著者らは、胚の図1におけるヘッドやヘルプや議論のための研究室の他のメンバーを説明するためのアイリーン·チョイに感謝したいと思います。この作品は、NIHの助成金DE012728（TW）でサポートされています

1。ディスパーゼIIを調製<ol><li>新鮮なHepes緩衝生理食塩水（HBS）（50mMのHEPES / KOH pH7.4の150mMのNaCl）を準備します。 16.8ミリリットル純H 2 Oに2ミリリットル、0.5M HEPES / KOH pH7.4及び1.2ミリリットル2.5 M NaClを追加</li><li> 10 mg / mlのディスパーゼIIを作る。 0.2グラムディスパーゼII（Roche社、カタログ番号4942078001）を計量し、20ミリリットルHBSに溶解する。長期保存のために-20℃で、個々のエッペンドルフチュー​​ブとストアに1.0mlのアリコートを準備します。 </li><li>使用前にPBS中の10 mg / mlのディスパーゼII 1:10に希釈します。氷の上に希釈ディスパーゼIIを入れてください。 </li></ol> 2。妊娠中の女性からのCD-1 E10.5胚を解剖全ての動物実験は、コロラド州デンバー大学（UCD）の動物のケアと使用委員会によって承認されたプロトコールに従って行った。 CD-1（ICR）マウスをハーランラボラトリーズ（インディアナポリス、インディアナ州）から入手した。 <ol><li> 70％エタノールを用いて解剖エリアやツールを清掃してください。 </li><li> approvを使用して妊娠CD-1雌を安楽死させるedのプロトコル。 </li><li>腹部を開いた後、70％エタノールで腹部をスプレーしてください。子宮から子宮間膜を引き離すために、子宮と別のを引っ張るために鉗子の1セットを使用します。体内から子宮を取り出します。腹部の反対側の子宮角にこの手順を繰り返します。 </li><li>簡単に言えば血液を洗い流す10cmのペトリ皿に氷冷PBSで子宮をすすいでください。 </li><li>氷冷PBSで新しいシャーレに子宮を転送します。注入部位の間に切断することにより1-胚断片に分離します。 </li><li>実体顕微鏡下で氷冷PBS中に1つの胚を転送します。卵黄嚢と胚を露出するように切断部位の1つで始まって子宮の筋肉壁をはがすように細かい鉗子を使用しています。卵黄嚢と羊膜を剥がします。氷上のPBSで6 cmのシャーレに解剖した胚を転送します。すべての胚がこのように収集されるまで進んでください。 </li></ol> 3。ディスパーゼIIの治療<ol><li> 6 cmのシャーレにPBSですべての胚を洗浄します。置き換えるPBS中で10ミリリットル希釈ディスパーゼIIを含むPBS。 25分間37℃でペトリ皿とインキュベートをカバー-細菌インキュベーターまたは暖かい部屋で十分です-私たちは、このステップのために、CO 2組織培養インキュベーターを使用していない。 </li><li>ペトリ皿を取り出し、実体顕微鏡下で胚を観察します。外胚葉が緩んでいるはずですが、この組織層と下層の間充織との間に明確なギャップに代表される。されていない場合は、37℃でさらに5分間℃のインキュベート次いで氷上でペトリ皿を置く。 </li></ol> 4。無傷の顔の際立ちを解剖する<ol><li>使い捨てガラスピペット（VWR、カタログ番号14672から380）を使用して、 実体顕微鏡下で1ディスパーゼIIが6 cmのペトリ皿に氷冷PBS中に胚を扱わ転送します。 </li><li>胚を保持するために鉗子のペアを1つ使用します。ヘッド（ 図1に取り付けられて顔面プロミネンスの境界線をカットするために慎重細かい鉗子の別のペアを使用しての境界を示していますフェイシャルプロミネンス）。 MXPその後、MNPの一方の側から起動して、FNPを持続。そのまま外に顔のプロミネンス（ 図2A）を解剖し、反対側にも同じことを行います。 </li></ol> 5。間葉とは別の顔の外胚葉<ol><li> 3倍の作動距離を持つ実体顕微鏡下で顔面プロミネンスを観察します。優しく上下に5回フェイシャルプロミネンスをピペットに長いガラスパスツールピペットを使用しています。この後、外胚葉は剥がれやすいです。 </li><li>顔のプロミネンスを保持するために鉗子のペアを1つ使用します。非常にゆっくりと外胚葉剥がし鉗子の他のペアを使用します。 図2Bは、顔の外胚葉の分離シートを示しています。 </li><li>氷上のPBSで1.5mlのエッペンドルフチュー​​ブに顔面外胚葉を転送するために新しい長さのガラスパスツールピペットを使用しています。氷上のPBSで別の1.5mlエッペンドルフチュー​​ブに間充織を転送します。 </li><li>各解剖のために新しい氷冷PBSを使用しています。サンプルを収集します。 </li><li>トンを洗うPBSで彼がサンプル。 4℃、3分間、500×gで遠心分離します。 </li><li>長いガラスパスツールピペットを用いてPBSを吸引除去。サンプルはChIPアッセイに使用する場合は、6.1に進みます。 RNA抽出のために、6.2に進みます。タンパク質の抽出には、6.3に進みます。 </li></ol> 6。さらなる実験のためのプロセスのサンプル<ol><li> ChIPアッセイ用サンプル<ol><li>架橋〜1％ホルムアルデヒドで1mlのPBSを追加します。サンプルをホモジナイズする。 </li><li>回転に室温で10分間インキュベートする。 </li><li>未反応のホルムアルデヒドをクエンチするために各チューブに1.25 Mグリシンの110μlを添加する。 </li><li>混ぜ、室温で5分間静置します。 </li><li> 3分間、4℃で500×gで遠心する。上清を除去します。 </li><li>組織を洗浄するために1ミリリットルの冷PBSを追加します。 </li><li> 3分間、4℃で500×gで遠心する。 </li><li> PBSを除去し、PBSで一度洗浄を繰り返す。 </li><li>上清を除去し、液体窒素で組織を凍結スナップ。 SAを保存-80℃で、その後のプーリングおよび処理するためのmples。 </li></ol></li><li> RNA抽出用サンプル<ol><li> RNAlaterで（Ambion）を含むチューブの顔面外胚葉と間充織を置きます。 </li><li>一晩4 RNAlaterでソリューションでサンプルをインキュベート℃で組織の徹底的な浸透を可能にする。 </li><li>後続のプーリングおよび処理するため-20℃で保存。 </li></ol></li><li>タンパク質抽出用のサンプル</li></ol>その後の処理のために試料に溶解バッファーを追加します。または液体窒素で組織を凍結スナップ。 -80℃で、その後のプーリングして処理するためのサンプルを保管してください。 注釈<ol><li>時間の制約。第6節の初めに至るまで解剖の開始4から6時間を計画します。 </li><li>ときに堪能、それはセクション4および5（プロミネンス解離および外胚葉分離）を実行するために胚当たり約10〜15分かかります。 </li><li>サンプルは、RNA単離、毎solutioのためにしている場合nは、第ジエチルで治療すべきである。また鉗子＆アウェイRNaseの作業領域をきれいにしてください。 </li><li>ディスパーゼIIの治療および解剖を除いてすべての回でサンプルを氷上で保管してください。解剖に使用PBSは寒いであることを確認するために各郭の新しい氷冷PBSに変更します。 </li><li>シャープ鉗子は、正確な切開を得ることが重要です。任意の汚染を防ぐために完全に鉗子を清掃してください。 </li><li>異なるプロミネンスからサンプルを収集するために、ディスパーゼIIの治療後に、個別に各プロミネンスを解剖。その後、外胚葉と間充織を分離します。 </li><li>ディスパーゼIIで処理した後、顔の外胚葉が剥がれやすいです。他の外胚葉からの汚染に特別な注意を払う。すべての汚染された可能性のある組織を捨ててください。 </li><li>一度サンプルがセクション6で適切なポイントに到達するまで停止しないと、起動します。早くすることができますようにすべての胚の顔面外胚葉と間葉の分離を完了します。 </li><li>電子ディスパーゼIIのnzyme活動は、バッチとブランドの間で異なる可能性があります。それは、さまざまなブランドとディスパーゼIIのバッチの処理時間を調整することが必要である。 </li></ol>

<ol>
	<li>Wilkie, A. O., Morriss-Kay, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+craniofacial+development+and+malformation.">Genetics of craniofacial development and malformation.</a> Nat. Rev. Genet. 2, 458-468 (2001).</li><li>Gritli-Linde, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+2+-+The+Etiopathogenesis+of+cleft+lip+and+cleft+palate:+usefulness+and+caveats+of+mouse+models.">Chapter 2 - The Etiopathogenesis of cleft lip and cleft palate: usefulness and caveats of mouse models.</a> Current Topics in Developmental Biology. 84, 37-138 (2008).</li><li>Schutte, B. C., Murray, 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=The+many+faces+and+factors+of+orofacial+clefts.">The many faces and factors of orofacial clefts.</a> Human Molecular Genetics. 8, 1-7 (1999).</li><li>Chai, Y., Maxson, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+craniofacial+morphogenesis.">Recent advances in craniofacial morphogenesis.</a> Dev. Dyn. 235, 2353-2375 (2006).</li><li>Jiang, R., Bush, J. O., Lidral, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+upper+lip:+Morphogenetic+and+molecular+mechanisms.">Development of the upper lip: Morphogenetic and molecular mechanisms.</a> Dev. Dyn. 235, 1152-1166 (2006).</li><li>Reid, B. S., Yang, H., Melvin, V. S., Taketo, M. M., Williams, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectodermal+WNT/&#946;-catenin+signaling+shapes+the+mouse+face.">Ectodermal WNT/&#946;-catenin signaling shapes the mouse face.</a> Developmental Biology. 349, 261-269 (2011).</li><li>Feng, W., 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=Spatial+and+temporal+analysis+of+gene+expression+during+growth+and+fusion+of+the+mouse+facial+prominences.">Spatial and temporal analysis of gene expression during growth and fusion of the mouse facial prominences.</a> PLoS ONE. 4, e8066 (2009).</li><li>Chia, R., Achilli, F., Festing, M. F., Fisher, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origins+and+uses+of+mouse+outbred+stocks.">The origins and uses of mouse outbred stocks.</a> Nat. Genet. 37 (11), 1181-1186 (2005).</li><li>Nagai, T., Aruga, J., Takada, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+expression+of+the+mouse+Zic1,+Zic2,+and+Zic3+gene+suggests+an+essential+role+for+Zic+genes+in+body+pattern+formation.">The expression of the mouse Zic1, Zic2, and Zic3 gene suggests an essential role for Zic genes in body pattern formation.</a> Developmental Biology. 182, 299-313 (1997).</li><li>Britsch, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcription+factor+Sox10+is+a+key+regulator+of+peripheral+glial+development.">The transcription factor Sox10 is a key regulator of peripheral glial development.</a> Genes Dev. 15 (1), 66-78 (2001).</li></ol>

著者らは、競合する利益またはこの記事の内容に関連した他の競合はありません。

このプロトコルは、初期ディスパーゼIIの処理工程に基づいてマウス胎児顔面外胚葉と間充織を分離するための簡単​​な方法を提供します。以前の研究で、我々はディスパーゼIIの治療の前に顔のプロミネンスの解剖を行ったが、我々は一貫して間充織は &quot;スティッキー&quot;と操作することがより困難になることを発見した。私たちの新しいプロトコルは、この問題を回避できます。ピ?...

<table>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <td>Dispase II (neutral protease, grade II)</td>
 <td>Roche</td>
 <td>4942078001</td>
 <td>Make 10 mg/ml Dispase II stock solution in HBS. Aliquot and store at -20 &deg;C . </td>
 </tr>
 <td>RNAlater</td>
 <td>Ambion</td>
 <td>AM7021</td>
 <td>&nbsp;</td>
 </tr>
</table>

separation of mouse embryonic facial ectoderm and mesenchyme

口腔顔面裂は、世界中の千新生児1,2で1.5に影響を与える最も頻繁に顔面の欠陥である。口腔顔面分裂文形成が異常な顔の開発3によって引き起こされます。ヒトとマウスでは、顔の初期成長とパターン形成は、組織のいくつかの小さな芽、4,5顔面プロミネンスに依存しています。ペアの前頭鼻プロセス（FNP）、上顎プロミネンス（MXP）及び下顎プロミネンス（MDP）：顔が6メインプロミネンスから派生しています。これらのプロミネンスは、上面を覆う上皮に収められて間葉の腫れで構成されています。複数の種における研究では、顔の外胚葉と間充織間のシグナリングのクロストークが面6を形成するための重要であることが示されている。しかし、これらのシグナル伝達に関与する遺伝子のリレーに関する機序の詳細は不足している。一遺伝子発現の包括的な理解を得るための方法、結合転写因子、およびdeveloに関連付けられたクロマチンマーク顔面外胚葉と間充織にpingを実行すると、分離された組織区分を隔離し、特徴付けることである。 ここでは、胎生（E）は10.5、プロミネンスの融合の前にマウスの顔の形成に重要な発達段階で顔の外胚葉と間充織を分離するための方法を提示します。本手法は、我々が以前に顔のプロミネンス7を解剖に使用してきたアプローチから適応されている。この株は遺伝学、ゲノミクスおよび顔面形態8のための標準となっているように、この以前の研究では、近交系のC57BL / 6マウスを採用していた。ここでは、しかし、可能な組織のより数量限定のため、弊社では一切の近交系より腹あたり近交系のCD-1購入したほうが安い株を、畜産のためのより堅牢で、より多くの胚を生成する傾向がある（12-18）を利用してきたマウス系統8。胚の分離に続いて、中性プロテアーゼディスパーゼIIは、全胚を治療するために使用されていました。その後、顔のプロミネンスは解剖されたED出て、顔の外胚葉が間葉から分離した。この方法は、顔の外胚葉と間充織の両方をそのまま保持します。この方法論を用いて得られた試​​料は、タンパク質検出、クロマチン免疫沈降（ChIP）アッセイ、マイクロアレイ研究、およびRNA-seqを含む技術に使用することができます。

ディスパーゼIIの治療後、胚の外胚葉が緩んでいる傾向にある。顔のプロミネンスは解剖（ 図2A）した後、無傷でなければなりません。隔離された顔の外胚葉は、はっきりと間葉組織（ 図2B）は無料です。プロトコルの有効性を決定するために、我々は前脳についてアッセイし、交差汚染を検出するZic3 9、外胚葉特定CDH1 5と逆転写PCRを用いた間葉系特...

Watch this Scientific Journal Video about Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme at JoVE.com

Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme

胚顔面外胚葉と間充織を分離するためのプロトコルが記載されている。我々は、最初の全体の胚を扱う全体フェイシャルプロミネンスを解剖してから、顔の外胚葉と間充織を分離するためにディスパーゼIIを使用。

マウス胚性外胚葉顔と間葉の分離

 Orofacial clefts are the most frequent  craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal ...

separation-of-mouse-embryonic-facial-ectoderm-and-mesenchyme

Research

JoVE Journal

Biology

10.8K ビュー.  University of Colorado Denver Anschutz Medical Campus. 胚顔面外胚葉と間充織を分離するためのプロトコルが記載されている。我々は、最初の全体の胚を扱う全体フェイシャルプロミネンスを解剖してから、顔の外胚葉と間充織を分離するためにディスパーゼIIを使用。

ビデオ: Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). 

In this video, we demonstrate a method for electrophoretic separation of proteins using poly-acrylimide gel electrophoresis (PAGE).

タンパク質の電気泳動分離

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

生物学

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 ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

生体イメージングおよび卵巣癌の同所性マウスモデルにおける治療的処置の

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction analysis. One particular method to achieve this conditional deletion is via the use of the Cre-loxP system. This method involves flanking the gene of interest with loxP sites, which are specific recognition sequences for the Cre recombinase protein. Exposure of the so-called floxed (flanked by loxP site) DNA to this enzyme results in a Cre-mediated recombination event at the loxP sites, and subsequent excision of the intervening gene3. Several different methods exist to administer Cre recombinase to the site of interest. In this video, we demonstrate the use of an adenovirus containing the Cre recombinase gene to infect primary mouse embryonic fibroblasts (MEFs) obtained from embryos containing a floxed Rac1 allele1. Our rationale for selecting Rac1 MEFs for our experiments is that clear morphological changes can be seen upon deletion of Rac1, due to alterations in the actin cytoskeleton2,5. 72 hours following viral transduction and Cre expression, cells were stained using the actin dye phalloidin and imaged using confocal laser scanning microscopy. It was observed that MEFs which had been exposed to the adeno-Cre virus appeared contracted and elongated in morphology compared to uninfected cells, consistent with previous reports2,5. The adenovirus method of Cre recombinase delivery is advantageous as the adeno-Cre virus is easily available, and gene deletion via Cre in nearly 100% of the cells can be achieved with optimized adenoviral infection.

In this video we use an adenovirus carrying the Cre recombinase gene to infect primary mouse embryonic fibroblasts carrying a floxed Rac1 allele.

培養一次マウス胚線維芽細胞におけるシグナル伝達分子のアデノウイルス媒介遺伝子除去

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

マウス胎児の腎臓の腎性帯からの細胞の単離と培養

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic development 1, 2. One of the signaling pathways involved in this regulation is the Wnt pathway, which is used many times during embryogenesis and critical for human disease3, 4, 5. Multiple molecular components of this pathway coordinately regulate signaling in a spatially-restricted manner, but the underlying mechanisms are not fully understood. Xenopus embryonic epithelial cells is an excellent system to study subcellular localization of various signaling proteins. Fluorescent fusion proteins are expressed in Xenopus embryos by RNA microinjection, ectodermal explants are prepared and protein localization is evaluated by epifluorescence. In this experimental protocol we describe how subcellular localization of Diversin, a cytoplasmic protein that has been implicated in signaling and cell polarity determination6, 7 is visualized in Xenopus ectodermal cells to study Wnt signal transduction8. Coexpression of a Wnt ligand or a Frizzled receptor alters the distribution of Diversin fused with red fluorescent protein, RFP, and recruits it to the cell membrane in a polarized fashion 8, 9. This ex vivo protocol should be a useful addition to in vitro studies of cultured mammalian cells, in which spatial control of signaling differs from that of the intact tissue and is much more difficult to analyze.

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Xenopus embryonic ectoderm has become an attractive model for studies of cell polarity. An assay is described, in which subcellular distribution of fluorescent proteins is assessed in ectoderm cells. This protocol will help address questions related to spatial control of signaling.

の蛍光タンパク質の偏転アフリカツメガエルWntシグナルへの対応に>外胚葉

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic ...

Electroporation of Craniofacial Mesenchyme

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse 1-10. However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme.
When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis 11-13. In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation.
Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers 14. Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (&tilde;100 million years closer) 15. Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.

Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo.

顔面間葉のエレクトロポレーション

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

培養ヒト胚および誘導多能性幹細胞に適したマウス胚性線維芽細胞の調製

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

のためのシステム<em&gt; ex vivoで</em胚膵臓&gt;培養

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

頭蓋間葉の細胞挙動を評価するための外植片アッセイ

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

Methods Article

マウス胚性外胚葉顔と間葉の分離