この作品は、R01 - DE018234とR01 - DE019638によって賄われていた。

1。ドナー組織の準備<ol><li>培地を準備し、ガラスのピンをシャープに、タングステンの針を強めています。 </li><li>シェルから胚を回収、氷冷PBSで洗う。 <ol class="lalpha"><li> 10mLのシリンジと18ゲージの針を使用すると、卵の殻の先端からアルブミン1.0 mLを削除します。 </li><li>はさみのポイントを使用してシェルの上に小さな穴を確認し、胚を露出させる円形の開口部をカット</li></ol></li><li> DMEM（無血清、室温）に頭と場所を削除します。 </li><li>胚（またはそのような上顎または下顎のプロセスなど、他のドナーサイト）からFrontonasalプロセスを細かく分析</li><li> 20分氷上でPBSの2.4U / mLのディスパーゼのダイジェスト</li><li>消化を停止するには、1％BSAを含むDMEMでティッシュカバー</li><li>別の外胚葉、間充織、および神経外胚葉に尖ったタングステン針を使用して、 </li><li>ホストは、移植の準備ができるまで、氷上でDMEM、1％BSAで移植組織を保存する</li></ol> 2。ホストの準備<ol><li>胚を公開<ol class="lalpha"><li> 10mLのシリンジと18ゲージの針を使用すると、卵の殻の先端からアルブミン1.0 mLを削除します。 </li><li>はさみのポイントを使用してシェルの上に小さな穴をあけます。穴の上のテープを配置して、胚を露出させる円形の開口部をカット。 </li></ol></li><li>鉗子の2組を使用して、羊膜を持ち、ゆっくりと穴を作るために引き裂く。 </li><li>右眼にピンセットを配置し、圧力をかけて頭を回転させる。頭を固定し、移植片を収容するために、ホスト外胚葉を除去するためにタングステン針を使用。 </li><li>ガラスピペットを使用してホストに移植片を移す。 </li><li>削除された外胚葉を交換する移植片を置きます。場所に移植片を固定するために移植された組織の各コーナーでガラスのピンを挿入します。ガラスのピンを作成するには、アルコールの炎の細かい点にマイクロキャピラリーチューブを引き出します。 </li><li>穴にしっかりとテープを配置し、分析のために適切な時間のためにインキュベーターに胚を戻す。 </li><li>も参照してください：9 </li></ol> 3。代表的な結果： キメラを評価するために、胚を回収し、ホストとドナー組織の分布の分析のために処理されなければなりません。ウズラ細胞の検出、QcPNの抗体を用いた免疫組織化学が（で説明されている：3）採用されている、とマウス細胞の検出のための、パラフィン切片上でin situハイブリダイゼーションで 6が使用されます。ドナー細胞は、上皮に限定すべきである、とドナー間葉系組織（図1）の証拠があってはならない。間充織での孤立したドナー細胞の存在は、顔の開発の多くの側面（例えば、10）上でこれらの細胞の影響により、任意の形態学的解釈を混乱させるだろう汚染神経堤細胞の存在を示している。一度移植技術は間充織の汚染がないことを確信し、さらに形態学的または分子のアウトカム指標は、キメラを評価するために使用することができます。我々の目的のために、我々は（図3）グラフト化された外胚葉への応答における移植組織（図2）、及び間葉組織中の分子の変化によって誘発された上顎の重複に対応する異所性軟骨や骨の存在を検出5,6。 <img src="/files/ftp_upload/2557/2557fig1.jpg" alt="図1" /> 図1。キメラ現象の評価 （）アンチQcPN染色は、ウズラ、ニワトリのキメラでウズラの細胞を検出するために使用されます。間葉で染色の証拠はない。 （B）in situハイブリダイゼーションは、マウス、ニワトリのキメラでSINEのB2の転写産物の発現を検出するために使用されます。式は、移植片を構成する外胚葉に制限されます。 <img src="/files/ftp_upload/2557/2557fig2.jpg" alt="図2" /> 図2。トリクローム染色では、（A）ウズラ-ニワトリキメラの上顎の骨格の複製、及び（B）マウス、ニワトリキメラを示しています。 <img src="/files/ftp_upload/2557/2557fig3.jpg" alt="図3" /> 図3外胚葉は、間充織での遺伝子発現をregultes。ニワトリ胚の間充織の（A）ノーマルBMP7発現（Adobe Photoshopの正の信号擬似色の赤とin situハイブリダイゼーション放射性）。 （B）BMP7の発現は、キメラ胚の移植片（矢印）に隣接する間充組織に誘導される。

<ol>
	<li>Noden, D. 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+Role+of+the+Neural+Crest+in+Patterning+of+Avian+Cranial+Skeletal,+Connective,+and+Muscle+Tissues.">The Role of the Neural Crest in Patterning of Avian Cranial Skeletal, Connective, and Muscle Tissues.</a> Developmental Biology. 96, 144-144 (1983).</li><li>Bronner-Fraser, M., Stern, C. <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+Mesodermal+Tissues+on+Avian+Neural+Crest+Cell+Migration.">Effects of Mesodermal Tissues on Avian Neural Crest Cell Migration.</a> Developmental Biology. 143, 213-213 (1991).</li><li>Schneider, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+can+form+cartilages+normally+derived+from+mesoderm+during+development+of+the+avian+head+skeleton.">Neural crest can form cartilages normally derived from mesoderm during development of the avian head skeleton.</a> Developmental Biology. 208, 441-441 (1999).</li><li>Couly, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+Hox-negative+cephalic+neural+crest+cells+and+the+foregut+endoderm+in+patterning+the+facial+skeleton+in+the+vertebrate+head.">Interactions between Hox-negative cephalic neural crest cells and the foregut endoderm in patterning the facial skeleton in the vertebrate head.</a> Development. 129, 1061-1061 (2002).</li><li>Evans, D. J., Noden, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+relations+between+avian+craniofacial+neural+crest+and+paraxial+mesoderm+cells.">Spatial relations between avian craniofacial neural crest and paraxial mesoderm cells.</a> Dev Dyn. , (2006).</li><li>Hu, D., Marcucio, R., Helms, J. 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+zone+of+frontonasal+ectoderm+regulates+patterning+and+growth+in+the+face.">A zone of frontonasal ectoderm regulates patterning and growth in the face.</a> Development. 130, 1749-1749 (2003).</li><li>Hu, D., Marcucio, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+organization+of+the+frontonasal+ectodermal+zone+in+birds+and+mammals.">Unique organization of the frontonasal ectodermal zone in birds and mammals.</a> Dev Biol. 325, 200-200 (2009).</li><li>Le Li&#232;vre, C. S., Le Douarin, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+derivatives+of+the+neural+crest:+analysis+of+chimaeric+quail+and+chick+embryos.">Mesenchymal derivatives of the neural crest: analysis of chimaeric quail and chick embryos.</a> Journal of Embryology and Experimental Morphology. 34, 125-125 (1975).</li><li>Bollag, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+repetitive+mouse+B2+element+to+identify+transplanted+mouse+cells+in+mouse-chick+chimeras.">Use of a repetitive mouse B2 element to identify transplanted mouse cells in mouse-chick chimeras.</a> Exp Cell Res. 248, 75-75 (1999).</li><li>Korn, M. J., Cramer, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Windowing+chicken+eggs+for+developmental+studies.">Windowing chicken eggs for developmental studies.</a> J Vis Exp. , (2007).</li><li>Eames, B. F., Schneider, R. 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利害の衝突は宣言されません。

この移植の方法を使用すると、私たちは外胚葉の背腹極性と上顎のproximodistal拡張を調節するシグナル伝達情報が含まれていることを決定することができました。ウズラやマウスの外胚葉、および多くの種6,11の間でこの組織の分子シグナルの保全を使用して結果の類似性は、これは脊椎動物間で高度に保存されたシグナル伝達の中心であることを示します。さらに、他の研究者らは、表面頭部外胚葉の異なる領域の信号特性をテストするために同様の技法を使用しているし、面12の形態形成に貢献する外胚葉に位置する複数のシグナル伝達の中心の存在を明らかにしました。このメソッドは、私たちは、異なる組織だけでなく、組織内の地域的な違いは、顔の開発時に果たす役割についての理解を促進することができます。最後に、鳥 - キメラシステムの強さとマウス遺伝学の強みを組み合わせることで我々は開発中の上皮 - 間葉相互作用の分子メディエーターを解明することができる、と我々はこれらの相互作用を調節するメカニズムを特定できるようになります開発。

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assessing signaling properties of ectodermal epithelia during craniofacial development

鳥類胚のアクセシビリティは、実験的な発生学者が開発し、パターニングし、脊椎動物の形態形成（例えば、1、2、3、4）規制組織の相互作用の役割の間に細胞の運命を理解して支援してきました。ここで、我々は、顔の開発中に外胚葉組織のシグナリングとパターニング特性をテストするには、このアクセ​​シビリティを悪用する方法を示しています。これらの実験では、我々は、同じ地域やニワトリ胚の異所性の領域のいずれかの上にウズラやマウスからの上顎を覆う表面頭部外胚葉を移植することにより、ウズラ、ニワトリ5またはマウス-ひよこ6キメラを作成します。雛への移植のドナー組織としてのウズラの使用は、このように研究者がホストとドナーの組織7を区別できるように、ウズラの存在ではなく、ニワトリの細胞核マーカーを活用するために開発されました。私たちはマウスニワトリキメラ8のホストとドナーの組織を区別できるようになる、同様に、反復的な要素は、マウスのゲノムに存在し、遍在的に発現される。これは、私たちは様々な変異体胚に由来する外胚葉のシグナル伝達の特性をテストすることができますので、ドナーの組織としてのマウスの外胚葉の使用は大幅に、これらの組織の相互作用の理解を拡張します。

Watch this Scientific Journal Video about Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development at JoVE.com

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

この記事では、頭蓋顔面の開発中にシグナリングと表面頭部外胚葉のパターニング特性をテストするために設計された組織移植の手法を説明します。

頭蓋顔面の開発時には外胚葉上皮のシグナリング特性を評価する

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue ...

assessing-signaling-properties-ectodermal-epithelia-during

Nanyang Technological University

Research

JoVE Journal

Biology

9.4K ビュー.  University of California San Francisco. この記事では、頭蓋顔面の開発中にシグナリングと表面頭部外胚葉のパターニング特性をテストするために設計された組織移植の手法を説明します。

ビデオ: Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

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. ...

生物学

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

髪の再生の間に上皮間葉シグナル伝達の迅速遺伝子解析

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

ゼブラフィッシュの開発時Photoconvertibleタンパク質を用いた細胞トラッキング

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

胚発生時に細胞運命のコミットメントをテストするために割球植

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

細胞単層に機械的刺激によって誘発されるカルシウム波の伝播：ウシ角膜内皮細胞の例

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

SOX10中の顔面の開発の可視化：楓トランスジェニックゼブラフィッシュラインタイムラプス共焦点顕微鏡を用いて

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

比較プロテオーム解析のための代謝標識および膜分画<em&gt;シロイヌナズナ</em&gt;サスペンション細胞培養

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

外植片培養での歯胚の開発に続いてのためのスライス培養法

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

ヒト多能性のための代替培養はセル生産、メンテナンス、および遺伝子解析幹

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to the histological and morphological development of vertebrate embryos. In particular, the use of chimeric embryos has established the importance of neural crest in directing the species-specific morphology of the craniofacial complex. The method described herein utilizes two avian species, duck and quail, with remarkably different craniofacial morphology. This method greatly facilitates the investigation of molecular and cellular regulation of species-specific pattern in the craniofacial complex.&#160;Experiments in quail and duck chimeric embryos have already revealed neural crest-mediated tissue interactions and cell-autonomous behaviors that regulate species-specific pattern in the craniofacial skeleton, musculature, and integument. The great diversity of neural crest derivatives suggests significant potential for future applications of the quail-duck chimeric system to understanding vertebrate development, disease, and evolution.

This article describes a method to generate chimeric embryos that are&#160;designed to test the species-specific contributions of neural crest and/or other tissues to craniofacial development.

ウズラ·アヒルキメラを使用して顔面発展に種特異的な貢献を評価する

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to ...