電しているので、それが負から正極に、感覚上皮に隣接するプローブのカソード端を配置することによって移行し、 DNAは、主に感覚上皮の表面を通過します。 </li><li>エレクトロを使用して、トンで24 mVでの9~10パルス、フットペダルスイッチと30ミリ秒のパルス持続時間を提供彼エレクトロ、その後にエレクトロ蝸牛に暖かい培養培地100μlを追加。 </li><li>繰り返しますが、すべての蝸牛外植のために、目的のすべての遺伝子のDNAに対する3.2.1-4ステップと、（ステップ2.7）をめっきするための基底膜マトリックスコートディッシュにエレクトロポ蝸牛を移す。その後、免疫細胞化学のために処理される時間の所望の長さのために37℃、5％CO 2の加湿インキュベーター中で培養すべてのエレクトロポ蝸牛。 注：CD1マウス（約8〜12匹）の全体のごみから蝸牛は、孤立顕微解剖、エレクトロポレーションおよび&lt;4時間でめっきすることができる。 </li></ol></li></ol> 4.分析蝸牛外植片の文化注：培養物は、通常の発達段階、P0と同等である6 DIVインキュベートする。 インビトロでのインキュベーション後、培養物を固定し、免疫細胞化学のために処理した。 <ol><li>株式会社の所望の長さの後にubation、培地を除去し、すぐにリン酸緩衝生理食塩水（PBS）中の蝸牛外植片をすすぎ、室温で15分間（PBS中で調製）を4％パラホルムアルデヒド中でインキュベートする。 </li><li> PBSを加えることによって固定液を洗浄し、15分間PBSで3回リンス。 </li><li> 10％ヤギ血清を含むPBS-Tでブロッキングする前に、30分間、PBS-T（PBS + 0.5％のTween）を用いて透過性。 </li><li> 4℃で一晩、1％ヤギ血清を含むPBS-T中で一次抗体を含む一次抗体溶液中でインキュベートする。 </li><li>適切なアレクサ結合二次抗体を加える前に、各PBS-Tの15分を使用して室温で蝸牛を3回洗浄します。 </li><li>蝸牛外植片、ガラスカバースリップ上で培養しているので、それは皿からカバースリップを分離し、スライド上に取り付けることにより、個別に処理することができます。これは、室温で少なくとも1時間OS30溶媒中​​で培養皿を浸漬することによって達成することができる。無菌のカミソリの刃を使用して、dからカバースリップを切り離すISHとスライド上にマウントし、蛍光顕微鏡下で可視化する。 </li></ol>

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No conflicts of interest declared.

、感覚非感覚と螺旋神経節ニューロンを含むマウス内耳の膜迷路内のすべての細胞はすべてE8 10-14の周りに、外胚葉の後脳に隣接して位置placodally由来の耳胞に由来している。 E11で、耳胞の腹側領域は蝸牛管を形成するように延びており、開発が進むにつれて、となる蝸牛内の上皮細胞、ならびに内耳胞の他の領域群は、その後に生じさせるprosensoryパッチとして指定機械感覚有毛細?...

The mammalian organ of Corti is comprised of a mosaic of specialized cell types, including two types of mechanosensory hair cells as well as at least four types of nonsensory supporting cells making it an ideal model system to study normal cellular processes like proliferation, fate specification, differentiation and patterning. In addition, the normal development of these different cell types is essential for normal hearing function. Hence, it is crucial to understand the factors, both molecular and cellular, that regulate their development. However, the small size of the mouse cochlea as well as its inaccessibility poses a particular challenge for gene expression studies. Moreover, most of the cell fate specification and patterning events occur during embryonic time periods and are mostly completed before birth. Therefore, identification and characterization of signaling events during embryonic time periods is essential to gain insight into the molecular basis of cochlear morphogenesis. 
Here, we demonstrate a method to culture intact cochlea in vitro from embryonic mouse inner ears. The rationale behind the use of this technique is that cultured cochleae maintain their molecular and morphological characteristics thereby providing a valuable model for investigating potential candidate genes and exploring the mechanisms involved in cochlear morphogenesis. Although transgenic mice can be used for gene expression studies, an in vitro system is often needed for monitoring specific gene functions. Moreover, cochlear cultures can be established from transgenic mouse embryos so that their in vitro development and response to various soluble factors and antagonists can be studied. Although embryos at day 13 (E13) are used in this protocol, cultures from E12 or E14 to early postnatal inner ears can give similar results. 
We also present a gene transfer technique in cultured embryonic cochlear explants using square wave electroporation. Following the isolation of the cochlear explants, electroporation can be used to express DNA plasmids of gene(s) of interest in individual cells within the cochlear duct. This technique serves as a complementary approach to studies utilizing transgenic mice to gain insight into the molecular pathways underlying cellular phenotype. Using this method of gene transfer, a variety of epithelial cell types within the embryonic cochlea are transfected, thereby enabling loss-and gain-of-function analyses at the single-cell level. In addition, electrophysiological studies can also be performed in cochlear explant cultures8. This method of in vitro electroporation is relatively simple and straightforward, combined with minimal damage to the tissue, has resulted in a rapid expansion of this technique.

<table><tbody><tr><td>HBSS</td><td>Gibco</td><td>14065-056</td><td></td></tr><tr><td>HEPES</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Medium</td><td>Gibco</td><td>12430-054</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>10082</td><td></td></tr><tr><td>N-2 Supplement (100x)</td><td>Gibco</td><td>17502-048</td><td></td></tr><tr><td>Ciprofloxacin hydrochloride</td><td>Cellgro</td><td>61-277-RF</td><td></td></tr><tr><td>Glass Dish 60 mm</td><td>Kimble Chase</td><td>23062-6015/23064-6015</td><td></td></tr><tr><td>Glass Dish 100 mm</td><td>Kimble Chase</td><td>23064-10015/23062-10015</td><td></td></tr><tr><td>Minutien pins</td><td>Fine Science Tools</td><td>26002-15</td><td></td></tr><tr><td>Dumont #5 forceps</td><td>Fine Science Tools</td><td>11251-10</td><td></td></tr><tr><td>Pulse generator</td><td>Protech International Inc</td><td>CUY21Vivo-SQ</td><td></td></tr><tr><td>Glass bottom culture dishes</td><td>MatTek</td><td>P35G-0-10-C</td><td></td></tr><tr><td>Matrigel matrix</td><td>BD Biosciences</td><td>356237</td><td></td></tr><tr><td>Culture dish, 60 x 15 mm</td><td>Becton Dickinson</td><td>353037</td><td></td></tr><tr><td>Tissue culture dishes</td><td>Greiner Bio-one</td><td>639160</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Gibco</td><td>10010-023</td><td></td></tr><tr><td>OS-30</td><td>Dow Corning</td><td>4021768</td><td></td></tr><tr><td>Fluoromount</td><td>Southern Biotech</td><td>0100-01</td><td></td></tr><tr><td>Conical tubes, 15 ml</td><td>Greiner Bio-one</td><td>188261</td><td></td></tr><tr><td>Myosin 6</td><td>Proteus Biosciences Inc</td><td>25-6791</td><td></td></tr><tr><td>Myosin 7a</td><td>Proteus Biosciences Inc</td><td>25-6790</td><td></td></tr><tr><td>TuJ1</td><td>Sigma</td><td>T2200</td><td></td></tr></tbody></table>

culture of embryonic mouse cochlear explants and gene transfer by electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We describe a method to isolate cochlea from embryonic inner ears and micro-dissect to expose the sensory epithelium. Once dissected, it may be plated and cultured as an intact cochlear duct (Figure 3) and analyzed by immunohistochemistry. The cultured cochlear explants provide a useful assay to examine the effect of variety of soluble factors and pharmacological drugs on cochlear development. Following dissection of cochlear explants, electroporation technique can be used to misexpress genes of interest...

Watch this Scientific Journal Video about Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation at JoVE.com

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

エレクトロポレーションによりマウス胎児蝸牛外植の文化と遺伝子導入

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

culture-embryonic-mouse-cochlear-explants-gene-transfer

Research

JoVE Journal

Developmental Biology

12.8K ビュー.  Medical University of South Carolina, College of Medicine. We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development. 

ビデオ: Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, cell culture has been widely used in developmental biology studies. However, it is important to determine whether in vitro cultured cells truly represent in vivo cell types. Grafting cells into embryos, followed by an assessment of their contribution during development is a useful method to determine the potential of in vitro cultured cells. In this study, we describe a method for grafting cells into a defined site of early postimplantation mouse embryos, followed by ex vivo culture. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing precise localization and adjustment of the number of cells receiving exogenous DNA with both high transfection efficiency and low cell death. These techniques, which do not require any specialized equipment, render experimental manipulations of the gastrulation and early organogenesis-stage mouse embryo possible, allowing analysis of commitment in cultured cell subpopulations and the effect of genetic manipulations in situ on cell differentiation.

We demonstrate a method for grafting cultured cells into defined sites of early mouse embryos to determine their in vivo potential. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing the precise delivery of exogenous DNA into a few cells in the embryos.

早期着床後のマウス胚への細胞やDNAの正確​​ローカライズ転送する方法

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, ...

発生生物学

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2&#180;-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr&#160;after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

チキン聴覚器官への遺伝子導入によって、<em&gt;卵内</em&gt;マイクロエレクト​​ロポレーション

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) has been shown to be a useful approach to local drug delivery into the inner ear. The goal of this article is to describe, in detail, the surgical techniques involved in canalostomy in both adult and neonatal mice. As indicated by fast-green dye and adeno-associated virus serotype 8 with the green fluorescent protein gene, the canalostomy facilitated broad distribution of injected reagents in the cochlea and vestibular end-organs with minimal damage to hearing and vestibular function. The surgery was successfully implemented in both adult and neonatal mice; indeed, multiple surgeries could be performed if required. In conclusion, canalostomy is an effective and safe approach to drug delivery into the inner ears of adult and neonatal mice and may be used to treat human inner ear diseases in the future.

Here we describe canalostomy procedure which allows local drug delivery into the inner ears of adult and neonatal mice through the semicircular canal with minimum damage to hearing and vestibular function. This method can be used to inoculate viral vectors, pharmaceuticals, and small molecules into the mouse inner ear.

成人および新生児マウス内耳への薬剤の局所投与への外科的アプローチとしての Canalostomy

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) ...

遺伝学

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 ...

神経科学

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 ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

初代培養およびコルチのマウス臓器のプラスミドエレクトロ。

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

生物学

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

マウス蝸牛外植片の長期的タイムラプスイメージング

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. Platforms have been recently developed for highly robust and prolonged ex utero culture of mouse embryos from egg-cylinder stages until advanced organogenesis. These platforms enable appropriate and faithful development of pregastrulating embryos (E5.5) until the hind limb formation stage (E11). Late gastrulating embryos (E7.5) are grown in rotating bottles in these settings, while extended culture from pregastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle cultures. In addition, sensitive regulation of O2 and CO2 concentration, gas pressure, glucose levels, and the use of a specific ex utero culture medium are critical for proper embryo development. Here, a detailed step-by-step protocol for extended ex utero mouse embryo culture is provided. The ability to grow normal mouse embryos ex utero from gastrulation to organogenesis represents a valuable tool for characterizing the effect of different experimental perturbations during embryonic development.

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

An enhanced platform for whole-embryo culture allows continuous and robust ex utero development of postimplantation mouse embryos for up to six days, from pregastrulation stages until advanced organogenesis. In this protocol, we detail the standard procedure for successful embryo culture using static plates and rotating bottle systems.

元ウテロ マウス胚の採虫前から高度器官形成までの培養

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. ...

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as the hair cell. Basic research in the inner ear has led to a deep understanding of how the hair cell functions, and how dysregulation can lead to hearing loss and vertigo. For this research, the mouse has been the pre-eminent model system. However, mice, like all mammals, have lost the ability to replace hair cells. Thus, when trying to understand cellular therapies for restoring inner ear function, complementary studies in other vertebrate species could provide further insights. The auditory epithelium of birds, the basilar papilla (BP), is a sheet of epithelium composed of mechanosensory hair cells (HCs) intercalated by supporting cells (SCs). Although the anatomical architecture of the basilar papilla and the mammalian cochlea differ, the molecular mechanisms of inner ear development and hearing are similar. This makes the basilar papilla a useful system for not only comparative studies but also to understand regeneration. Here, we describe dissection and manipulation techniques for the chicken inner ear. The technique shows genetic and small molecule inhibition methods, which offer a potent tool for studying the molecular mechanisms of inner ear development. In this paper, we discuss in ovo electroporation techniques to genetically perturb the basilar papilla using CRIPSR-Cas9 deletions, followed by dissection of the basilar papilla. We also demonstrate the BP organ culture and optimal use of culture matrices, to observe the development of the epithelium and the hair cells.

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The chick is a cost-effective, accessible, and widely available model organism for a variety of studies. Here, a series of protocols is detailed to understand the molecular mechanisms underlying avian inner ear development and regeneration.

鳥類の内耳の発達を研究するためのOvoおよびEx Ovoの方法

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

新生児マウス蝸牛移植法<em&gt;インビトロ</em&gt;聴覚研究のスクリーニングツール

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

エレクトロポレーションによりマウス胎児蝸牛外植の文化と遺伝子導入

要約

さらに動画を探す

この動画の章

早期着床後のマウス胚への細胞やDNAの正確ローカライズ転送する方法

チキン聴覚器官への遺伝子導入によって、

成人および新生児マウス内耳への薬剤の局所投与への外科的アプローチとしての Canalostomy

開発マウス網膜の in vivoエレクトロポレーションで

開発マウス内耳への遺伝子導入による

初代培養およびコルチのマウス臓器のプラスミドエレクトロ。

マウス蝸牛外植片の長期的タイムラプスイメージング

元ウテロマウス胚の採虫前から高度器官形成までの培養

鳥類の内耳の発達を研究するためのOvoおよびEx Ovoの方法

新生児マウス蝸牛移植法

エレクトロポレーションによりマウス胎児蝸牛外植の文化と遺伝子導入

要約

さらに動画を探す

この動画の章

早期着床後のマウス胚への細胞やDNAの正確​​ローカライズ転送する方法

チキン聴覚器官への遺伝子導入によって、

成人および新生児マウス内耳への薬剤の局所投与への外科的アプローチとしての Canalostomy

開発マウス網膜の in vivoエレクトロポレーションで

開発マウス内耳への遺伝子導入による

初代培養およびコルチのマウス臓器のプラスミドエレクトロ。

マウス蝸牛外植片の長期的タイムラプスイメージング

元ウテロ マウス胚の採虫前から高度器官形成までの培養

鳥類の内耳の発達を研究するためのOvoおよびEx Ovoの方法

新生児マウス蝸牛移植法

早期着床後のマウス胚への細胞やDNAの正確ローカライズ転送する方法

元ウテロマウス胚の採虫前から高度器官形成までの培養