作者要感谢迪尔德丽·尼尔森博士的有益的意见和批评性阅读的手稿。这项工作是由美国国立卫生研究院拨款DE019244，DE019197，和DE021841 ML，F32DE02098001圣雅各福群会，和C06 RR015464大学奥尔巴尼分校，纽约州立大学。

该协议包含四个主要的步骤，在图1中所描绘的。所有步骤都全面详细的描述。腺病毒载体建设和病毒纯化，应提前进行的活体摘取器官在基因转导的解剖上皮细胞的雏形。所有BSL-2标准的安全防范措施时，应遵循工作的腺病毒。 1。小鼠胚胎颌下腺（SMG）收获和显微切割<ol><li>安乐死定时的怀孕雌性小鼠（封闭群CD-1或ICR）使用CO 2昏迷状态，其次是颈椎错位和收获字符串的小鼠胚胎在胚胎13天（E13，3-5上皮芽）以下程序批准的机构IACUC委员会。阴道塞发现这一天被指定为E0。在E12阶段时，有一个单独的主芽和茎裂刚刚开始，唾液腺也有所收获，并培养启动。冲锋枪在此之前的阶段需要不同的培养条件下比在此间表示。 </li><li>转移胚胎字符串到无菌的10厘米组织文化的菜肴装有25毫升的DMEM /火腿的F12中（F12）无酚红（Life Technologies公司），辅以100ü/ ml青霉素，和100微克/毫升链霉素（笔，链球菌， Life Technologies公司）。 </li><li>用无菌手术刀（11片扇叶），镊子（＃5，精细的科学工具，11252-20），从囊中取出胚胎成一个独立的10厘米菜含有25毫升的DMEM/F12 +笔链球菌。 </li><li> SEVER的的胚胎头与下颚的正下方有角度的切割。 </li><li>下一个立体解剖显微镜与透射光底座（尼康SMZ645或同等学历）使用切割低于上颌骨，下颌骨的头低隔离。将顶部的一个额外的一块玻璃上，而不是直接在显微镜基础的玻璃成分的组织。 </li><li>收集下颌骨3毫升DMEM/F12 +笔链球菌在无菌的35 mm组织培养皿中。 </li><li>一个颚切片放置在解剖显微镜载物台上，使舌是切片的底部上，但指向12:00点钟位置。 </li><li>使用两个交叉钳在剪运动的一面，删除和丢弃的下1/3 路的下颌骨片。 </li><li>将片90°的右侧（右手），使用的钳子剪切一半切片，切下颌骨片的顶部（不切割的舌头）。 </li><li>剥离多余的组织，并删除它，露出下面的冲锋枪。会有一个舌根附近的每一侧上。 </li><li>用一个钳子按住组织的舌头，使用产钳的舌头挑逗SMG距离。重复的其他腺体。 </li><li>到一个新的无菌35mm组织分为3毫升的DMEM/F12 +笔-链球菌收藏报错显微切割的唾液腺培养皿中。注：较大的颌下腺（3-5上皮芽）与关连较小的舌下腺（1芽）是可以培养的完整跳过步骤4和执行步骤4.1，4.3，4.4，如前所述，2,8。 </li></ol> 2。 SMG上皮萌芽分离<ol><li>地点5（或更多）唾液腺腺体到玻璃底部的，50毫米直径微孔培养皿（MatTek公司，P50G-1.5-14-F）含有200微升的Hank氏平衡盐溶液（HBSS中缺乏钙+ +或镁+ +， Life Technologies公司），含有0.4％（体积/体积）分散酶（Life Technologies公司，目录号17105-041），并孵育15分钟，在37℃下</li><li>温育后，用200μl的无菌移液管尖，小心吸出分散酶溶液，而不会干扰腺体。立即加入200微升的DMEM/F12培养基含有5％（重量/体积）BSA（DMEM/F12-BSA用0.22μm的过滤器，预先灭菌的），以中和分离酶。 </li> &lt;李在解剖显微镜下，用钳子细提示（＃5 Dumostar，精细的科学工具，11295-20），轻轻分开松动的间充质细胞各地SMG上皮芽和管道，照顾离开上皮细胞的完整性。 </li><li>预湿的无菌200微升移液器吸头与DMEM/F12-BSA媒体，轻轻移液管的上皮细胞到一个新的50毫米直径的微孔菜含有200μl的DMEM/F12 +笔链球菌的雏形。使用高品质的200微升移液器吸头与平滑的边缘。 </li><li>在菜含有间质片，丢弃任何非间充质组织的舌下腺和颌下腺任何分离的芽。 </li></ol> 3。腺病毒感染的上皮雏形<ol><li> 200微升稀释在DMEM/F12 +笔链球菌在一个离心管中，使所得到的溶液的滴度为1×10 8 -10 9 PFU就能景点的腺病毒的病毒感染的媒体。 “不同的病毒所需的最佳的效价可能会有所不同。重要的是要使用氯化铯（CsCl）纯化的病毒制剂的最佳吸收效率。 </li><li>从盘子里取出媒体包含5个上皮细胞的雏形。快速加入200μl的感染媒介，在任何时候都保持潮湿的雏形。采取必要的预防措施，在处理病毒和处置受污染的枪头。 10％的漂白粉溶液消毒任何被病毒污染的表面。 </li><li>将上皮细胞的雏形远从彼此用钳子夹，以防止它们粘在一起，使他们能够解决的盘底。在病毒性媒体，在室温下的1小时的孵育雏形。在培育，分离的雏形，如果他们靠拢在一起。过度摇摆或运动将使腺体聚集在一起。 </li><li>培养结束后，温和地去除病毒的媒体和丢弃适当，小心不要破坏了Rudiments。加入200μlDMEM/F12 +笔链球菌。重复此清洗至少两次，并更换200μl的DMEM/F12 +笔链球菌媒体。这些洗是关键，以消除任何病毒，不连接的上皮和间质，以防止感染。 </li><li>孵育上皮雏形与200毫升的DMEM/F12中含有2％（体积/体积）的基质胶（BD Biosciences公司，356231）20分钟。我们发现，这在Matrigel的潜伏期短，最大限度地减少了现有裂在最初的文化回归，但这个步骤缺一不可，如果不希望暴露在Matrigel的。 </li></ol> （4） 体外培养重组冲锋枪<ol><li>放置每个雏形，一次一个地，使用预湿的200μl的移液管尖，在顶部的切口（小切口）13毫米，0.1微米核孔履带蚀刻膜过滤器（Whatman）超过200μl的培养基（漂浮入门/过滤器）在玻璃底的微孔板。文化传媒是：含有50微克/毫升转铁蛋白和150微克/毫升的抗坏血酸，如前所述2,8的DMEM/F12 +笔-链球菌。转铁蛋白是众所周知的刺激的生存和分支的唾液腺等器官外植体的形态发生9。添加抗坏血酸已证明刺激SMG分支。10是已知的作为一个辅助因子在许多羟化反应的代谢途径（如脯氨酸羟化胶原纤维形成11期间），也刺激其他器官的外植体中的纤连蛋白和层粘连蛋白的生产12。 </li><li>后移除尽可能多的媒体之上的过滤器尽可能增加每个上皮雏形。太多媒体的过滤器上，可能会导致过滤器下沉，而较少的媒体位置的雏形和间充质更容易。可以去除多余的介质，使用了一套吸头或用钳子夹住媒体之间的技巧。 </li><li>地点间充质件的来回米3至4个腺体的顶部和/或围绕每个上皮雏形。删除任何额外的介质，周围的腺体，间质，以避免运动距离的雏形。间充质来自更腺体的是没有好处的，但不太可能会损害上皮雏形增长。 </li><li>孵育重组冲锋枪可增湿培养箱中（95％空气/ 5％CO 2）在37℃下为48 -72小时，根据需要。 </li><li>收购明场和/或荧光图像的活腺体0小时（如果需要），重组后2小时，每24小时后倒置，直立，或立体光学显微镜配备了一台数码相机使用了4倍或5倍放大倍率的目标是在一个单一的框架来看，捕捉整个重组腺。唾液腺的图像显示使用明场设置或适当的相环的光路（上完全自动化的显微镜，这是不可能的）部分进入最佳的对比度。标准相差不吨必要的，因为腺体很厚，产生的对比。 </li><li>在期望的时间点可以是固定的20分钟制成的2％多聚甲醛（PFA）的1X PBS中含有5％（重量/体积）蔗糖（从而更好地保持的保持的细胞在细胞内部结构与固定液，重组冲锋枪等渗状态）。不同于4％PFA，2％PFA将维持一个显着量的GFP信号，如果固定剂固定后完全除去。腺体，可存储长达1周1X PBS在黑暗中，在4°C，直到整个安装进行免疫细胞化学和激光共聚焦成像，如先前报道3,6,13-15。理想的情况下，免疫细胞化学和成像应尽快进行固定后，以获得最佳的图像质量。腺体也可以在RIPA缓冲液中溶解，然后通过Western印迹法分析进行SDS-PAGE。 </li></ol>

<ol>
	<li>Tucker, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salivary+gland+development.">Salivary gland development.</a> Seminars in cell &amp; developmental biology. 18, 237-244 (2007).</li><li>Sakai, T., Onodera, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+organ+culture.">Embryonic organ culture.</a> Curr. Protoc. Cell Biol. Chapter 19, Unit 19 (2008).</li><li>Patel, V. N., Rebustini, I. T., Hoffman, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salivary+gland+branching+morphogenesis.">Salivary gland branching morphogenesis.</a> Differentiation. 74, 349-364 (2006).</li><li>Sequeira, S. J., Larsen, M., DeVine, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+and+growth+factors+in+salivary+gland+development.">Extracellular matrix and growth factors in salivary gland development.</a> Frontiers of oral biology. 14, 48-77 (2010).</li><li>Zheng, C., 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=Transient+detection+of+E1-containing+adenovirus+in+saliva+after+the+delivery+of+a+first-generation+adenoviral+vector+to+human+parotid+gland.">Transient detection of E1-containing adenovirus in saliva after the delivery of a first-generation adenoviral vector to human parotid gland.</a> J. Gene Med. 12, 3-10 (2010).</li><li>Larsen, M., 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=Role+of+PI+3-kinase+and+PIP3+in+submandibular+gland+branching+morphogenesis.">Role of PI 3-kinase and PIP3 in submandibular gland branching morphogenesis.</a> Developmental biology. 255, 178-191 (2003).</li><li>Hoffman, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiles+of+mouse+submandibular+gland+development:+FGFR1+regulates+branching+morphogenesis+in+vitro+through+BMP-+and+FGF-dependent+mechanisms.">Gene expression profiles of mouse submandibular gland development: FGFR1 regulates branching morphogenesis in vitro through BMP- and FGF-dependent mechanisms.</a> Development. 129, 5767-5778 (2002).</li><li>Rebustini, I. T., Hoffman, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ECM+and+FGF-dependent+assay+of+embryonic+SMG+epithelial+morphogenesis:+investigating+growth+factor/matrix+regulation+of+gene+expression+during+submandibular+gland+development.">ECM and FGF-dependent assay of embryonic SMG epithelial morphogenesis: investigating growth factor/matrix regulation of gene expression during submandibular gland development.</a> Methods Mol. Biol. 522, 319-330 (2009).</li><li>Partanen, A. M., Thesleff, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transferrin+and+tooth+morphogenesis:+retention+of+transferrin+by+mouse+embryonic+teeth+in+organ+culture.">Transferrin and tooth morphogenesis: retention of transferrin by mouse embryonic teeth in organ culture.</a> Differentiation. 34, 25-31 (1987).</li><li>Naka, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+branching+morphogenesis+of+fetal+mouse+submandibular+gland+by+sodium+ascorbate+and+epigallocatechin+gallate.">Modulation of branching morphogenesis of fetal mouse submandibular gland by sodium ascorbate and epigallocatechin gallate.</a> In Vivo. 19, 883-888 (2005).</li><li>Bornstein, P., Traub, W., Neurath, H., Hill, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemistry+and+biology+of+collagen.">The chemistry and biology of collagen.</a> The Proteins. 4, 412-632 (1979).</li><li>Zhou, L., Higginbotham, E. J., Yue, B. Y. <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+ascorbic+acid+on+levels+of+fibronectin,+laminin+and+collagen+type+1+in+bovine+trabecular+meshwork+in+organ+culture.">Effects of ascorbic acid on levels of fibronectin, laminin and collagen type 1 in bovine trabecular meshwork in organ culture.</a> Curr. Eye Res. 17, 211-217 (1998).</li><li>Daley, W. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROCK1-directed+basement+membrane+positioning+coordinates+epithelial+tissue+polarity.">ROCK1-directed basement membrane positioning coordinates epithelial tissue polarity.</a> Development. 139, 411-422 (2012).</li><li>Daley, W. P., Gulfo, K. M., Sequeira, S. J., Larsen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+mechanochemical+checkpoint+and+negative+feedback+loop+regulating+branching+morphogenesis.">Identification of a mechanochemical checkpoint and negative feedback loop regulating branching morphogenesis.</a> Developmental biology. 336, 169-182 (2009).</li><li>Sequeira, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulation+of+focal+adhesion+complex+formation+and+salivary+gland+epithelial+cell+organization+by+nanofibrous+PLGA+scaffolds.">The regulation of focal adhesion complex formation and salivary gland epithelial cell organization by nanofibrous PLGA scaffolds.</a> Biomaterials. 33, 3175-3186 (2012).</li><li>Nogawa, H., Mizuno, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+control+over+elongating+and+branching+morphogenesis+in+salivary+gland+development.">Mesenchymal control over elongating and branching morphogenesis in salivary gland development.</a> Journal of embryology and. 66, 209-221 (1981).</li><li>Sakai, T., Larsen, M., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibronectin+requirement+in+branching+morphogenesis.">Fibronectin requirement in branching morphogenesis.</a> Nature. 423, 876-881 (2003).</li><li>Daley, W. P., Kohn, J. M., Larsen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+focal+adhesion+protein-based+mechanochemical+checkpoint+regulates+cleft+progression+during+branching+morphogenesis.">A focal adhesion protein-based mechanochemical checkpoint regulates cleft progression during branching morphogenesis.</a> Developmental dynamics : an official publication of the American Association of Anatomists. 240, 2069-2083 (2011).</li><li>Knox, S. M., 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=Parasympathetic+innervation+maintains+epithelial+progenitor+cells+during+salivary+organogenesis.">Parasympathetic innervation maintains epithelial progenitor cells during salivary organogenesis.</a> Science. 329, 1645-1647 (2010).</li><li>Larsen, M., Wei, C., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+and+fibronectin+dynamics+during+branching+morphogenesis.">Cell and fibronectin dynamics during branching morphogenesis.</a> J. Cell Sci. 119, 3376-3384 (2006).</li><li>Wei, C., Larsen, M., Hoffman, M. P., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-organization+and+branching+morphogenesis+of+primary+salivary+epithelial+cells.">Self-organization and branching morphogenesis of primary salivary epithelial cells.</a> Tissue Eng. 13, 721-735 (2007).</li><li>Zheng, C., Baum, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+viral+and+mammalian+promoters+for+use+in+gene+delivery+to+salivary+glands.">Evaluation of viral and mammalian promoters for use in gene delivery to salivary glands.</a> Mol. Ther. 12, 528-536 (2005).</li><li>Hsu, J. C., 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=Viral+gene+transfer+to+developing+mouse+salivary+glands.">Viral gene transfer to developing mouse salivary glands.</a> J. Dent. Res. 91, 197-202 (2012).</li></ol>

没有利益冲突的声明。

上皮-间质体外重组技术首次公布于1981年16颌下唾液腺。在这个协议中，我们扩大后，原来的方法，使用腺病毒感染的操作范围内的重组腺上皮细胞基因的表达。我们表明，与腺病毒感染的上皮细胞的百分比，而被感染的细胞的百分比取决于病毒启动子的性质，病毒滴度，和病毒纯度。我们已经发现，有必要使用CsCl梯度纯化的高效上皮细胞转导，其中不在这里描述的纯化的病毒。既...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>无酚红DMEM /火腿的F12中</td><td> Life Technologies公司</td><td> 21041-025 </td><td></td></tr><tr><td>青霉素和链霉素</td><td> Life Technologies公司</td><td> 15070-163 </td><td> 10倍的股票</td></tr><tr><td>中性蛋白酶</td><td> Life Technologies公司</td><td> 17105-041 </td><td>冻结一次性使用等份-20C </td></tr><tr><td> BSA </td><td>西格玛</td><td> A2934-100G </td><td>分数V，低内毒素</td></tr><tr><td>腺-X-GFP的</td><td> BD Biosciences公司</td><td> 8138-1 </td><td>应该是高滴度的（1×10 10 pfu /毫升）。 CsCl纯化病毒是更有效地比在该测定中的柱纯化病毒。 </td></tr><tr><td> 16％的多聚甲醛</td><td&gt;电子显微学</td><td> 15710 </td><td>在PBS中稀释至2％，用5％蔗糖（w / v的） </td></tr><tr><td> 1X磷酸盐缓冲盐水（PBS） </td><td> Life Technologies公司</td><td> 70011-044 </td><td>准备从10倍的股票</td></tr><tr><td> Hank氏平衡盐溶液</td><td> Life Technologies公司</td><td> 14175095 </td><td>没有钙，镁，没有酚红</td></tr><tr><td>转</td><td>西格玛</td><td> T8158 </td><td> 25毫克/毫升股票在DMEM/F12媒体解决方案。冻结一次性使用的分装-20C </td></tr><tr><td> L-抗坏血酸（维生素C） </td><td>西格玛</td><td> A4403 </td><td> 75毫克/毫升原液在DMEM/F12 media.Freeze的单次使用的等份-20C </td></tr><tr><td></td><td></td><td></td><td> 表1列出的，为SMG重组协议所需的试剂。 </td></tr><tr><td></td><td></td><td></TD&gt; <td></td></tr><tr><td> 10厘米无菌塑料菜</td><td>康宁</td><td> 430167 </td><td>非组织培养处理的板，也可以被使用。 </td></tr><tr><td>立体解剖显微镜透射光底座</td><td>尼康</td><td> SMZ645 </td><td>可以使用具有任何立体解剖显微镜的透射光基础。 </td></tr><tr><td> 35 mm组织培养皿</td><td>鹰</td><td> 353001 </td><td>非组织培养处理的板，也可以被使用。 </td></tr><tr><td> 50毫米直径的微孔菜</td><td> MatTek公司</td><td> P50-G-1.5-14F </td><td></td></tr><tr><td>核孔径迹蚀刻膜过滤器</td><td> Whatman公司</td><td> 110405 </td><td> 13毫米直径，0.1毫米孔径</td></tr><tr><td>宽视场荧光显微镜</td><td>卡尔·蔡司，美国</td><td> Axio上观察Z1 </td><td>任何荧光显微镜（正直，我nverted或立体解剖显微镜）可以与一个附加的数码相机，用于监视在低放大倍率的GFP表达。 </td></tr><tr><td>共聚焦显微镜</td><td>徕卡显微系统</td><td> TCS SP5 </td><td>激光共聚焦显微镜是要看到详细的细胞结构。在任何可以使用共聚焦显微镜。 </td></tr><tr><td>定时怀孕的雌性小鼠，CD-1菌株或ICR </td><td>查尔斯河实验室</td><td></td><td>收获胚胎第13天（天插头发现指定为第0天）。 </td></tr><tr><td>手术刀＃11 </td><td>精细科学工具</td><td> 10011-00 </td><td></td></tr><tr><td>手术刀手柄＃3 </td><td>精细科学工具</td><td> 10003-12 </td><td></td></tr><tr><td>杜蒙＃5镊子抗氧化合金，0.05mm的X0.02毫米</td><td>精细科学工具</td><td> 11252-20 </td><td>从胚胎收获腺体的理想选择</td></tr><tr><td&gt;杜蒙第5的镊子dumostar合金，0.05mm的X0.01毫米</td><td>精细科学工具</td><td> 11295-20 </td><td>精细的技巧都需要消除从上皮细胞的间充质。也可以使用钨针。 </td></tr><tr><td></td><td></td><td></td><td> 表2。使用的设备，在SMG重组协议。 </td></tr></tbody></table>

genetic modification and recombination of salivary gland organ cultures

分支形态发生发展过程中的许多器官和胚胎小鼠颌下腺（SMG），是一个经典模型的研究分支形态。在发展中国家SMG，这个过程涉及迭代步骤的上皮芽管形成，最终上升到一个复杂的腺泡和导管，分支网络的服务产生和修改/运输唾液，分别进入口腔1 - 3。上皮相关的基底膜和间充质室，包括间充质细胞，生长因子和细胞外基质，由这些细胞产生的方面，分支机构是至关重要的，但如何协调细胞和分子事件仍然知之甚少4 。这项研究的分子机制驾驶上皮形态的进步，我们的发展机制的理解和洞察可能雷格纳积极行动的医学方法。这些研究的唾液腺上皮细胞进行遗传操作的有效方法由于缺乏而受到阻碍。目前，腺病毒转导针对成年腺体的上皮细胞在体内 5表示的最有效方法。然而，在胚胎外植体，致密的间充质细胞和上皮细胞周围的基底膜阻碍病毒获得的上皮细胞。如果间质被除去，被转染的上皮细胞可以使用腺病毒，和上皮雏形可以恢复分支形态存在的Matrigel或层粘连蛋白-111 6,7。间充质-上皮雏形的增长还需要额外的补充可溶性生长因子，并不能完全概括分支形态，因为它发生在完整的腺体8。在这里，我们描述了一种技术，这有利于腺病毒转导的上皮细胞和转染的电子文化pithelium相关的间充质。胚胎冲锋枪，去除间质，用含GFP-腺病毒感染的上皮细胞显微切割之后，我们显示，上皮细胞与未感染的间充质细胞自发重组，扼要完整SMG腺体结构和分支形态。使用标准的荧光显微镜方法，可以很容易地监控转基因上皮细胞人口，如果荧光标记的腺病毒结构。这里描述的组织重组方法是目前最有效的和可访问的方法，与野生型或突变型的矢量在一个复杂的三维组织结构，不需要产生转基因动物的上皮细胞的转染。

流动的主要的实验步骤在图1中概述。在图2中所示的一个例子的一个完整的SMG，一个孤立的上皮雏形，和其相应的间充质细胞。固定和成像的明视场图像重组冲锋枪，继续进行培养体外时的指定的时间，在图3中所示的分支形态，生长48小时表达上皮的GFP的图4中所示的重组质粒腺体的重组冲锋枪的共聚焦图像随后通过免疫细胞化学的72小时培...

Watch this Scientific Journal Video about Genetic Modification and Recombination of Salivary Gland Organ Cultures at JoVE.com

Genetic Modification and Recombination of Salivary Gland Organ Cultures

一个基因操纵技术上皮细胞内全<em&gt;体外</em培养的胚胎小鼠颌下腺（冲锋枪）病毒转基因。这种方法利用的先天能力，SMG上皮细胞和间充质，自发重组后的分离和感染的上皮与腺病毒载体的雏形。

基因改造和重组唾液的腺体器官培养

Branching  morphogenesis occurs during the development of many organs, and the embryonic  mouse submandibular gland (SMG) is a classical model for the ...

genetic-modification-recombination-salivary-gland-organ

Research

JoVE Journal

Biology

12.8K 次观看.  University at Albany, SUNY. 一个基因操纵技术上皮细胞内全体外

视频: Genetic Modification and Recombination of Salivary Gland Organ Cultures

Dissection of Midgut and Salivary Glands from Ae. aegypti Mosquitoes

The mosquito midgut and salivary glands are key entry and exit points for pathogens such as Plasmodium parasites and Dengue viruses. This video protocol demonstrates dissection techniques for removal of the midgut and salivary glands from Aedes aegypti mosquitoes. 



The mosquito midgut and salivary glands are key entry and exit points for vector pathogens like Plasmodium falciparum and the dengue virus. This video demonstrates the dissection techniques for removing the midgut and salivary glands from Aedes aegypti mosquitoes.

从AE肠和唾液腺的解剖。埃及伊蚊蚊

The mosquito midgut and salivary glands are key entry and exit points for pathogens such as Plasmodium parasites and Dengue viruses. This video protocol ...

科研

生物学

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

文化早期鸡胚体外（新文化）的方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

双整装原位杂交技术在早期鸡胚

Preparation of 2-dGuo-Treated Thymus Organ Cultures

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

This video demonstrates the dissection and removal of the fetal thymus as well the preparation of ex vivo cultures of 2-dGuo-treated thymus.

2 dGuo治疗胸腺器官培养的制备

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

鼠标涎腺干细胞的分离

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

DNA Methylation: Bisulphite Modification and Analysis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5'-CpG-3' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.
This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing.
DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.1 and Clark et al.2, methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.

The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.

DNA甲基化：亚硫酸氢修改和分析

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene ...

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.

另类文化的人多能干细胞的生产，维护和遗传分析

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

DNA Electroporation, Isolation and Imaging of Myofibers

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently labeled proteins in the flexor digitorum brevis (FDB) muscle. Conditions have been optimized to provide a large number of high quality myofibers expressing the electroporated plasmid while minimizing muscle damage. The method employs fluorescent tags on various proteins. Combining this expression method with high resolution confocal microscopy permits live cell imaging, including imaging after laser-induced damage. Fluorescent dyes combined with imaging of fluorescently-tagged proteins provides information regarding the basic structure of muscle and its response to stimuli.

This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure.

DNA电穿孔，肌纤维的分离和成像

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...

Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit

Cyanobacteria are a diverse group of prokaryotic photosynthetic organisms that can be genetically modified for the renewable production of useful industrial commodities. Recent advances in synthetic biology have led to development of several cloning toolkits such as CyanoGate, a standardized modular cloning system for building plasmid vectors for subsequent transformation or conjugal transfer into cyanobacteria. Here we outline a detailed method for assembling a self-replicating vector (e.g., carrying a fluorescent marker expression cassette) and conjugal transfer of the vector into the cyanobacterial strains Synechocystis sp. PCC 6803 or Synechococcus elongatus UTEX 2973. In addition, we outline how to characterize the performance of a genetic part (e.g., a promoter) using a plate reader or flow cytometry.

Here, we present a protocol describing how to i) assemble a self-replicating vector using the CyanoGate modular cloning toolkit, ii) introduce the vector into a cyanobacterial host by conjugation, and iii) characterize transgenic cyanobacteria strains using a plate reader or flow cytometry.

使用CyanoGate模块化克隆工具包进行共生对蓝藻的遗传改造

Cyanobacteria are a diverse group of prokaryotic photosynthetic organisms that can be genetically modified for the renewable production of useful ...

Dissection and Immunostaining of Larval Salivary Glands from Anopheles gambiae Mosquitoes

Mosquito salivary glands (SGs) are a requisite gateway organ for the transmission of insect-borne pathogens. Disease-causing agents, including viruses and the Plasmodium parasites that cause malaria, accumulate in the secretory cavities of SG cells. Here, they are poised for transmission to their vertebrate hosts during a subsequent blood meal. As adult glands form as an elaboration of larval SG duct bud remnants that persist beyond early pupal SG histolysis, the larval SG is an ideal target for interventions that limit disease transmission. Understanding larval SG development can help develop a better understanding of its morphology and functional adaptations and aid in the assessment of new interventions that target this organ. This video protocol demonstrates an efficient technique for isolating, fixing, and staining larval SGs from Anopheles gambiae mosquitoes. Glands dissected from larvae in a 25% ethanol solution are fixed in a methanol-glacial acetic acid mixture, followed by a cold acetone wash. After a few rinses in phosphate-buffered saline (PBS), SGs can be stained with a broad array of marker dyes and/or antisera against SG-expressed proteins. This method for larval SG isolation could also be used to collect tissue for in situ hybridization analysis, other transcriptomic applications, and proteomic studies.

Dissection and Immunostaining of Larval Salivary Glands from Anopheles gambiae Mosquitoes

The adult mosquito salivary gland (SG) is required for the transmission of all mosquito-borne pathogens to their human hosts, including viruses and parasites. This video demonstrates efficient isolation of the SGs from the larval (L4) stage Anopheles gambiae mosquitoes and preparation of the L4 SGs for further analysis.

冈 比亚按蚊 幼虫唾液腺的解剖和免疫染色

Mosquito salivary glands (SGs) are a requisite gateway organ for the transmission of insect-borne pathogens. Disease-causing agents, including viruses and ...