Name: trans-inner cell mass injection of embryonic stem cells leads to higher chimerism rates
Uploaded: 2018-05-29T13:00:00.000+00:00
Duration: 5 min 53 s
Description: Watch this Scientific Journal Video about Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates at JoVE.com

この研究は、NIH、国立環境衛生研究所の学内研究プログラムによって [一部] 支えられました。統計分析のために Shyamal Peddada 感謝します。私たちはまた彼の継続的なサポートやアドバイスをこの原稿とフランコ DeMayo のレビュー ハンフリー八尾、ゲイリー鳥と結城荒尾を感謝したいと思います。

すべての操作はマウスを NIEHS ノック中核施設の通常の操作の一部として行われました。すべてのマウスが、12:12 で保たれた h: 明暗サイクル NIH 31 と水の食事をしたとアドリブ。動物のケアおよび使用委員会の国立環境衛生研究所に従ってすべての実験を行った。
1. 動物の準備
<ol><li>ドナー マウス、B6 を繁殖 (Cg)-Tyr < c 2 j > B6 に当然のことながら年齢の 8-10 週で/J 雌マウス (Cg)-Tyr < c 2 j >/J 生後 8、26 週齢の雄マウス。視覚的に次の朝 (E0.5) のプラグを確認します。</li>
	<li>受信者のマウス、6 と 9 週齢の雌マウス自然精管スイスウェブ スター マウスを飼育しているスイスのウェブスターを繁殖させます。視覚的に次の朝 (E0.5) のプラグを確認します。</li>
</ol>2 ES 細胞作製
<ol><li>129 硼素 AB2.2 ES 細胞で 15 に DMEM で細胞の培養 %fbs、1% のグルタミン、1% ペン/連鎖球菌および 0.1% の β-メルカプトエタノール。ES 細胞供給の指示に、その推奨されるプロトコルを使用して他のすべての ES 細胞を培養します。</li>
</ol>3. 胚のコレクション
<ol><li>
動物解剖
	<ol>
<li>60 mm の料理をいくつか、5 マウス プラス 3 余分な 10 mL ブラストメディア コレクション (BCM) あたりおよそ 1 (10 %fbs の DMEM)、1 つ 4 ウェルあたり 0.5 mL BCM も皿と。37 ° c、5% CO2平衡します。</li>
		<li>B6 を安楽死させる (Cg)-Tyr < c 2 j > 10% の流量と CO2窒息を使用して E3.5 で/J 雌マウス。</li>
		<li>安楽死させる後、仰臥位の位置にマウスを配置、70% エタノールで十分に腹部を濡れています。</li>
		<li>できるだけ大きく開口部を作り、腹部の中間点で最初だけ、皮膚切開を行います。次にできるだけ大きく切開を再度作る腹部壁を開きます。 削減または排除毛皮汚染注意: 2 つの手順でこれを行うことができます。</li>
		<li>鈍い鉗子が付いて卵巣の脂肪パッドを押し、卵巣から始まる卵巣、卵管、子宮、脂肪組織を最小限に保つために余分な世話を慎重に取り外します。1 つの単一ユニットとして、または次の手順の変更なしで、個々 の角と子宮を削除します。</li>
		<li>除去した後、コレクションのブラスト媒体を含む 60 mm ディッシュに子宮を配置します。</li>
	</ol>
</li>
	<li>
胚が除去
	<ol>
<li>BCM でいっぱい 10 mL 注射器を準備し、25 G 針と合います。</li>
		<li>一度に 1 つの子宮 BCM の持株皿からそれらを削除、余分な血液を削除し、低倍率を解剖顕微鏡の下で 60 mm ディッシュに配置組織にそれらのしみ。</li>
		<li>ゆっくりと慎重に、鉗子を解離性大 (近くに輸卵管) 遠位端のすぐ下の子宮を保持している間並列可能な平面としての子宮の中心に針を挿入します。鉗子の先端を指し示す針を挿入します。</li>
		<li>鉗子針に子宮を保持するためにいくつかの圧力を適用し、BCM の子宮の角を約 1 mL を追放します。 注: 注意が必要この段階で注射器にあまりにも多くの圧力により、メディアの手に負えないストリームを引き起こすのでよく料理を残してします。</li>
		<li>60 mm ディッシュあたり最大 5 全体子宮子宮をフラッシュした後のコレクションまでインキュベーターに料理を配置します。</li>
	</ol>
</li>
	<li>
胚のコレクション
	<ol>
<li>手描きガラス ピペットと口のピペットなどの器具を使って、25 X 35 X と設定、切り裂くスコープ下で胚盤胞を収集します。(図 1)。 注: 口ピペット装置で構成されフレキシブル チューブの 30-40 cm、内径 5 mm 未満を挿入、1 000 μ L ピペット チップ、最初のポイント 1 つの端に、あるガラス ピペットを貼る。シリンジ フィルターを添付 1 cc シリンジ バレルと、口の部分として機能するないのプランジャーのもう一方の端に追加します。</li>
		<li>インキュベーターから料理を削除し、格子パターンを用いた胚の料理のすべての部分を確認します。胚を選んだが個別にし、セカンダリの皿に BCM のドロップを配置。</li>
		<li>すべての料理から収集した後新鮮な BCM のドロップで胚をもう一度洗うし、注射を待つ 4 よく皿に入れます。</li>
	</ol>
</li>
</ol>4. 胚の注入
<ol><li>注射針の確認に基づいて個別に ES 細胞をピックアップします。<ol>
<li>5-7 mL 注入メディアを追加することによって注入皿を準備 (DMEM、10 %fbs と 20 mM HEPES) ガラス下部注入皿に。注射は、部屋の温度で行われます。</li>
		<li>空気またはオイル マイクロインジェクターを使用して、ES 細胞を描画する、注射針に真空を適用します。セル間可能な限り小さなスペースとして保つために世話をします。ここでは、または注入の手順では、ES 細胞をカウントします。</li>
		<li>細胞、滑らかな表面と液胞など明らかな欠陥なしの一般集団と比較してサイズの小さい媒体は、ES 細胞を選択します。 注: ES 細胞集団は形態学的品質で大きく変化して使用される標準は、それらを調整する必要があります。</li>
	</ol>
</li>
	<li>約 15 の ES 細胞 (図 1) で胚を注入します。<ol>
<li>ホールディング ピペットを添付 2 番目の空気マイクロインジェクターにチップし、ホルダーの口近く胚の位置します。ホルダーに胚をセキュリティで保護するために真空を適用します。</li>
		<li>Z 軸の上下ホールディング ピペットを移動して、胚は正にこの帯で細かいピント合わせに続いて、スライドして調整します。次に、注射針の Z 軸を先端近くの ES 細胞は、フォーカスに調整します。</li>
		<li>上部または下部、ICM の目的の位置に回転で注射針から優しく押して今胚を調整します。</li>
		<li>帯と ICM または企業が制御のプッシュを使用して、絨毛を注射針を挿入します。胞胚腔が崩壊しないようにするため注射針のガラスシリンジにわずかな圧力を適用されます。</li>
		<li>針、胞胚腔に侵入して、一度は、ES 細胞を胚の胞胚腔に転送するマイクロインジェクターから圧力を適用します。針の傾斜面として簡単に一時停止は、細胞を維持しながら、正常に戻ります胚で圧力を許可する胚を終了を開始します。 注: 参照グループの ES 細胞を通常投与、次の伝統的な ES 細胞注入プロトコル9ICM に触れないように世話します。ES 細胞も、直接を通じて、ICM (TICM) (図 2、補足映画) 針を進めて、ICM を注入しました。</li>
	</ol>
</li>
</ol>5. eEmbryos の転送
<ol><li>製造の手順ごとの非外科的胚転送デバイスを使用して E2.5 をスイスウェブ スター マウスの胚を転送します。麻酔や手術後の特別なケアは、この非外科的処置は必要ありません。家のマウス胚と配信とその後離乳後個別に。</li>
</ol>

<ol>
	<li>Cong, 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=Multiplex+Genome+Engineering+Using+CRISPR/Cas+Systems.">Multiplex Genome Engineering Using CRISPR/Cas Systems.</a> Science. 339 (6), 819-823 (2013).</li><li>Mali, P., 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=RNA-guided+human+genome+engineering+via+Cas9.">RNA-guided human genome engineering via Cas9.</a> Science. 339 (6), 823-826 (2013).</li><li>Friedel, R., Seisenberger, C., Kaloff, C., Wurst, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EUCOMM+the+European+Conditional+Mouse+Mutagenesis+Program.">EUCOMM the European Conditional Mouse Mutagenesis Program.</a> Brief Funct Genomic Proteomic. 6 (3), 180-185 (2007).</li><li>Austin, C. P., 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+knockout+mouse+project.">The knockout mouse project.</a> Nat Genet. 36 (9), 921-924 (2004).</li><li>Coleman, J. L., Brennan, K., Ngo, T., Balaji, P., Graham, R. M., Smith, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Knockout+and+Reporter+Mouse+Line+Generation+and+Breeding+Colony+Establishment+Using+EUCOMM+Conditional-Ready+Embryonic+Stem+Cells:+A+Case+Study.">Rapid Knockout and Reporter Mouse Line Generation and Breeding Colony Establishment Using EUCOMM Conditional-Ready Embryonic Stem Cells: A Case Study.</a> Front Endocrinol (Lausanne). 30, 105 (2015).</li><li>Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W., Roder, 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=Derivation+of+completely+cell+culture-derived+mice+from+early-passage+embryonic+stem+cells.">Derivation of completely cell culture-derived mice from early-passage embryonic stem cells.</a> Proc Natl Acad Sci U S A. 90 (18), 8424-8428 (1993).</li><li>Eggan, K., 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=Male+and+female+mice+derived+from+the+same+embryonic+stem+cell+clone+by+tetraploid+embryo+complementation.">Male and female mice derived from the same embryonic stem cell clone by tetraploid embryo complementation.</a> Nat Biotechnol. 20 (5), 455-459 (2002).</li><li>Eggan, K., 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=Hybrid+vigor,+fetal+overgrowth,+and+viability+of+mice+derived+by+nuclear+cloning+and+tetraploid+embryo+complementation.">Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation.</a> Proc Natl Acad Sci U S A. 98 (11), 6209-6214 (2001).</li><li>Eakin, G. S., Hadjantonakis, A. K., Papaioannou, V. E., Behringer, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+potential+and+behavior+of+tetraploid+cells+in+the+mouse+embryo.">Developmental potential and behavior of tetraploid cells in the mouse embryo.</a> Dev Biol. 288 (1), 150-159 (2005).</li><li>Huang, J., 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+production+of+mice+from+embryonic+stem+cells+injected+into+four-+or+eight-cell+embryos+by+piezo+micromanipulation.">Efficient production of mice from embryonic stem cells injected into four- or eight-cell embryos by piezo micromanipulation.</a> Stem Cells. 26 (7), 1883-1890 (2008).</li><li>Poueymirou, W. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=F0+generation+mice+fully+derived+from+gene-targeted+embryonic+stem+cells+allowing+immediate+phenotypic+analyses.">F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses.</a> Nat Biotechnol. 25 (1), 91-99 (2007).</li><li>Behringer, R., Gertsenstein, M., Nagy, K. V., Nagy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the Mouse embryo. , (2014).</li><li>Pease, S., Saunders, 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=.">.</a> Advanced Protocols for Animal Transgenesis. , (2011).</li><li>Young, M. K., 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=Effects+of+mechanical+stimulation+on+the+reprogramming+of+somatic+cells+into+human-induced+pluripotent+stem+cells.">Effects of mechanical stimulation on the reprogramming of somatic cells into human-induced pluripotent stem cells.</a> Stem Cell Res Ther. 8 (1), 139 (2017).</li></ol>

著者は開示する競合する興味を持ってないです。

ICM の妨害につながることを死亡、実際には、現在の胚盤胞マイクロインジェクション プロトコルはまだこの12,13の警告それが長く考えられてしまった。我々 がここで示されているものは、マイクロインジェクション ICM を通して胎児に悪影響はないとキメラの収量を増加させます。
この効果の正確なメカニズムは特定されて?...

約 25 年間、遺伝子ターゲットのマウスの作成は生殖系列キメラを送信の世代のため、胚盤胞 ES 細胞インジェクション段階でしばしばボトルネックによって妨げ、ゆっくりとしたプロセスをされています。CRISPR/Cas91,2 ES 細胞と高スループット ES のターゲットを含む、最近の進歩は携帯サイト制作のような生成コンソーシアム、遺伝子組み換え ES 細胞の生成/可用性を改善しています。ただし、生殖巣キメラの生成は、これらの ES 細胞3,4から遺伝子改変マウスの作成にボトルネック残ります。高スループット ES 細胞世代プロジェクトは、生殖系列キメラの作成に成功のために重大である ES 細胞質の高い変動に悩まされています。さらに、一般利用の ES 細胞株の高い異数性と生殖系列キメラ主務5の困難な生産の知られています。
いずれか高いキメラとマウスを作成するための多くの方法が開発されている、または完全に ES 細胞マウス6,7,8,9,10。これらの各システムは、独自のメリットが、また自分の欠点があります。非効率的、四倍体キメラ、マウス由来 100 %es 細胞の生成間の世代でありザイモグラム行5,6に一般に限られました。桑実胚に注入する新しい方法は、100% に近づいている高パーセントキメラをもたらすが、一般的に重要な追加機器 (レーザーまたは piezos) とトレーニング10,11が必要です。研究所は、これらの技術が既に場所で、この新しい手法が必要場合がかもしれません。
本研究の目的は、後続のマウスのコロニーを確立する突然変異体の対立遺伝子の生殖細胞感染の機会が増え、キメラ世代の率を高めるための一般的な技法と容易に利用可能な機器を使用する方法を見つけることだった。

<table><tbody><tr><td>ES Cell injection needle</td><td>Humagen</td><td>MSC-20-0</td><td></td></tr><tr><td>NSET device</td><td>Paratechs</td><td>60010</td><td></td></tr><tr><td>DMEM</td><td>Gibco (life technologies)</td><td>11965-092</td><td></td></tr><tr><td>FBS</td><td>Gibco (life technologies)</td><td>10439-024</td><td>lots should be individually tested for ES Cell compatibility.</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H0887</td><td></td></tr><tr><td>&amp;#946;-mercaptoethanol</td><td>Sigma</td><td>M7522</td><td></td></tr><tr><td>Micro manipulator</td><td>Leica</td><td>Micro manipulator</td><td></td></tr><tr><td>micro injector</td><td>Eppendorf AG</td><td>Cell Tram Air 5176</td><td></td></tr><tr><td>Microscope</td><td>Leica</td><td>DM IRB</td><td></td></tr><tr><td>4 well dish</td><td>Thermo Scientific</td><td>176740</td><td></td></tr><tr><td>60 mm dish</td><td>Sarstedt</td><td>83.3901</td><td></td></tr><tr><td>B6(Cg)-Tyr&amp;#60;c-2J&amp;#62;/J</td><td>Jackson Labs, Bar Harbor, Maine, USA</td><td>000058</td><td></td></tr><tr><td>Swiss Webster mice</td><td>Taconic, USA</td><td>Tac:SW</td><td></td></tr><tr><td>Injection Dish</td><td>MatTek Corp.</td><td>P50G-0-30-F</td><td></td></tr></tbody></table>

trans-inner cell mass injection of embryonic stem cells leads to higher chimerism rates

ES 細胞の方法論による遺伝子改変マウスの作成の効率を上げるために、現在の胚盤胞の注入のプロトコルへの適応を提案する.単純な回転胚とトランス内部細胞塊 (TICM) を注射が追加の機器の 50% 31% からキメラマウスの割合を増加またはさらに特殊な訓練を行った。26 の異なるクローンを近交系し、9 ヶ月の期間にわたって 35 合計クローンを注入しました。伝統的な注入技術と TICM 間妊娠率や胚の回収率に有意差はありませんでした。したがって、注入プロセスと胚盤胞の単純なポジショニングと ICM を注入することですべての主要な変更、なし胞胚腔腔に ES 細胞を解放可能性があります向上できますキメラの生産、その後量生殖。

ランダム効果として細胞株を扱う、混合効果線形モデルのソフトウェア (例えば SAS (バージョン 9.2)) のデータを分析していました。各解析転送胚とセルの行の数を制御します。生存仔数の制御も 2 番目の分析。
この研究のため各 ES 細胞とクローンは従来の両方が注入されたと TICM テクニック、交互にそれぞれの胚の後 2 ?...

Watch this Scientific Journal Video about Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates at JoVE.com

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

ここで新しい機器を使用せずキメラを増産するためのプロトコルを提案する.胚の注入のための簡単な方向の変更は生産、胚の数を増やすし、潜在生殖するタイムラインを削減できます。

トランス内部細胞のキメリズム率が高いため、胚性幹細胞の大量注入

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

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10.2K ビュー.  NIEHS/NIH. ここで新しい機器を使用せずキメラを増産するためのプロトコルを提案する.胚の注入のための簡単な方向の変更は生産、胚の数を増やすし、潜在生殖するタイムラインを削減できます。

ビデオ: Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged antigen-specific T cell receptor (TCR) have been an indispensable tool to study T cell development and function. However, such TCRs are usually isolated from the relevant T cells after long-term culture often following repeated antigen stimulation, which unavoidably selects for T cells with high affinity. Random genomic integration of the TCR &alpha;- and &beta;-chain and expression from non-endogenous promoters can lead to variations in expression level and kinetics.
Epigenetic reprogramming via somatic cell nuclear transfer provides a tool to generate embryonic stem cells and mice from any cell of interest. Consequently, when SCNT is applied to T cells of known specificity, these genetic V(D)J rearrangements are transferred to the SCNT-embryonic stem cells (ESCs) and the mice derived from them, while epigenetic marks are reset. We have demonstrated that T cells with pre-defined specificities against Toxoplasma gondii can be used to generate mouse models that express the specific TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

に対する事前定義されたT細胞受容体の特異性をもつTransnuclearマウストキソプラズマ SCNTを介して取得

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

免疫・感染学

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

Combinational Treatment of Trichostatin A and Vitamin C Improves the Efficiency of Cloning Mice by Somatic Cell Nuclear Transfer

Somatic cell nuclear transfer (SCNT) provides a unique opportunity to directly produce a cloned animal from a donor cell, and it requires the use of skillful techniques. Additionally, the efficiencies of cloning have remained low since the successful production of cloned animals, especially mice. There have been many attempts to improve the cloning efficiency, and trichostatin A (TSA), a histone deacetylase inhibitor, has been widely used to enhance the efficiency of cloning. Here, we report a dramatically improved cloning method in mice. This somatic cell nuclear transfer method involves usage of Hemagglutinating virus of Japan Envelope (HVJ-E), which enables easy manipulation. Moreover, the treatment using two small molecules, TSA and vitamin C (VC), with deionized bovine serum albumin (dBSA), is highly effective for embryonic development. This approach requires neither additional injection nor genetic manipulation, and thus presents a simple, suitable method for practical use. This method could become a technically feasible approach for researchers to produce genetically modified animals from cultured cells. Furthermore, it might be a useful way for the rescue of endangered animals via cloning.

We describe a dramatically improved method for mouse cloning using trichostatin A, vitamin C, and deionized bovine serum albumin. We show a simplified, reproducible protocol that supports efficient development of cloned embryos. Hence, this method could become a standardized procedure for mouse cloning.

トリコスタチン A とビタミン C の組合せ治療が体細胞核移植によるマウスのクローニングの効率を向上します。

Somatic cell nuclear transfer (SCNT) provides a unique opportunity to directly produce a cloned animal from a donor cell, and it requires the use of ...

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

組織標的胚キメラ：ゼブラフィッシュの原腸胚細胞移植

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

生物学

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances early in the gestation. The rationale behind IUT for therapeutic purposes is based on the small size of the fetus, the fetal immunologic immaturity, the accessibility and proliferative nature of the fetal stem or progenitor cells, and the potential to treat a disease or the onset of symptoms prior to birth. Taking advantage of these normal developmental properties of the fetus, the delivery of hematopoietic stem cells (HSC) via an IUT has the potential to treat congenital hematologic disorders such as sickle cell disease, without the required myeloablative or immunosuppressive conditioning required for postnatal HSC transplants. Similarly, the accessibility of progenitor cells in multiple organs during development potentially allows for a more efficient targeting of stem/progenitor cells following an IUT of viral vectors for gene therapy or genome editing. Additionally, IUT can be used to study normal developmental processes including, but not limited to, the development of immunologic tolerance. The murine model provides a valuable and affordable means to understanding the potential and limitations of IUT prior to pre-clinical large animal studies and an eventual clinical application. Here, we describe a protocol for performing an IUT in the murine fetus through intravenous and intra-amniotic routes. This protocol has been used successfully to elucidate the necessary conditions and mechanisms behind in utero hematopoietic stem cell transplantation, tolerance induction, and in utero gene therapy.

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

We describe a protocol for performing an in utero transplantation (IUT) through intravenous and intra-amniotic routes of injection in the murine model. This protocol can be used to introduce cells, viral vectors, and other substances into the unique immune-tolerant fetal environment.

マウス モデルにおける静脈内投与と胎嚢内子宮内移植

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances ...

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

鳥胚胚心筋細胞の生体内注入にマイクロインジェクション ベース システム

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...

Dechorionation of Medaka Embryos and Cell Transplantation for the Generation of Chimeras

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of the vertebrate genome functions. Manipulation of medaka embryos, such as dechorionation, mounting embryos for imaging and cell transplantation, are key procedures to work on both medaka and zebrafish in a laboratory. Cell transplantation examines cell autonomy of medaka mutations. Chimeras are generated by transplanting labeled cells from donor embryos into unlabeled recipient embryos. Donor cells can be transplanted to specific areas of the recipient embryos based on the fate maps 3 so that clones from transplanted cells can be integrated in the tissue of interest during development. Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. In this video, we show detailed procedures to manipulate medaka embryos.

Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. This video shows step-by-step procedures for how to manipulate medaka embryos, including dechorionation, mounting in agarose for imaging and cell transplantation for the production of chimeras. These procedures are essential to use medaka and zebrafish in a laboratory to take full advantage of their complementary features for the genetic dissection of vertebrate genome functions.

キメラの生成のためのメダカ胚および細胞移植のDechorionation

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in ...

Xenotransplantation of Human Stem Cells into the Chicken Embryo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage1-6. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.

In this paper we present a method for transplanting human stem cells into various regions of the central nervous system of the chicken embryo. This provides an in vivo model for assessing the proliferation and differentiation of various types of human stem cells in embryonic tissue environments.

ニワトリ胚へのヒト幹細胞の異種移植

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.

鶏胚における単一細胞のトランスフェクション

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the efficiency in developing gene-knockout mice by inducing small indel mutation would be sufficient enough, the efficiency of embryo genome editing for making large-size DNA knock-in (KI) is still low. Therefore, in contrast to the direct KI method in embryos, gene targeting using embryonic stem cells (ESCs) followed by embryo injection to develop chimera mice still has several advantages (e.g., high throughput targeting in vitro, multi-allele manipulation, and Cre and flox gene manipulation can be carried out in a short period). In addition, strains with difficult-to-handle embryos in vitro, such as BALB/c, can also be used for ESC targeting. This protocol describes the optimized method for large-size DNA (several kb) KI in ESCs by applying CRISPR/Cas9-mediated genome editing followed by chimera mice production to develop gene-manipulated mouse models.

Here we present a protocol for developing genetically modified mouse models using embryonic stem cells, especially for large DNA knock-in (KI). This protocol is tuned up using CRISPR/Cas9 genome editing, resulting in significantly improved KI efficiency compared with the conventional homologous recombination-mediated linearized DNA targeting method.

CRISPR/Cas9を介した胚性幹細胞における高効率遺伝子ターゲティングによる遺伝子操作マウスモデルの開発

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the ...

トランス内部細胞のキメリズム率が高いため、胚性幹細胞の大量注入

要約

さらに動画を探す

この動画の章

に対する事前定義されたT細胞受容体の特異性をもつTransnuclearマウストキソプラズマ SCNTを介して取得

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

トリコスタチン A とビタミン C の組合せ治療が体細胞核移植によるマウスのクローニングの効率を向上します。

組織標的胚キメラ：ゼブラフィッシュの原腸胚細胞移植

マウスモデルにおける静脈内投与と胎嚢内子宮内移植

鳥胚胚心筋細胞の生体内注入にマイクロインジェクションベースシステム

キメラの生成のためのメダカ胚および細胞移植のDechorionation

ニワトリ胚へのヒト幹細胞の異種移植

鶏胚における単一細胞のトランスフェクション

CRISPR/Cas9を介した胚性幹細胞における高効率遺伝子ターゲティングによる遺伝子操作マウスモデルの開発

トランス内部細胞のキメリズム率が高いため、胚性幹細胞の大量注入

要約

さらに動画を探す

この動画の章

に対する事前定義されたT細胞受容体の特異性をもつTransnuclearマウストキソプラズマ SCNTを介して取得

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

トリコスタチン A とビタミン C の組合せ治療が体細胞核移植によるマウスのクローニングの効率を向上します。

組織標的胚キメラ：ゼブラフィッシュの原腸胚細胞移植

マウス モデルにおける静脈内投与と胎嚢内子宮内移植

鳥胚胚心筋細胞の生体内注入にマイクロインジェクション ベース システム

キメラの生成のためのメダカ胚および細胞移植のDechorionation

ニワトリ胚へのヒト幹細胞の異種移植

鶏胚における単一細胞のトランスフェクション

CRISPR/Cas9を介した胚性幹細胞における高効率遺伝子ターゲティングによる遺伝子操作マウスモデルの開発

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

マウスモデルにおける静脈内投与と胎嚢内子宮内移植

鳥胚胚心筋細胞の生体内注入にマイクロインジェクションベースシステム