Name: genetic modification and recombination of salivary gland organ cultures
Uploaded: 2013-01-28T12:00:00
Description: Watch this Scientific Journal Video about Genetic Modification and Recombination of Salivary Gland Organ Cultures at JoVE.com

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?...

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	<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>

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 ...

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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 ...

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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 ...

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

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The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the ...

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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 ...

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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 ...