作者要感谢艾琳在图1和实验室的其他成员的帮助和讨论，说明胚胎头彩。这项工作是由美国国立卫生研究院授予DE012728（TW）

1。准备Dispase酶II <ol><li>制备新鲜的盐水（HBS）的Hepes缓冲液（50mM HEPES / KOH pH为7.4，150mM的NaCl）。添加到16.8毫升超纯水H 2 O 2毫升0.5 M羟乙基哌嗪/ KOH pH值7.4和1.2毫升2.5 M氯化钠</li><li> 10毫克/毫升中性蛋白酶II。中性蛋白酶称取0.2 g II（罗氏，猫＃4942078001），和溶于20毫升哈佛商学院。准备在个别的Eppendorf管中并储存在-20℃的1.0毫升等分试样用于长期存储。 </li><li> 10毫克/毫升的中性蛋白酶II 1:10在PBS中稀释后再使用。将稀释后的中性蛋白酶II冰。 </li></ol> 2。解剖CD-1的E10.5胚胎发育从怀孕的女所有的动物实验，按照美国科罗拉多州丹佛大学（UCD）动物护理和使用委员会批准的协议。 CD-1（ICR）小鼠由哈伦实验室（印第安纳波利斯，IN）。 <ol><li>用70％乙醇清洁解剖区域和工具。 </li><li>安乐死怀孕CD-1女批准编辑协议。 </li><li>腹部喷用70％的乙醇，然后再打开腹部。使用一组产钳拉子宫，另一个系膜及子宫撕裂。从身体中取出子宫。重复另一侧子宫角的腹部。 </li><li>简单地用冰冷的PBS冲洗子宫，在10厘米的培养皿中洗出的血液。 </li><li>子宫转移到一个新的培养皿中，用冰冷的PBS。独立成一个胚胎碎片切割之间植入部位。 </li><li>在立体显微镜下，转移到冰冷的PBS中的一个胚胎。用细镊子撕断子宫肌壁的切割位点暴露的胚胎卵黄囊开始在。卵黄囊和羊膜剥离。解剖胚胎转移到6厘米的陪替氏培养皿，用PBS在冰上。继续，直到所有的胚胎已经以这种方式收集。 </li></ol> 3。中性蛋白酶II处理<ol><li>洗，PBS 6厘米的培养皿中的胚胎。更换PBS用10毫升在PBS中稀释的中性蛋白酶II。覆盖培养皿中，在37°C孵育25分钟-一个细菌培养箱或温暖的房间应该足够了-我们还没有使用的CO 2组织培养的孵化器这一步。 </li><li>培养皿中，在立体显微镜下观察胚胎。外胚层的应变得松散，此组织层和底层的间充质细胞为代表的一个明确的差距。如果没有，一个额外的5分钟，在37℃下孵育然后把培养皿放在冰块上。 </li></ol> 4。解剖出完整的面部日珥<ol><li>使用一次性的玻璃吸管（VWR目录号14672-380） 传送1 Dispase酶II处理胚胎到冰冷的PBS中在6厘米的陪替氏培养皿，在立体显微镜下</li><li>用一个钳子持有的胚胎。使用另一对细钳子仔细切割，附着在鱼头面部日珥（ 图1示出的边界的边界面部突起）。从MnP的一侧的，那么MXP，和最后的FNP开始。到另一侧做同样的解剖出完整的面部突起（ 图2A）。 </li></ol> 5。独立的脸外胚层分化而来间充质<ol><li> 3倍工作距离体视显微镜下观察面部的突起。使用长玻璃巴斯德吸管轻轻地移液的面部突起向上和向下的5倍。在此之后，外胚层易于剥离。 </li><li>用一个钳子持有的面部突起。使用其他对镊子剥离外胚层非常缓慢。 图2B示出面部外胚层分离的片材。 </li><li>使用一个新的长玻璃巴斯德吸管面部外胚层转移到1.5 ml Eppendorf管中，用PBS在冰上。的间充质细胞转移到另一个1.5 ml Eppendorf管中，用PBS在冰上。 </li><li>使用新的冰冷的PBS为每个解剖。收集样本。 </li><li>洗净吨他样品用PBS。离心机在4℃，500×g离心3分钟。 </li><li>使用长玻璃巴氏吸管吸取PBS。如果用于芯片检测样品，进行步骤6.1。对于RNA的提取，进行步骤6.2。对于蛋白质的提取，进行步骤6.3。 </li></ol> 6。进一步的实验过程中样品<ol><li> ChIP实验样品<ol><li>加入1毫升的PBS，用1％甲醛的交联。均质样品。 </li><li>与旋转的10分钟，在室温下孵育。 </li><li>在每个管中加入110微升的1.25M的甘氨酸，淬灭未反应的甲醛。 </li><li>混合，并在室温下孵育5分钟。 </li><li>附带在4℃下在500×g离心3分钟。除去上清液。 </li><li>加入1ml冷PBS到洗的组织。 </li><li>附带在4℃下在500×g离心3分钟。 </li><li>删除PBS和重复PBS洗一次。 </li><li>去除上清液，糟糕冻结组织在液氮中。存储SAmples在-80℃下为后续池和处理。 </li></ol></li><li>样品提取RNA <ol><li>将面部外胚层和间充质在RNAlater溶液（Ambion公司），管中含有。 </li><li>孵育在RNAlater溶液4℃过夜，以允许彻底渗透的组织样本。 </li><li>随后池和处理在-20℃下储存。 </li></ol></li><li>蛋白提取样品</li></ol>裂解缓冲液加入到样品中，供后续处理。急速冷冻在液氮中的组织。将样品储存在-80℃下为后续池和处理。 笔记<ol><li>时间限制。计划在4-6小时，从一开始就通过解剖开始的第6条。 </li><li>当精通，约需10-15分钟，每胚胎执行第4和第5（突出解剖和外胚层分离）。 </li><li>如果样品是RNA分离，每个搜寻解决方案n应该被视为第一焦碳酸二乙酯。同时清洁钳和工作区用RNase客场。 </li><li> Dispase酶II的治疗和解剖除了冰在任何时候都保持样品。每个解剖，以确保用于解剖的PBS是冷的，切换到新的冰冷的PBS。 </li><li>夏普的的镊子是关键，以获得精确的解剖。完全清洁的钳子，以防止任何污染。 </li><li>从不同的日珥收集样本，分别剖析每一个突出中性蛋白酶II处理后。然后分离外胚层和间充质细胞。 </li><li>治疗后中性蛋白酶II，面部外胚层容易脱落。要特别注意从其他外胚层污染。放弃所有可能受污染的组织。 </li><li>一旦你开始，不要停止直到样品达到第6章中的适当位置。完成所有的胚胎分离的面部外胚层和间充质迅速，你可以。 </li><li>电子nzyme，Dispase酶II的活动可能会有所不同批次之间和品牌。这是必要的，调整的处理时间为不同品牌和批次的中性蛋白酶II。 </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>

作者有没有利益冲突或其他冲突与本文内容相关的。

该协议提供了一个简单的方法来分离面部小鼠胚胎外胚层和间充质的基础上Dispase酶II的初步处理步骤。在以前的研究中，我们进行了解剖的面部突起之前，中性蛋白酶II处理，但我们一直发现，间充质变成“粘”，更难以操纵。我们的新的协议，避免了这个问题。结合中性蛋白酶II处理与柔和的身体的力量，通过上下吹打分离的外胚层和间充质更容易。此外，该协议还可以很好地用于分离的胚胎外...

<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

口面裂是最常见的颅面畸形，从而影响1.5在1000新生儿全球1,2。颜面部clefting引起的脸部发育异常3。在人类和小鼠中，最初的增长和图案的脸依赖于几个小芽组织，面部突起4,5。面对来自6个主要的日珥：配对正面的鼻流程（FNP），上颌骨突起（MXP）和下颌骨突起（MDP）。这些日珥包括装在覆上皮细胞肿胀，间质细胞，。在多个物种的研究表明，信号之间的串扰面部外胚层和间充质是至关重要的塑造脸部6。然而，机械的细节关于这些信号继电器中所涉及的基因所缺乏的。的方式来获得一个全面的了解基因表达的转录因子结合，及相关的染色质标记的开发与平面外胚层和间充质细胞的分离和鉴定分离的组织车厢。 在这里，我们提出了一个方法，分离面外胚层和间充质细胞在胚胎天（E）10.5鼠标的面部的形成，融合的日珥之前，一个关键的发展阶段。我们的方法是适用的方法，我们以前用于解剖面部突起7。在这个早期的研究中，我们采用近交系C57BL / 6小鼠作为这株已成为标准的遗传学，基因组学和面部形态。在这里，虽然，由于可用组织更有限的数量，我们已利用远交的CD-1菌株，购买更便宜，更健壮的畜牧业，并趋向于产生更多的胚胎（12-18）比任何自交系每窝小鼠品系8。在胚胎隔离，中性蛋白酶中性蛋白酶用于治疗II全胚胎。然后，面部突起剖析编出来，面部外胚层分离的间充质细胞。这种方法保持完整的面部外胚层和间质。可用于蛋白质检测，染色质免疫沉淀法（ChIP）的检测，基因芯片的研究，和RNA-seq的技术，包括使用这种方法获得的样品。

中性蛋白酶II处理后的胚胎的外胚层往往是松散的。经过解剖（ 图2A）的面部突起应该是完整的。孤立的面部外胚层是明确的，免费的间充质组织（ 图2B）。的协议，以确定疗效和检测交叉污染检测前脑表达Zic3 9，外胚层具体的CDH1和间质特定的SOX10 10采用逆转录聚合酶链反应。我们的研究证实，这两个面部外胚层和间充质细胞表达预期的基因和无交叉污染...

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

外胚层和间充质细胞的胚胎面部分离的协议。我们使用的是中性蛋白酶II对待整个胚胎首先，剖析整个面部突起，然后分离的面部外胚层和间充质细胞。

分离的小鼠胚胎面外胚层和间充质

 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 次观看.  University of Colorado Denver Anschutz Medical Campus. 外胚层和间充质细胞的胚胎面部分离的协议。我们使用的是中性蛋白酶II对待整个胚胎首先，剖析整个面部突起，然后分离的面部外胚层和间充质细胞。

视频: 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).

电泳分离蛋白质

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

科研

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.

小鼠胚胎生殖细胞的分离和推导

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.

活体成像和治疗卵巢癌的原位小鼠模型

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.

腺病毒介导的基因去除信号分子在原代培养的小鼠胚胎成纤维

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.

从小鼠胚胎肾肾区细胞的分离和培养

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.

偏光易位荧光蛋白爪蟾外胚层响应Wnt信号

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.

颅面间质细胞的电

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

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51&plusmn;0.8% of differentiation efficiency (n = 3; p &lt; 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

高效的小鼠胚胎干细胞分化成运动神经元

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...

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.

适用于培养人类胚胎和诱导多能干细胞的小鼠胚胎成纤维细胞的制备

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.

一个系统<em&gt;体外</em培养的胚胎胰腺

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