私たちは、原稿を読んで、編集するためのブラッドLackfordに感謝します。この研究は国立環境健康科学研究所、保健学内研究プログラム国立研究所Z01ES102745（GHまで）によってサポートされていました。

1。 Oct4GiPマウスES細胞の維持 Oct4GiP細胞は、親切に博士はオースティンスミスによって提供されました。彼らは、Oct4の-GFPiresPacトランス4,5を運ぶ129/Olaマウスから得られた。彼らはESGRO完全プラスクローングレード培地（ミリポア社製）、またはM15培地でゼラチンコート組織培養プレートに保持されます：DMEM（Invitrogen社）は、15％FBS、1000 U / mlのESGRO（ミリポア社製）、1Xを添加した非必須アミノ酸（Invitrogen社製）、1X EmbryoMax Nucleasides（ミリポア社製）、および10μMβ-メルカプトエタノール。 <ol><li> 30分間室温で0.1％ゼラチン（シグマ）（0.1ミリリットルゼラチン溶液/ cm 2）を持つコートプレート。 </li><li> 〜2で、ゼラチンコートしたプレートのプレートOct4GiPセル×10 4 / cm 2である 。 </li><li> 0.05％トリプシンですべての二日間のセルを分割します。 </li></ol> 2。 Oct4GiP細胞におけるsiRNAトランスフェクション Oct4GiPレポーターアッセイは、最もconvenienです。M15培地中でまたは384ウェルプレート（CorningまたはBD） - TLYゼラチンでコーティングされた平坦な底部96に実施した。全体的な手順は、 図1に概説されています。 <ol><li> siRNAを脂質複合体のアセンブリ： <ol><li> 96ウェルプレートでトランスフェクションのために、U底96ウェルプレートでのsiRNA-脂質複合体を組み立てる。 </li><li>各ウェルに、10μlののOptiMEM（Invitrogen社製）、0.3μlのリポフェクトアミン2000（Invitrogen）を混合し、室温で5分間インキュベートします。 </li><li>脂質のOptiMEM混合物に5 pmolのsiRNAを追加し、さらに15分間インキュベートします。 siRNAは：6ul：脂質比は100 pmolのです。 </li><li>マルチチャンネルピペットを用いてゼラチンでコーティングされた平底プレートにU-底板からのOptiMEM中のsiRNA-脂質複合体を転送します。 </li><li> 384ウェルプレートでトランスフェクションのために、0.1μlのリポフェクタミン2000および10μlのOptiMEM 2 pmolのsiRNAを用いたsiRNA-脂質複合体を準備します。 </li></ol></li></ol>注：最終siRNA濃度でトランスフェクションは50nmである。 3月4日の生物学を行うそれぞれのsiRNAトランスフェクションのために複製されます。平底ゼラチンコートプレートにU-底板とアリコート中のsiRNA-脂質複合体のマスター混合物を設定します。 <ol start="2"><li> Oct4GiP細胞播種とトランスフェクション： <ol><li> 96ウェルプレートでトランスフェクションのために、Oct4GiP細胞を収集し、9×10 4細胞/ mlに、新鮮なM15培地に再懸濁します。 </li><li>よくマルチチャンネルピペットを用いて各細胞懸濁液100μlを添加します。 </li><li>上下に3-5回のピペッティングでよくで事前に分注したsiRNA-脂質複合体と細胞懸濁液を混ぜる。異なるsiRNAのトランスフェクションするためのヒントを変更します。 </li><li> 384ウェルプレート、再懸濁しOct4GiPセル〜6×10 4細胞/ ml、アリコート30μlの細胞懸濁液中のトランスフェクションのために各ウェルに。 </li></ol></li></ol>注：最適な細胞播種密度は通常3×10 5細胞/ cm 2であるが、メートルAYは劇的に細胞の増殖または生存に影響を及ぼすsiRNAをさらに最適化する必要があります。低播種密度は、トランスフェクション時に悪い細胞の生存につながる。高いメッキ密度が高い細胞合流分化誘導による高いバックグラウンドの原因になります。 2から4×10 5細胞/ cm 2の間に必要に応じて、テストの播種密度。 <ol start="3"><li>メディア変更： <ol><li>マルチチャンネルアスピレーター（コーニング）または真空ワンド（VP-Scientific）を使用して、古い培地を除去します。プレートの下部にある細胞を傷つけないように注意してください。 </li><li>毎日のマルチチャンネルピペットを用いて新鮮なES細胞用培地（96ウェルプレートに100μlと384ウェルプレートで30μl）を持つ細胞を供給します。 </li></ol></li></ol> 3。 FACS分析<ol><li>細胞解離： <ol><li>四日間トランスフェクション後、マルチチャンネルアスピレーター（コーニング）または真空ワンド（VP-Scientific）を用いて培地を除去します。 </li><li> 96ウェルプレートの場合は、100μで一度細胞をリンスリットルPBS。 </li><li>よくマルチチャンネルピペットを用いて、それぞれに25μlの0.25％トリプシンを追加します。 </li><li>時折撹拌しながら5分間室温でインキュベートし、視覚的に細胞の完全な分離を確保するために点検してください。 </li><li>トリプシンを不活性化し、繰り返しピペッティングによって単一細胞懸濁液に細胞を解離するために10％FBSを含むPBS90μlのを追加します。 </li><li> 384ウェルプレートの場合は、10％FBS、30μlのPBSと10μlのトリプシンとクエンチで細胞を解除します。 </li></ol></li><li> FACS分析： <ol><li> HTS装置または他の似たようなFACSアナライザ（例えばAccuriまたはIntellicytなど）を搭載したBD LSRII FACSアナライザのGFP蛍光を分析する。 </li><li> HTSの高スループットモードを使用し、細胞懸濁液10μlを分析します。 1.0×10 4細胞にカウントしきい値を設定します。 </li><li>前方に対側の散布図の細胞をキャプチャするためにPMT電圧としきい値を調整します。 FORWのライブ細胞集団にゲートARD対側の散布図。 </li><li> GFPチャネルのヒストグラムプロットを作成します。細胞の約10％は、モックまたはコントロールsiRNAをトランスフェクトした細胞ではGFP陰性であることが表示されるように、GFPチャネルのゲートを設定します。 </li><li> ％分化した細胞=％GFP陰性細胞：各治療から％GFP陰性細胞を決定します。 2両側t検定を用いた実験およびコントロールsiRNAをトランスフェクションの間に％分化した細胞を比較し、p値&lt;0.01と正のヒットを獲得。 </li></ol></li></ol> 4。代表的な結果 OCT4、Nanogの、とSox2はES細胞の自己複製6,7,9-11の維持に重要な役割を果たして3遺伝子である。 図2は、Oct4GiPレポーターアッセイが容易にこれらの要因をサイレンシングによって引き起こされる分化を検出することができますOct4GiP ES細胞。 図2Aは、ESとして維持するときOct4GiP ES細胞がGFP陽性であることを示して<html> 図2Bの細胞が前方に対側の散布図と制御またはOct4の-siRNAでトランスフェOct4GiP細胞のGFPチャネルのヒストグラムを示しています。死んだ細胞や破片はGFP陰性であり、バックグラウンドを増加させるとして、前方対側の散布図でのライブセルのゲートに必要である。一方、可能な細胞塊を除外するために二重の差別は常に必要ではありません。コントロールsiRNAをトランスフェクトした細胞において、細胞の大半はGFP陽性でなければなりません。明らかにGFP陰性集団はコントロールsiRNAをトランスフェクトしたウェル中に存在する場合、開始Oct4GiP細胞やトランスフェクション手順が損なわれている可能性があり、その結果が解釈されない場合があります。 図2Cは、コントロール、Oct4の - 、Nanogの、、とSox2-siRNAをトランスフェクトした細胞から分化した細胞％（％分化した細胞=％GFP陰性細胞）のバーグラフを示しています。 <html> d/3987/3987fig1.jpg &quot;/&gt; 図1。 Oct4GiPレポーターアッセイの概要 。 <img alt="図2" src="/files/ftp_upload/3987/3987fig2.jpg" /> 図2。 Oct4GiPレポーターアッセイは、Nanog、Oct4のか、Sox2のサイレンシングによって引き起こされるES細胞の分化を検出することができます。 A）E14Tg2a（野生型、黒線）とOct4GiP（グリーンライン）細胞をFACSで解析した。 GFP-チャンネルのヒストグラムはOct4GiP細胞がGFP陽性であることを示している。B）Oct4GiP細胞はトランスフェクション4日後に分析し、コントロールまたはOct4の-siRNAとFACSで96ウェルプレートでトランスフェクトした。側方散乱プロットとトランスフェクトした細胞のGFP-チャンネルのヒストグラムが示されている前方に対。C）Oct4GiP細胞が制御- 、Nanogの- 、Oct4の-、または-Sox2のsiRNAでトランスフェクトした。 ％分化細胞は4日間トランスフェクション後の％のGFP陰性細胞から決定され、+ /意味としてプロットした - 平均の標準誤差（N = 4）。F = &quot;http://www.jove.com/files/ftp_upload/3987/3987fig2large.jpg&quot;ターゲット= &quot;_blank&quot;&gt;拡大図を表示するには、ここをクリックしてください。

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利害の衝突が宣言されません。

我々は上記の記述Oct4GiPレポーターアッセイは、定量的に自己再生対分化の程度を測定することができます。そのような形態をベース12と増殖/生存性アッセイなど他の利用可能な方法に比べ、より高い感度とスループットだけでなく、ES細胞の状態のより直接的な測定を提供しています。したがって、大規模な画面や遺伝的エピスタシス解析のために適している。実際、我々と他の成?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td> ESGRO完全プラスクローングレード媒体</td><td>ミリポア</td><td> SF001-500P </td></tr><tr><td> DMEM（高グルコース1X） </td><td>インビトロジェン</td><td> 11965 </td></tr><tr><td> 0.25％トリプシン-EDTA </td><td>インビトロジェン</td><td> 25200 </td></tr><tr><td>リポフェクタミン2000 </td><td>インビトロジェン</td><td> 1001817 </td></tr><tr><td> OPTI-MEM（血清培地を減らす） </td><td>インビトロジェン</td><td> 31985 </td></tr><tr><td> ESGRO mLIF（10 7 units/1ml） </td><td>ミリポア</td><td> DAM1776540 </td></tr><tr><td> MEM NEAA（非必須アミノ酸） </td><td>インビトロジェン</td><td> 11140 </td></tr><tr><td> ES細胞のために100倍ヌクレオシド</td><td>ミリポア</td><td> 10620から1 </td&gt; </tr><tr><td> 2 - メルカプトエタノール</td><td>シグマ</td><td> M7522-100ミリリットル</td></tr><tr><td> ES-修飾されたウシ胎児血清</td><td>インビトロジェン</td><td> 10437 </td></tr><tr><td> NanogのsiRNAを</td><td>インビトロジェン</td><td> MSS231181 </td></tr><tr><td> Oct4のsiRNAを</td><td> Dharmacon </td><td> D-046256から02 </td></tr><tr><td> Sox2のsiRNAを</td><td> Dharmacon </td><td> M-058489から01 </td></tr></tbody></table>コントロールsiRNA：Dharamconによって合成されたホタルルシフェラーゼ（5&#39;-CGTACGCGGAATACTTCGA）を標的とするsiRNAの二重。

oct4gip reporter assay to study genes that regulate mouse embryonic stem cell maintenance and self-renewal

多能性と自己複製は、胚性幹細胞（ES細胞）の2つの定義特性です。基本的な分子機構を理解することは非常に発生生物学研究、疾患モデル、創薬、再生医療（1,2日で）のES細胞の使用を容易にします。 ES細胞の維持と自己複製の新たな調節因子の同定と特徴付けを促進するために、我々は定量的にOct4GiPセル3を用いたマウスES細胞における自己複製の状態を測定する蛍光レポーターベースのアッセイを開発しました。 Oct4GiP細胞は4,5 Oct4の遺伝子のプロモーター領域の制御下にある緑色蛍光タンパク質（GFP）を発現する。 Oct4のは、ES細胞の自己複製に必要とされており、非常にES細胞で発現し、急速に分化6,7の間にダウンレギュレートされています。その結果、レポーター細胞におけるGFPの発現と蛍光が忠実に相関ES細胞のアイデンティティー5と、蛍光活性化セルソーティング（FACS）で分析が密接に単一細胞レベル3,8の細胞の自己複製状態を監視するために使用することができます。 RNAiのと相まって、Oct4GiPレポーターアッセイは、すぐにES細胞の維持と自己複製3,8のレギュレータを識別し、研究するために使用することができます。自己複製を分析するための他の方法に比べ、それ以上の、便利な高感度、定量的、かつ低コストである。それは、96に実施することができる - または、ハイスループットスクリーニングや遺伝子のエピスタシス解析などの大規模研究のための384ウェルプレート。最後に、他の系統特異的レポーターES細胞株を使用することによって、我々はここで説明するアッセイはまた、ES細胞の分化の間に運命の仕様を勉強するように変更することができます。

Watch this Scientific Journal Video about Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal at JoVE.com

Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

我々はすぐに識別し、マウス胚性幹細胞の維持と自己複製を制御する遺伝子を特徴付けるための蛍光レポーターアッセイを説明します。

マウス胚性幹細胞の維持と自己複製を制御する遺伝子を研究するためにOct4GiPレポーターアッセイ

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will ...

oct4gip-reporter-assay-to-study-genes-that-regulate-mouse-embryonic

Research

JoVE Journal

Biology

10.4K ビュー.  National Institute of Environmental Health Sciences. 我々はすぐに識別し、マウス胚性幹細胞の維持と自己複製を制御する遺伝子を特徴付けるための蛍光レポーターアッセイを説明します。

ビデオ: Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

マウス胚性幹細胞から造血幹細胞の導出

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

生物学

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

免疫手術でヒト胚性幹細胞の導出

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects.

A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin.

To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. 

In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

in vitroおよびヒト胚性幹細胞のin vivoでの生物発光レポーター遺伝子イメージングの

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

ヒト胚性幹細胞の文化とメンテナンス

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

The use of SC1 (Pluripotin) to Support mESC Self-renewal in the Absence of LIF

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and activation of the LIF/JAK/STAT3 pathway is not absolutely required to maintain the self-renewal state.2 The SC1 small molecule may be an economical alternative to LIF. SC1 functions through dual inhibition of Ras-GAP and ERK1.3 Illustration of its mechanism of action makes it a useful tool to study the fundamental molecular mechanism of self-renewal. Here we demonstrate the procedure for culturing mouse ES cells in the presence of SC1 and show that they are able to maintain self-renewal in the absence of LIF. Cells cultured with SC1 showed similar morphology compared to cells maintained with LIF. Both exhibited typical mouse ES morphology after five passages. Expression of typical pluripotency markers (Oct4, Sox2, Nanog, and SSEA1) was observed after five passages in the presence of SC1. Furthermore, SC1 caused no overt toxicity on mouse ES cells.

The use of SC1 Pluripotin to Support mESC Self-renewal in the Absence of LIF

SC1 functions through dual inhibition of Ras- GAP and ERK1. We tested the function of SC1 in supporting mouse ES cell self-renewal in the absence of LIF and showed that SC1 is able to maintain self-renewal of mouse ES cell cultures.

LIFの不在下で自己再生MESCをサポートするためのSC1（Pluripotin）の使用

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

マウスES胚細胞の単離と導出

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

胚肢の皮膚の血管系のためのホールマウント免疫組織化学的解析：胚における血管分岐形態形成を研究するモデルシステム

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

Modified Mouse Embryonic Stem Cell based Assay for Quantifying  Cardiogenic Induction Efficiency

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways. One of the hurdles to its understanding has been the varied methods in correlating changes of key signaling events to differentiation efficiency. We describe here the use of a mouse embryonic stem (ES) cell based assay to identify critical time windows for Wnt/&beta;-catenin and BMP signal activation during cardiogenic induction. By scoring for contracting embryonic bodies (EBs) in a 96-well plate format, we can quickly quantify cardiogenic efficiency and identify crucial time windows for Wnt/&beta;-catenin and BMP signal activation in a time course following specific modulator treatments. The principal outlined here is not limited to cardiac induction alone, and can be applied towards the study of many other cell lineages. In addition, the 96-well format has the potential to be further developed as a high throughput, automated assay to allow for the testing of more sophisticated experimental hypotheses.

We describe the use of a mouse ES cell based assay to identify critical time windows for Wnt/&#946;-catenin and BMP signal activation during cardiogenic induction. The method provides a standardized platform that reliably quantifies cardiogenic efficiency, and it is applicable to the study of other cell lineages.

心原性誘導効率を定量化するために修飾されたマウス胚性幹細胞ベースのアッセイ

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways.  One of the hurdles ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

初期段階のマウス胚を用いて組織特異的遺伝子のためのクロマチン免疫沈降アッセイ

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

人工多能性幹細胞に由来するマウスの作製

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...