这项工作是由美国国立卫生研究院NRSA 1F32CA14208701（SXA）和国立卫生研究院资助AR052785 AR046786（AEO和W-MW）的支持。

1。准备0〜2日龄新生小鼠的皮肤解剖<ol><li>安乐死鼠标幼仔使用CO 2室为至少20分钟。离开幼崽在冰上了一个小时，直到解剖。移植产量较低，结果在P0-P2小鼠的，强烈建议准备从年纪较大的老鼠的细胞祖能力降低。准备一盘70％乙醇（1.3）和三道菜的清洗液（步骤3）当准备进行皮肤解剖。解冻中性蛋白酶的解决方案，并保持在4°C.颈椎脱臼的幼崽后，CO 2曝光过度，确保安乐死。 </li><li>将安乐死的幼崽在冰上转移到无菌的流罩在培养皿中。 </li><li>洗净幼仔通过简要浸渍在70％乙醇和无菌培养皿上的地方。 </li></ol> 2。解剖小鼠皮肤<ol><li>在该基地的躯干，用无菌剪刀切断每一个四肢和尾巴。 </li><li>牢牢把握身体之间的对铜rved钳和，沿着背部皮肤做一个切口从头部到尾部使用手术刀，不渗透的基础筋膜。 </li><li>小心剥离的皮肤远离中线的鼠标。 </li><li>牢牢抓住暴露的鼠标的长边弯钳和插入另一对皮肤下的鼠标在后端弯钳，轻轻拉向腹侧半的鼠标臀部皮肤。 </li><li>小心地剥开皮完全关闭鼠标在一个流畅的动作，丢弃的尸体。 </li></ol> 3。清洗皮肤和孵育中性蛋白酶解决方案<ol><li>将皮肤的真皮侧的洗涤液中的第一道菜。传播的皮肤，并，搅拌用钳子。解剖下的皮肤，使肌肤的第一道菜的洗涤液。 </li><li>放置下一个解剖皮肤的洗涤溶液中的第一道菜后，现有的皮肤转移到洗涤溶液中的第二盘。保持皮肤在日E二次洗涤液，直到收集所有的皮肤。将所有的皮肤，最后洗，搅拌简要介绍。 </li><li>无菌的10厘米的培养皿中加入10毫升冰冷的中性蛋白酶。传输的皮肤中性蛋白酶的解决方案和展平皮肤的真皮侧。 </li><li>浮动皮肤8-16小时，在4°C（替代方案:1小时，在37°C）。 </li></ol> 4。独立的真皮和表皮<ol><li>皮肤转移到一个新的无菌培养皿中，并展平皮肤的真皮侧。从一个角落里，用手术刀小心地按住真皮。 </li><li>抓住镊子，慢慢地剥离表皮从真皮。表皮应剥离为一块。表皮是白色的，薄的，应该是褐色的，更厚，胶质和真皮。 </li><li>分开的两个组织成单独的无菌培养皿中。 <ol class="lalpha"><li>为了使真皮特定基因敲除/过度表达移植，真皮和剁碎成非常小的碎片与T禾手术刀。丢弃的表皮。在一天的感染（步骤7），解剖另一组的小鼠的皮肤，以制备新鲜的，未经处理的表皮细胞结合与慢病毒感染的皮肤细胞。 </li><li>为了使表皮细胞的特定基因敲除/过度表达移植，每一个表皮切片为6 - 8个更小的碎片。舍弃真皮。在一天的感染（步骤7），解剖另一组的小鼠的皮肤，以制备新鲜的，未经处理的皮肤细胞与慢病毒感染的表皮细胞结合。 </li></ol></li></ol> 5。游离小鼠原代的真皮细胞（MDCS），肝等组织<ol><li>游离MDCS培养真皮片和新鲜的的胶原酶溶液在37°C 1小时。使用鼠标小狗的胶原酶溶液每10毫升。 </li><li>轻轻地混合溶液真皮每10分钟。在显微镜下检查的离解的程度;消化的细胞悬浮液应该是大多是单细胞。该解决方案应该从明确到多云从真皮真皮细胞解离。 </li><li>添加FBS的胶原酶解含有真皮胶原酶的活性降低至10％最终体积。 </li><li>通过细胞悬液通过一个70微米到一个新的50ml管中去除大量未消化的团块和整个毛囊细胞过滤器。 DMEM/10％FBS中加入10毫升每30毫升的流量通过，以进一步稀释的胶原酶。 </li><li>离心管中，在桌面上离心5分钟，以进一步除去整个毛囊在30×g离心含有消化真皮。 </li><li>小心地将上清转移到一个新的50ml管。通过在200×g离心5分钟，在台式离心机离心沉淀细胞。 </li><li> DMEM/10％FBS（1毫升每只幼犬）重悬细胞并计数细胞。板细胞Amniomax C-100感染的媒体（步骤7），或结合慢病毒感染的KC，用于接枝（步骤9）。典型的产量是20×10 6细胞每只幼犬。 </li></ol> 6。游离主角质细胞（KC），肝等组织<ol><li>通过表皮片与新鲜解冻的0.05％胰蛋白酶-EDTA在37℃下孵育15分钟，游离KC。使用胰蛋白酶-EDTA每毫升鼠标小狗。 </li><li>轻轻旋转解决方案，包含表皮，每隔5分钟。 </li><li>和溶液加入等体积的胰蛋白酶-EDTA含表皮中的胰蛋白酶的活性降低。 </li><li>通过在200×g离心5分钟，在台式离心机离心沉淀细胞。剩余未消化的组织，浮在上面。 </li><li>小心倒出上清液与未消化的组织。除去剩余的上清液与10毫升的移液管，而不会干扰细胞沉淀。用PBS重悬细胞沉淀。如果在角质形成是不完全沉淀，倒出上清液，并，，直到大多数细胞沉淀重复离心。 </li><li>菌株细胞悬浮液通过70μm的细胞过滤网进新管中，以除去剩余的组织或团块。 </li><li>颗粒细胞离心在200×g离心5分钟，在台式离心机。 </li><li> CNT-07的媒体或PBS（1毫升每只幼犬）和计数细胞的悬浮细胞。使用CNT-07的电镀感染的细胞（步骤7），用PBS结合的慢病毒感染的mDC移植术（步骤9）。典型的产量为3-10×10 6个细胞的小狗。 </li></ol> 7。感染原代细胞的慢病毒载体的shRNA或cDNA 天感染解剖另一组的小鼠的皮肤以制备新鲜的，未经处理的主枯否或的mDC到与天的接枝（步骤9）的病毒感染的细胞结合。 <ol><li><ol class="lalpha"><li>有关的mDC，板4×10 6个，每10厘米板的mDC AmnioMAX-C100媒体和孵育在37°C + 5％CO 2的50％的细胞密度在第二天感染。一般3-4板的mDC被用于产生一种接枝，并总共10-12板都需要在一个实验中产生三个移植。或者，让MDCS连接和扩展F或至少在37℃下2小时+，5％CO 2，并执行感染当天细胞镀。 MDCS成纤维细胞样形态。 </li><li> KC，板12×10 6 KC每10厘米的板CNT-07的媒体和孵化，在37°C + 5％CO 2次日感染。或者，让KC附加和扩散为至少2小时，在37°C + 5％CO 2，并在同一天进行感染。一般3-4板的KC被用于产生一种接枝。共10-12板需要以产生三个移植一个实验。的枯否将有铺路石样形态。 </li></ol></li><li>垫上预温育5-10分钟的细胞8微克/毫升聚凝胺在37℃下解+，5％CO 2。 </li><li>交换介质，并添加慢病毒+ 8微克/毫升聚凝胺的解决方案。慢病毒的滴度会有所不同，这取决于使用的结构和前应确定感染。 </li><li>在200 XG离心板在台式离心机1小时，在32℃，C慢病毒感染的细胞。 </li><li>删除慢的媒体和增添新的媒体。对于KC，用PBS洗涤细胞一次，然后再添加新鲜的媒体。一般感染的细胞恢复过夜，在37℃下，5％CO 2。或者，允许至少两个小时的恢复，在37°C + 5％CO 2的胰酶消化细胞移植前。继续培养感染的细胞中的一小部分，以检查代表结果部分中所描述的感染效率。 </li></ol> 8。准备受感染的细胞移植<ol><li>新鲜的mDC或KCS隔离皮肤使用步骤4-6。在冰冷的无菌PBS悬浮细胞。计数细胞。 </li><li><ol class="lalpha"><li>对于皮肤的特定的基因敲除/过度接枝，游离从板8分钟，在37℃下用0.05％胰蛋白酶-EDTA的感染的mDC。 </li><li>表皮细胞的特定基因敲除/过度表达移植，游离受感染的KC板USI纳克0.125％胰蛋白酶-EDTA在37℃下15分钟。 </li></ol></li><li>中性化胰蛋白酶的活性，用DMEM +10％FBS（MDCS）或中和剂溶液（KC）。胰蛋白酶处理细胞转移到一个50毫升的试管。 </li><li>通过在200×g离心5分钟，在台式离心机离心沉淀细胞。 </li><li>在冰冷的无菌PBS悬浮细胞。计数细胞。 </li><li>结合与新鲜制备的未处理对应的信元类型的病毒感染的细胞。对于一个移植物，结合7-10×10 6的mDC 7-10×10 6个角质形成在冰冷的无菌PBS和颗粒的细胞浆在台式离心机中在4℃下在50元-100μL冰冷的无菌PBS悬浮细胞。保持细胞置于冰上，直到nu / nu小鼠室准备。 </li></ol> 9。创建返回nu / nu小鼠的伤口床和修复商会到接枝的前一天，灭菌用高压釜中，无菌硅腔的外科手术工具，通过浸泡在70％乙醇中。更换70％的ETHA在使用前腔北环线，用无菌PBS。 <ol><li>麻醉nu / nu小鼠（7-12周龄雌性）（8毫克/毫升）使用氯胺酮/赛拉嗪（1.6毫克/毫升），经腹腔注射（100μl/10g体重）或其他优选的方法。应用眼睛润滑油。将加热鼠标垫下。 </li><li>裸鼠背部皮肤用碘酒消毒，并用酒精棉签清洁。 </li><li>加入1-3滴0.25％布比卡因切口部位的局部。在基地的脖子用钳子捏住裸小鼠背部皮肤。 </li><li>在捏皮肤，用无菌剪刀剪了一个小的圆形孔（直径在1厘米）。 </li><li>将无菌室到伤口床，盖上腔的边缘与周围皮肤。 </li><li>安全室分成室边缘的缝合线沿每个腔室（6针），如在图2A中示出的地方。 </li><li>室都准备好了，轻轻摇动细胞浆，并绘制成1毫升注射器23号针头。如果细胞浆液已凝结在底部，轻轻重悬画进针前，用1毫升移液枪枪头。 </li><li>穿刺针，慢慢地滴细胞到伤口室。 </li><li>将小鼠到干净，蒸压笼。楼2小鼠每笼和饮水中添加抗生素和泰诺（3毫克/毫升）。根据我们的经验，并没有导致不良反应室的创伤，移植物，或动物的雌性小鼠的每笼。 </li><li>将变暖垫下半笼，观察小鼠，直到麻醉消退。 </li></ol> 10。删除后7-9天会<ol><li>麻醉腔nu / nu小鼠在步骤9.1。应用眼睛润滑油。 </li><li>消毒室，碘和周围的皮肤用酒精棉签清洁。 </li><li>精心裁剪的针和删除从一个房间。 </li><li>轻轻删除腔鼠标。如果新的皮肤移植枝室，抬起室室使用镊子轻轻分开移植。保持移植的过程中去除室。移植物应该厕所K表枯燥和图2B不透明的。 </li><li>应用非粘附垫入到位（2针）的开放的伤口和缝合线的抗生素对用薄膜。 </li><li>围绕鼠标缠绕绷带，以保护垫，保护伤口。缝合包扎到位。 </li><li>的硅腔室是通过用温和的肥皂擦洗清洁，并用去离子水彻底冲洗。腔浸泡在70％乙醇过夜，空气干燥，和存储。 </li></ol> 11。检查鼠标对头发的生长<ol><li>取出绷带和垫后3天室去除。移植应干燥，不透明的褐色的颜色。 </li><li>定期检查小鼠对头发的生长。头发应在移植后第16天开始显示。 </li></ol>步骤概述 1天（2-3小时）:牺牲新生幼崽，去除角质，并留下分离酶在4℃过夜。 第2天（2-3小时）:从皮肤隔离的初级细胞和板推荐的细胞密度。 第3天（3-4小时）:用慢病毒感染的细胞。删除从另一组的新生幼崽离开皮肤上分离酶在4℃过夜。 第4天（6-8小时）:从皮肤中分离出原代细胞，计数，和留在冰上。恢复慢病毒感染的细胞用胰蛋白酶消化，离心，计数，并结合每移植到50-100μl的PBS 7-10×10 6真皮细胞和角质形成细胞。创建对小鼠的伤口床，连接室，应用细胞混合物的伤口床。 替代过程概述替代程序的轮廓将缩短程序从四天三年。 <ol><li>结合第1天，第2天治疗小鼠皮肤在分散酶，在37℃下为一小时（估计时间5-7小时）。 </li><li>结合第2天，第3天，通过电镀细胞两小时（估计时间5-7小时）后，用慢病毒感染细胞。 </li></ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembly+of+dermal+papilla+cells+into+inductive+spheroidal+microtissues+on+poly(ethylene-co-vinyl+alcohol)+membranes+for+hair+follicle+regeneration.">Self-assembly of dermal papilla cells into inductive spheroidal microtissues on poly(ethylene-co-vinyl alcohol) membranes for hair follicle regeneration.</a> Biomaterials. 29, 3521-3530 (2008).</li><li>Zheng, Y., Du, X., Wang, W., Boucher, M., Parimoo, S., Stenn, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organogenesis+from+dissociated+cells:+generation+of+mature+cycling+hair+follicles+from+skin-derived+cells.">Organogenesis from dissociated cells: generation of mature cycling hair follicles from skin-derived cells.</a> J. Invest. Dermatol. 124, 867-876 (2005).</li></ol>

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头发重建试验研究的从头毛囊形成的机制提供了一个独特的器官再生模型。在这里，我们描述了修改后的腔头发检测，可用于确定毛囊形成的基因的功能。我们的测定是基于报告室头发重建测定5星，6,11，这是我们修改引入慢病毒的表达技术来实现的真皮或表皮细胞类型的基因过表达或击倒在。我们的分析提供了一个快速的替代品产生组织特异性基因敲除。此法使我们能够在短短的三个星期内，用慢病毒感染原代细胞移植收集在毛囊形成的基因功能演示。我们的检测可扩展，以解决使用组合短发夹/慢病毒的cDNA交付到一个单元格12型的遗传相互作用，以及允许信令之间的真皮和表皮细胞典型的检查ES。 我们的头发测定是最适合于检测强的增益或损失的函数在毛囊再生的表型，而微妙的缺陷是难以观察到。我们已经成功地证明的几个基因，用我们的头发再生实验1,12的真皮特定基因的功能。基于GFP标记的共表达的慢病毒载体shRNA表达10，我们表明，再生的毛发移植供体皮肤细胞的shRNA表达坚持。 GFP阳性细胞中存在的（ 图3）的再生毛囊真皮乳头（DP）。 DP细胞可能包括不同程度的基因敲除细胞不同程度的GFP表达，从而观察到毛囊型范围从遗传hypomorph，为空。 此法的改进之一将是建立一个快速和有效的选择的shRNA表达细胞前graftin克如使用流式细胞仪，净化强烈的绿色荧光蛋白表达细胞。另一种方法是在此试验中9替代突变或条件性敲除细胞。另一方面，培养系统，可以保留DP细胞的功能是非常有用的，作为DP一旦主真皮细胞传代，逐渐失去效力。潜在的文化系统，包括使用生物材料来自人工的利基或聚集文化2,7,8,13。另一项改进将是产生一个可靠的表皮细胞的冷冻法具有高细胞的回收率，因为这将减少使用鼠标幼仔（步骤7）的第二组以产生新鲜的角质形成细胞，在真皮特定的损耗或增益功能的研究。 该室头发分析产生类似正常小鼠皮肤毛囊的密度和质量，但头发的生长是略低于同步和卵泡定位更随机。有一些限制的头发再生的基因检测含e是需要大量的慢病毒和细胞的数量，这是非常劳力密集的手术方法相比，其他形式的头发重建试验，如补丁和襟翼检测3,4,14。然而，毛囊和时间表的质量检测头发的生长优于其他实验4。我们的目标毛发再生现有的遗传模型分析提供了一个方便快捷的同伴。

<table border="1"><tbody><tr><td> 名称 </td><td> 公司 </td><td> 产品编号 </td><td> 评论 </td></tr><tr><td> PBS </td><td> Invitrogen公司</td><td> 14190-144 </td><td></td></tr><tr><td> HBSS </td><td> Invitrogen公司</td><td> 14170-122 </td><td></td></tr><tr><td> DMEM </td><td> Invitrogen公司</td><td> 11995-065 </td><td></td></tr><tr><td> 0.25％胰蛋白酶-EDTA </td><td> Invitrogen公司</td><td> 25200-056 </td><td></td></tr><tr><td> CNT-07 </td><td>小区n TEC </td><td> CNT-07 </td><td></td></tr><tr><td> AminoMAX-C100 </td><td> Invitrogen公司</td><td> 17001-074 </td><td></td></tr><tr><td> AminoMAX-C100补充</td><td> Invitrogen公司</td><td> 12556-015 </td><td></td></tr><tr><td> FBS </td><td> Invitrogen公司</td><td> 26140-079 </td><td></td></tr><tr><td>青霉素/链霉素</td><td> Invitrogen公司</td><td> 15140-122 </td><td></td></tr><tr><td>两性霉素B </td><td> Invitrogen公司</td><td> 15290-018 </td><td></td></tr><tr><td>庆大霉素</td><td> Invitrogen公司</td><td> 15710-064 </td><td></td></tr><tr><td>中性蛋白酶</td><td>罗氏公司</td><td> 4942078001 </td><td></td></tr><tr><td> 1型胶原酶， </td><td>西格玛</td><td> C-0130 </td><td></td></tr><tr><td>聚凝胺</td><td>西格玛</td><td> 107689-10G </td><td></td></tr><tr><td>组织培养皿</td><td> Fisher Scientific则</td><td> 08-772E </td><td></td></tr><tr><td>解剖剪</td><td> Fisher Scientific则</td><td> 11-999 </td><td></td></tr><tr><td>镊子</td><td> Fisher Scientific则</td><td> 10-275 </td><td>弯曲</td></tr><tr><td> Scalpal </td><td> Medex公司供应</td><td> GRF-2975＃10 </td><td>大小没有。 10 </td></tr><tr><td> 70微米的细胞染色机</td><td> VWR </td><td> 21008-952 </td><td></td></tr><tr><td>的15毫​​升锥形离心管中</td><td> Fisher Scientific则</td><td> 14-959-49D </td><td></td></tr><tr><td>酒精棉签</td><td> Fisher Scientific则</td><td> 1368063 </td><td></td></tr><tr><td> 23号针</td><td> Fisher Scientific则</td><td> 14-821-13F </td><td></td></tr><tr><td>眼睛润滑剂</td><td> CVS </td><td> 8883660 </td><td></td></tr><tr><td> Providone碘棉签</td><td> Fisher Scientific则</td><td> NC0116841 </td><td></td></tr><tr><td>硅钱伯斯</td><td>伦纳有限公司</td><td> F2U，30268 </td><td></td></tr><tr><td>缝合线</td><td> Acuderm </td><td> SUP3524 </td><td></td></tr><tr><td>弹性绷带</td><td> Fisher Scientific则</td><html><td> 22-363-100 </td><td></td></tr><tr><td>非粘性敷料</td><td> TELFA </td><td> 1050 </td><td></td></tr><tr><td></td><td></td><td></td><td> 物料 </td></tr><tr><td></td><td></td><td></td><td> 洗涤液 PBS 500微克/毫升青霉素/链霉素 12.5微克/毫升两性霉素B 100微克/毫升庆大霉素 中性蛋白酶的解决方案 HBSS 2.5毫克/毫升的最终Dispase酶 100微克/毫升庆大霉素 中和媒体 HBSS 15％（V / V）FBS的 胶原酶溶液 0.25％（重量/体积）的胶原酶，1型ontent"&gt; 100微克/毫升青霉素/链霉素 2.5微克/毫升两性霉素B 50微克/毫升庆大霉素 聚凝胺的解决方案 HBSS 8微克/毫升（重量/体积）聚凝胺</td></tr></tbody></table>

rapid genetic analysis of epithelial-mesenchymal signaling during hair regeneration

毛囊形态发生是一个复杂的过程，需要上皮细胞衍生的角质形成细胞和底层的间充质细胞之间的相互作用，是一个有吸引力的模型系统来研究器官发育和组织特异性的信号。虽然毛囊的发展是听话的基因，快速，重现性的分析，这个过程中的重要因素仍然是一个挑战。在这里，我们描述一个过程来产生有针对性的过表达或短发夹环介导的击倒的因素，使用慢病毒中的组织特异性的方式。使用修改后的版本毛发再生模型5，6，11，我们可以实现强大的增益或损失的小鼠原代角质细胞或皮肤细胞的功能分析，以方便上皮-间质的信号转导途径，导致毛囊形态发生的研究。我们描述了如何隔离新鲜小鼠角质形成细胞和真皮细胞，它含有毛乳头细胞和它们的前体，提供慢病毒含有宁shRNA或cDNA的一个细胞种群，细胞结合起来，以产生完全形成的裸小鼠的背部上的毛囊。这种方法允许需要在三个星期内产生毛囊组织的具体因素分析，现有的遗传模型提供了一个方便快捷的同伴。

在图1中，我们显示的结果真皮特定的慢病毒shRNA击倒的平滑（SMO），一个关键的Shh信号组件，在为期3周的再生头发移植。单独使用慢病毒载体的控制移植强大的头发生长（ 图1A）。相比之下，真皮特异性抑制毛发的生长（ 图1B）的损失的Smo结果。苏木精和曙红染色示出的阶段，在对照组和实验条件（ 图1C，D） ​​的毛囊。从类似的处理头发的生长表型可以是可变的在实验中，与慢病毒载体单独感染包括阳性对照。因此，积极控制移植应始终包含在每一个实验作为参考30％的移植失败的结果疤痕或不完整的移植附件。在测试两个因素，如过度的因素之一的cDNA之间的相互作用的情况下重新短发夹介导的击倒的scue的另一个因素，每个人都应该包括击倒单独的移植到明显的损失的功能在每个实验结果。这将提供一个必要的范围内，以确定一个特定基因如何扰动毛囊再生途径和两个基因如何交互。 去除室（ 图2A），必须小心。新成立的移植应该是稍微暗淡表面的不透明的（ 图2B）。移植室，所以轻轻地举起一面坚持是很重要的，以确保移植物保持连接的伤口床。移植是相当稳定的三天后取出敷料时，应当出示什么困难。鲁棒的头发生长，应观察到三个星期后（ 图2C）。省略无论是MDCS或KCS将导致恢复的伤口部位没有任何毛发（ 图2D）11。之一中的细胞死亡或细胞损失细胞类型的查询结果在没有头发，相反，减少毛发再生，如在图1B中所示的真皮Smo的击倒。 为了确保一个成功的试验，这是至关重要的实现大于90％的病毒感染的细胞，用适当的过表达或击倒效率接枝前。由于移植手术的时间限制的，我们建议开始前一个嫁接试验的故障排除病毒感染效率，并始终保存的一小部分细胞移植手术当天，以验证损失或增益的表达。慢病毒的滴度会有所不同，这取决于使用的结构，应确定感染前达到90-100％的效率。我们监测与GFP共表达的慢病毒载体病毒感染效率。我们定期进行定量PCR和/或Western印迹击倒或过度表达程度来决定。利用双表达载体与荧光标记，标记病毒感染的细胞，提供了一个方法来报告perdurance表达我们构建在成熟移植的细胞的信号和数目。我们使用由CMV启动子驱动的，从pSicoR-CMV-GFP组合Smo的短发夹10的慢病毒载体的绿色荧光蛋白。在图3中，我们展示了3周的皮肤特异性移植，其中GFP的慢病毒载体表达有代表性的毛囊的毛乳头。 <img alt="图1" src="/files/ftp_upload/4344/4344fig1.jpg" /> 图1。毛囊生长的组织学分析。（A）的控制毛发再生术与空慢病毒载体和未经处理的角质细胞与皮肤细胞感染。（B）磨平了击倒使用在皮肤细胞的慢病毒shRNA结合，与未经处理的角质形成细胞在毛囊损失。 （C）苏木精和曙红染色显示生长期发氟ollicles在控制移植到真皮层向下延伸（D）磨平了击倒毛囊继续发育不良，基本上不存在。所有图片均为3周后移植。比例尺表示100微米。 <img alt="图2" src="/files/ftp_upload/4344/4344fig2.jpg" /> 图2。嫁接原代细胞。（A）nu / nu小鼠与缝合室住房主要皮肤细胞和角质形成细胞（B）新成立的皮肤去除室公开是枯燥和不透明的字符。（C）完全成长的毛发移植野生输入主要的皮肤细胞和角质形成细胞在三个星期后移植。（D）增加的主要皮肤角质形成细胞的细胞没有导致无毛，后三个星期。类似的结果被认为加入初级的角质形成细胞无真皮细胞。 图3。维护GFP表达在真皮乳头。共聚焦图像从3周龄再生移植的毛乳头。 GFP的表达在真皮细胞种群从慢病毒载体，在真皮乳头（绿色）检测。聚糖（红色）划定的毛乳头和细胞核用Hoechst（蓝色）的标签。注意GFP的真皮细胞中特异表达的，但不是在周围的角质细胞。比例尺是指20微米。

Watch this Scientific Journal Video about Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration at JoVE.com

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

组织特异性分析的毛囊再生模型，利用慢病毒介导增益或损失的功能。

在毛发再生的上皮 - 间充质信号的快速遗传分析

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

rapid-genetic-analysis-epithelial-mesenchymal-signaling-during-hair

Research

JoVE Journal

Biology

13.6K 次观看.  Stanford University School of Medicine. 组织特异性分析的毛囊再生模型，利用慢病毒介导增益或损失的功能。

视频: Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) has become a popular organism for research in diverse areas of cell biology and genetics because of its simple life cycle, ease of growth and manipulation for genetic analysis, genomic resources, and transformability of the nucleus and both organelles. Mating strains is a common practice when genetic approaches are used in Chlamydomonass, to create vegetative diploids for analysis of dominance, or following tetrad dissection to ascertain nuclear vs. organellar inheritance, to test allelism, to analyze epistasy, or to generate populations for the purpose of map-based cloning. Additionally, genetic crosses are routinely used to combine organellar genotypes with particular nuclear genotypes. Here we demonstrate standard methods for gametogenesis, mating, zygote germination and tetrad separation. This protocol consists of an easy-to-follow series of steps that will make genetic approaches amenable to scientists who are less familiar with Chlamydomonas. Key parameters and trouble spots are explained. Finally, resources for further information and alternative methods are provided.

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

Mating and tetrad separation are required for genetic analysis in Chlamydomonas reinhardtii. Here we demonstrate standard methods for gametogenesis, mating, zygote germination and tetrad dissection. This protocol consists of an easy-to-follow series of steps that will make genetic approaches amenable to scientists who are less familiar with Chlamydomonas.

交配和四分体分离衣藻遗传分析

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) has become a popular organism for research in diverse areas of cell biology and ...

科研

生物学

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

从成年小鼠海马齿状回的剖析

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

一个反向遗传学方法测试胚胎发育过程中的功能冗余

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

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 ...

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue interactions that regulate patterning and morphogenesis of vertebrates (e.g., 1, 2, 3, 4). Here, we illustrate a method that exploits this accessibility to test the signaling and patterning properties of ectodermal tissues during facial development. In these experiments, we create quail-chick 5 or mouse-chick 6 chimeras by transplanting the surface cephalic ectoderm that covers the upper jaw from quail or mouse onto either the same region or an ectopic region of chick embryos. The use of quail as donor tissue for transplantation into chicks was developed to take advantage of a nucleolar marker present in quail but not chick cells, thus allowing investigators to distinguish host and donor tissues 7. Similarly, a repetitive element is present in the mouse genome and is expressed ubiquitously, which allows us to distinguish host and donor tissues in mouse-chick chimeras 8. The use of mouse ectoderm as donor tissue will greatly extend our understanding of these tissue interactions, because this will allow us to test the signaling properties of ectoderm derived from various mutant embryos.

This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of surface cephalic ectoderm during craniofacial development.

评估外胚层上皮细胞在颅面发育的信号属性

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

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a 'trunk' fragment is cut from an intact worm, a new 'head' will regenerate anteriorly and a 'tail' will regenerate posteriorly restoring the original 'head-to-tail' polarity of body structures prior to amputation (Figure 1A).
Regeneration is driven by planarian stem cells, known as 'neoblasts' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity2-9.
The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters10, represent an important system for studying evolution of the central nervous system11, 12 and have behavioral screening potential13, 14. 
Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs8, 9, 15. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

An attractive model for studying stem cell differentiation within a live animal is the planarian flatworm. Regeneration is studied by simple amputation experiments that are easily performed in a basic laboratory and are amenable to pharmacological and genetic (in vivo RNAi) manipulation as detailed by protocols in this article.

药理和功能的遗传分析处理涡虫的再生日本三角涡虫</em

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

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.

代谢标记和膜分馏的比较蛋白质组学分析<em&gt;拟南芥</em&gt;悬浮细胞培养

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

Extraction and Analysis of Cortisol from Human and Monkey Hair

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and validated a method for CORT extraction and analysis from rhesus monkey hair and subsequently adapted this method for use with human scalp hair. In contrast to CORT &#34;point samples&#34; obtained from plasma or saliva, hair CORT provides an integrated measure of hypothalamic-pituitary-adrenocortical (HPA) system activity, and thus physiological stress, during the period of hormone incorporation. Because human scalp hair grows at an average rate of 1 cm/month, CORT levels obtained from hair segments several cm in length can potentially serve as a biomarker of stress experienced over a number of months.
In our method, each hair sample is first washed twice in isopropanol to remove any CORT from the outside of the hair shaft that has been deposited from sweat or sebum. After drying, the sample is ground to a fine powder to break up the hair&#39;s protein matrix and increase the surface area for extraction. CORT from the interior of the hair shaft is extracted into methanol, the methanol is evaporated, and the extract is reconstituted in assay buffer. Extracted CORT, along with standards and quality controls, is then analyzed by means of a sensitive and specific commercially available enzyme immunoassay (EIA) kit. Readout from the EIA is converted to pg CORT per mg powdered hair weight. This method has been used in our laboratory to analyze hair CORT in humans, several species of macaque monkeys, marmosets, dogs, and polar bears. Many studies both from our lab and from other research groups have demonstrated the broad applicability of hair CORT for assessing chronic stress exposure in natural as well as laboratory settings.

Cortisol (CORT) accumulates in the growing hair shaft of humans and nonhuman primates. We describe methods for extracting and analyzing hair CORT with high precision and sensitivity. Measurement of hair CORT is particularly well-suited for assessing chronic stress over periods of weeks to months.

提取与分析皮质醇从人与猴的毛

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

快速分析和荧光显微镜图像的探索

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various physiological and pathological processes. Here we describe a strategy to detect alternative splicing during EMT using an inducible EMT model by expressing the transcription repressor Twist. EMT is monitored by changes in cell morphology, loss of E-cadherin localization at cell-cell junctions, and the switched expression of EMT markers, such as loss of epithelial markers E-cadherin and &#947;-catenin and gain of mesenchymal markers N-cadherin and vimentin. Using isoform-specific primer sets, the alternative splicing of interested mRNAs are analyzed by quantitative RT-PCR. The production of corresponding protein isoforms is validated by immunoblotting assays. The method of detecting splice isoforms described here is also suitable for the study of alternative splicing in other biological processes.

Alternative splicing regulation has been shown to contribute to the epithelial-mesenchymal transition (EMT), an essential cellular program in various physiological and pathological processes. Here we describe a method utilizing an inducible EMT model for the detection of alternative splicing during EMT.

在毛发再生的上皮 - 间充质信号的快速遗传分析

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