RSF has received funding from the European Union's - Seventh Framework Programme (FP7/2007-2013]) under grant agreement n&#176; 618432 &#8211; MC&#8212;Epi-Patho-Stem, as well as by the Israeli Science Foundation (218/14).

伦理声明:在本研究中的动物护理和使用都符合的ARVO声明对动物的眼科和视觉研究使用。所有实验均经当地伦理委员会。 1.选择合适的Cre重组酶表达的小鼠品系<ol><li>出的种类繁多的可购买26和取决于靶组织的Cre转基因系进行调查，选择其中Cre重组酶基因是由一个启动子，其特异于感兴趣的组织控制的酶Cre转基因小鼠品系。如果时间控制是必要的，选择一个诱导酶Cre线。 注:我们选择了他莫昔芬诱导K14-Cre重组ERT转基因特异性标记角膜缘和角膜上皮干/祖细胞24。注意，可能存在品系间差异，狄吉罗拉莫和同事报道角膜缘特异性无角膜表达他们K14-Cre重组酶ERT 22。使用可诱导的转基因的Cre小鼠的优点是，它允许谱系的感应跟踪在多功能时间帧。 </li></ol> 2.选择合适的溴小鼠品系<ol><li>选择合适的溴卡带根据研究问题和可用的Cre线16,18,20。 <ol><li>如果非诱导的Cre小鼠品系时，选择的Br盒不允许连续的DNA倒位/删剪，从而产生死端产物（例如Br1.0或Br1.1）16。 </li></ol></li></ol> 3.交叉利息的转基因品系<ol><li>对于这里显示的协议，越过纯合子R26R-五彩纸屑株（Br2.1 / Br2.1）与纯合子酶Cre ERT株（Cre重组酶ERT / Cre重组酶ERT）获得半合子后代（Br2.1 / +;酶Cre / +）这是准备单等位基因谱系追踪，因为它们都含有一个Br2.1等位基因和单Cre重组酶等位基因。 注意:为了提高标记的效率，人们可以产生含有两个酶Cre等位基因（Br2.1 / +;酶Cre /酶Cre）的小鼠，而2 Br2.1等位基因（Br2.1 / Br2.1;酶Cre / +）将增加色彩组合（如在22详细说明）。 </li></ol> 4. Cre重组酶诱导注意:他莫昔芬在这些实验的给药如下进行每日腹膜内注射，为3-4天的持续时间，以诱导的酶Cre重组酶的ERT的易位至角膜缘和角膜基底K14阳性干细胞/祖的核上皮细胞。以下方案可用于注射4只小鼠。可替代地，他莫昔芬的局部施用到眼表面产生较高的效率。的他莫昔芬溶液可保持在黑暗中在4℃下长达一周。热身之前使用的解决方案。 <ol><li>权衡三苯氧胺100毫克，溶于5毫升玉米油与形成20毫克/毫升三苯氧胺溶液。 </li><li>注入200微升三苯氧胺（4毫克）溶液腹膜内至8周大的小鼠，连续3-4天。 注意:他莫昔芬溶液可保持在黑暗中在4℃下长达一周。热身之前使用的解决方案。 </li><li>用于局部应用:称量60毫克三苯氧胺和溶解在1ml玉米油，以形成一个60毫克/毫升三苯氧胺溶液。涡旋并设置保持在65的溶液在振荡水浴 °C，直到它变得清晰留在洗澡，直到使用。 <ol><li>仔细应用100微升三苯氧胺溶液向小鼠的各眼表面。 </li></ol></li></ol> 5.组织准备成像<ol><li>下面的他莫昔芬注射和col使用异氟醚牺牲动物（2％汽化氧气）麻醉（确认正确的麻醉由缺乏反射的），其次是CO 2吸入在适当的时间点择相关的组织。 <ol><li>对于这些实验，剜除眼睛在以下时间点:1，4，8，12，和16周后注射他莫昔芬。用弯钳实现眼球摘除。按造成眼球弹出眼眶的眼睑。将眼球控股视神经背后的镊子。轻轻拉动眼球和分离。 注:在此阶段，如下所述，组织可以用于标记细胞wholemount分析制备。常规的冷冻组织切片也可以制备，固定在PFA和成像如下所述（参见第6 和 22）。 </li></ol></li><li>将每个眼睛在60mm培养皿填充有PBS。 </li><li>下一个立体显微镜使用镊子保持邻近视神经眼球与角膜朝下，并使用手术刀以除去视神经，肌肉和结缔组织，并作一小切口在眼睛的后部。 </li><li>使用弹簧剪刀小心地放大切让镜头里蹦出来，用钳子取出光圈。 </li><li>通过从左路用手术刀周边的中心开始4-5放射状切口压平角膜。 </li><li>用手术刀切割多余的外周组织以外的缘。 </li><li>修正了PFA 4％，角膜10分钟，然后简要用PBS洗涤。 </li><li>孵育样品用DAPI 10分钟，并用PBS简要洗净。 </li><li>角膜山玻片上的上皮面朝上。将一滴安装媒体在角膜上，盖上盖玻片的顶部。让幻灯片干O / N在RT。保持幻灯片在4℃。 </li></ol> 6.共聚焦显微镜和影像学分析<ol><li>使用光谱共聚焦系统，使YFP / GFP / CFP排放的分离。组荧光激发和取决于溴盒的荧光蛋白的发射波长。选择最合适的激发波长为每个蛋白旨在最小化交叉励磁。为了最小化来自不同蛋白质的信号的泄漏选择窄的发射范围。 <ol><li>对于Br2.1录像带，请使用以下设置荧光激发（EX）和发射（EM）为出发点:DAPI - 前405纳米/ EM 420-480纳米，CFP - 前458纳米/ EM 470-500纳米，GFP - 前488纳米/ EM 497-508纳米，YFP - 前514纳米/ EM 529-540纳米，RFP - 前561纳米/ EM 575-615纳米。 </li></ol></li><li>为了形象化大型组织区域具有高分辨率，获得瓦成像。对于图2A-B中，图象通过获得12×12图像的阵列的整个角膜上， 即在所示的结果，使用在各领域4荧光通道采集144字段（512×512）。共收集576图像，然后合并。 </li><li>探索标记细胞在不同深度和所述组织的结构的定位，获取感兴趣不同区域的Z堆叠图像。在订购TO映像角膜的整个深度，获取3微米的间隔8个光部分。收集以及基于最大强度图像投影的断面正交视图可使用常规的成像软件24来构造。 </li></ol> 7.角膜损伤伴他莫昔芬诱导<ol><li>麻醉小鼠用异氟烷（在气化氧为2％）和管理止痛剂（10毫克/公斤Carpofen，皮下注射）。确认适当的麻醉通过无反应到脚趾捏。 </li><li>称重1克三苯氧胺粉末与溶解在5毫升无菌DMSO中以产生200毫克/毫升浓度的溶液。 </li><li>暖三苯氧胺溶液至65℃的水浴中，直到形成澄清溶液。请槽液直到使用。 </li><li>应用眼膏，以未经处理的对侧眼，以防止在麻醉过程中脱水。不要让动物无人看管，直到它重新获得足够的骗子sciousness保持胸骨斜卧。不要返回经历了眼伤到其他老鼠的公司，直到完全恢复小鼠。小鼠应密切监测下的伤害。在情况下，他们失去其体重的10％以上，从角膜糜烂，或不适挨，实验应当停止。重复镇痛剂给药在整个实验，每24小时。 </li><li>使用无菌注射器，没有连接到它的针;应用200微升三苯氧胺溶液局部过度的各小鼠的一只眼睛的整个角膜表面。 注意:液体溶液迅速固化在眼睛表面在RT。角膜损伤的效果取决于伤口的严重性。下列的他莫昔芬/ DMSO溶液1应用没有不利影响，在小鼠中观察到。若是重复应用的角膜混浊可以观察到。 </li><li>继续组织编制和成像（见第离子5和6）在相应的时间点。对于此处所示的结果，牺牲小鼠后一周伤人（图2和24）（如在阶段5.1详述）。 </li></ol>

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The authors have nothing to disclose.

谱系追踪试验已进行了许多年。最近的技术改进和五彩纸屑小鼠品系的出现，开辟了新的途径进行研究。今天，研究人员可以受益于大量的可商购的​​组织特异性的Cre线，敲除小鼠和转基因小鼠。这为解决基本的专业知识的科学问题，避免了艰苦的练习设计多功能实验系统的机会，同时提供强大而可靠的数据。 R26R-五彩纸屑小鼠是一种优雅的工具，高效跟踪和研究细胞的性质和行为在活生物体。该系统是容易建立，它允许单细胞的非侵入性的谱系追踪胚胎发生过程中，干细胞再生下稳态，或在不同情况下，如损伤，炎症和癌症。可获得的数据提供了一个强大的不伦不类离子靶细胞，克隆细胞扩张和细胞迁移的生存，以计量的方式的。一个关键步骤是选择合适的基因小鼠品系，可以可靠地在精确的发育阶段允许高效率的组织特异性标记。这个系统的一个限制是，用于监测活生物体，组织必须是可访问和透明的。同样地，内活体动物跟踪一个厚的组织可以仅通过双光子或多光子显微镜促进。然而，细胞追踪有可能在事后组织切片，也许之后它已经转型成为透明的CLARITY方法32,33完整的组织可能会进行研究。另一个限制要考虑的是，虽然邻近的细胞表达同样的荧光团可能属于相同的无性系，它也有可能是它们可以从独立细胞来源可随机表达了相同的荧光基因衍生。这possibili使用多溴等位基因时，让无数的色彩组合TY变得不太可能。 在未来，结合五彩纸屑系统具有可用遗传工具（即敲除，敲入和转基因小鼠模型）将允许基因及其在健康和病理学突变的研究。同样，谱系下不同系统的扰动（即，干细胞小生境的破坏，激光介导的细胞消融，药物治疗）追踪将带来新的见解上的信令在细胞命运决定途径的参与。最终，组合使用荧光​​和生物发光标记，信号通路和尖端显微镜的遗传记者会为研究人员提供一个强大的系统，了解细胞如何单独到其周围的母语环境做出反应。

在胚胎发育期间，组织和器官形态发生通过可以在细胞增殖，细胞迁移和谱系规范1-4来描述一个精确程序。这些生物过程也参与维持成人组织的平衡，这需要干细胞的再生。谱系追踪，识别和细胞群的特定类型的后代的跟踪，为研究胚胎及干细胞的体内平衡的重要工具。实际上，人们越来越多地用于研究许多领域。特别是，谱系追踪成为确定的起源和干细胞命运的稳态和肿瘤发生，发展的重要工具。这是因为它可以提供有关如何在细胞的行为与完整的组织或生物体的上下文重要信息，以最少的实验介入，而相比之下，细胞分离和体外培养或transplantati对可能导致不希望的偏差。 传统上，染料如脂溶性羰花青，其中并入细胞的质膜，被用于谱系追踪。尽管这些类型的实验已经成功地导致重大发现和形状精液概念在发育生物学5,6，它们有一定的局限性，其中包括的难度，以控制目标细胞的特异性，所述染料对细胞行为的不希望的影响，并稀释随着时间的推移的标签。核苷酸类似物标记方法已启用记录"标签滞留细胞"这大概是对应于静止期的干细胞7,8慢骑自行车的存在。然而，虽然该技术可以证明在一个有限的时间周期的基础上增殖动力学慢循环细胞的存在它不能提供关于干细胞的功能方面有显见解。最佳的谱系追踪甲基odology应尽量减少标签上标记的细胞，它的后代，或邻国的性能的影响。此外，这将是有益的对标签感应控制，即，以具有在一个阶段和时间依赖性，以及在单细胞（克隆）的方式来标记的目标细胞群的能力。即到创始人单元格的所有后代传递一个稳定的，不可逆的标记方法很重要，这样的标签将保留一段时间，而不是转移到相邻小区。 最近，开发了细胞标记基因的方法。在这些系统中，细胞特异性启动子可用于触发Cre重组酶的表达，以除去一个loxP的侧翼路障并允许报告基因的表达，例如绿色荧光蛋白或Β半乳糖苷酶报告基因9-13这种方法不仅使细胞及其后代的跟踪，但也允许细胞是olation和分子特征。在单细胞水平细胞跟踪由感应一个单个荧光报道基因的表达成为可能。该系统的一个重要的限制是，只有当细胞的比例很低标记可以分离和跟踪个别克隆。为了克服这个限制多色记者都可以使用。当使用多色标记，相邻小区的在相同的小生差动命运的跟踪可以有效地14-17实现。最近，多种Brainbow（BR）等位基因谱系追踪实验被开发，使得能够利用酶Cre / lox系统的多个荧光报告基因的随机表达。在的Cre / lox系统包括Cre重组酶，其识别并结合特定的DNA序列，如loxP位点的。 Cre重组酶的以下的结合，位点特异性重组发生，这会导致去除loxP序列的侧翼序列resultin在随机表达不同的荧光蛋白质的克（的机理如下所述）。各种各样的溴系都可以使用（见评论16,18,19）。该溴菌株之间的主要差异包括在包含在磁带盒荧光基因的数目（一）的差异，从2（溴2.0），3（Br1.0）至4（Br1.1，Br2.1）荧光基因，（ⅱ）的往复取向lox位点的存在，以允许基因反转（Br2.0-2.1），用来lox位点的（ⅲ）（ⅳ）之前的表达或荧光的基因表达沉默的Cre激活的类型，和。最近设立BR3提供了神经元的多色成像的改进方法。一个此转录物的主要特征是，它含有一组新的耐光法呢荧光蛋白，这使均匀染色的最好的神经元的处理20。 重要的是，不同的版本溴盒可含有神经元特异性Thy1肾炎公关omoter或遍在表达CAG病毒（CMV）启动子。最后，虽然一些溴转基因小鼠可以包含单个溴转基因，其他人可能由多个转基因拷贝，由此允许生产的颜色组合的可能性的巨大数目被表达每个小区中（例如参见16）。 在我们的研究中，我们使用R26R，五彩纸屑鼠标包含溴2.1 盒 21。 Br2.1的独特设计允许的Cre介导的DNA倒位和由于loxP位点（图1）的倒数取向不仅删剪。因此，这些反转/切除事件，从而导致颜色变化可以继续下去，只要酶Cre存在16。出于这个原因，可诱导的颞Cre的表达系统是用于使用Br2.1可靠细胞跟踪必不可少。 如图。 1，Br2.1包含一个无处不在的CAG启动之后loxP的两侧新霉素抗性-casseTTE，其作为转录路障，即后跟4荧光报告基因，编码为绿色（GFP），黄色（YFP），红色（RFP）和青色（CFP）荧光蛋白，定位在2串联物。之前Cre重组酶激活没有荧光表达。感应Cre重组酶的使除去新-R的带盒使随机表达的4荧光蛋白之一的在给定的细胞。第一段含有loxP的侧翼GFP和一个颠倒YFP，而第二个段包含loxP的侧翼RFP和一个颠倒的CFP。这些DNA区段可以连续反转（使用loxP位即以相反的方向），或切出，只要Cre重组酶的存在。因此，一过性和受控的Cre转基因应该被使用。该Br2.1磁带提供了一个很好的系统，在信号的位置重叠的排放量区分:YFP的表达，RFP是细胞质，绿色荧光蛋白是核和CFP绑定到细胞膜。 GFP和RFP排放通过常规荧光显微镜容易地分离。为了分离YFP / GFP / CFP排放，需要一种光谱共焦成像系统。总而言之，Br2.1盒允许在每个靶细胞之一的四个不同的荧光基因随机的，可诱导的，和组织特异性表达。实际上，当需要的颜色组合的数量较多，可以产生含有2个等位基因Br2.1的纯合小鼠中，从而导致在2色标记为每个小区中的表达，以及在提高可能的颜色组合的数量，以10 22，部分溴小鼠品系的启用的更大数目的可能的颜色组合的表达，因为带盒的多个拷贝被整合到其基因组16。然而，在大多数情况下，由单个Br2.1等位基因提供四种颜色的组合是足够的。以下在此提供的协议，可以使用SPECTR的一个相对简单的设置建立此方法人用显微镜检查，如果一点任何需要进行校准。 在这里，我们提供了一个适应性强的协议为使用包含单个Br2.1等位基因R26R-五彩纸屑小鼠，对角膜上皮谱系追踪实验。这种转基因小鼠品系已被用于研究结肠21,23和角膜上皮干细胞22,24的起源和命运，干细胞活性的肠癌症25的存在，并且用于在局灶节段性肾小球硬化表征肾足细胞（FSGS） 17。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>R26RConfetti transgenic mice homozygous for the Brainbow 2.1 cassette.</td>
			<td>Jackson</td>
			<td>strain #013731</td>
			<td></td>
		</tr>
		<tr>
			<td>K14CreERT2 mice</td>
			<td>Jackson</td>
			<td>strain #005107</td>
			<td></td>
		</tr>
		<tr>
			<td>tamoxifen</td>
			<td>Sigma</td>
			<td>T5648</td>
			<td></td>
		</tr>
		<tr>
			<td>isoflurane USP Terrell</td>
			<td>Piramal Critical Care</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carprieve 30</td>
			<td>b.wt</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td>used at concentration of 4&#181;g/ml</td>
		</tr>
		<tr>
			<td>Immu Mount</td>
			<td>Thermo Scientific</td>
			<td>9990402</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM 510 Meta laser scanning confocal system hooked to an inverted motorized microscope (Axiovert 200M) equipped with a motorized stage .</td>
			<td>Zeiss</td>
			<td></td>
			<td>The spectral overlap between GFP, YFP and CFG requires the use of spectral imaging which allows the separation of the emission spectra of the fluorescent proteins. Here we use the Zeiss LSM 510 Meta spectral confocal microscope. The Meta detector included in this system has 32 detector elements each collecting emission from a 10-nanometer waveband. This technique termed &#34;spectral fingerprinting&#34; enables the user to set the most adequate limits for waveband filtering. There is a variety of spectral confocal systems that can be used, including Leica SP5(ref #21), SP8, Zeiss LSM 780 (ref #22).</td>
		</tr>
		<tr>
			<td>Zen 2009 Imaging Software</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Duratears-eye ointment</td>
			<td>Alcon</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a method for lineage tracing of corneal cells using multi-color fluorescent reporter mice

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied for many decades, revealing seminal findings in developmental biology. More recently, it was adopted by stem cell biologists to identify and track different stem cell populations with minimal experimental intervention. The recent developments in mouse genetics, the availability of a large number of mouse strains, and the advancements in fluorescent microscopy allow the straightforward design of powerful lineage tracing systems for various tissues with basic expertise, using commercially available tools. We have recently taken advantage of this powerful methodology to explore the origin and fate of stem cells at the ocular surface using R26R-Confetti mouse. This model offers a multi-color genetic system, for the expression of 4 fluorescent genes in a random manner. Here we describe the principles of this methodology and provide an adaptable protocol for designing lineage tracing experiments; specifically for the corneal epithelium as well as for other tissues.

最近，我们的目的是确定干细胞和角膜上皮24祖细胞的起源，生存和迁移。越来越多的证据支持了角膜上皮是由在角膜缘小生，一个环形区在角膜周边位于完全干细胞再生的教条。这一理论是通过体外 和体内数据，并通过所提供的基础成功开拓角膜缘干细胞疗法27-29临床证据的支持。然而，在本主题仍然高度由于基于角膜缘移植实验发现表明鼠标缘不参与角膜续期30的一项研究辩论近年来。为了验证这一有趣的假设，使用R26R，五彩纸屑小鼠中进行，世系追踪实验遵循角膜缘和角膜上皮干/祖细胞的稳态条件下的命运长达120 天 ，每天24。摘出眼睛，并配有安装角膜是由光谱激光扫描共聚焦荧光显微镜分析10和120天后他莫昔芬注射。正如所料，荧光细胞的小簇零星观察到在整个眼表面注射后10天，而1-4个月后（图2B 和 24），细长条纹，从朝向角膜中心角膜缘延伸发展缓慢。这一结果表明，角膜是由从朝向角膜的中心角膜缘移植细胞再生。接下来，伤人条件下，角膜再生进行了检查。为了根据伤人条件而诱导有效的细胞的跟踪测试K14阳性缘/角膜细胞的命运，我们溶解三苯氧胺二甲基亚砜（DMSO），这将导致在局部应用角膜损伤。这样，我们设法诱导温和伤人如果单个局部施用涂布（图2C），莫<html>减免伤害，如果额外的DMSO单独处理是在他莫昔芬诱导或DMSO时，独显在局部连续2天前他莫昔芬感应应用重伤的前一天使用。 在对比的是发展缓慢和达到4个月内稳态条件下角膜中心的薄缘条纹，细胞迁移损伤后迅速。值得注意的是，在这些条件下已经发展于7天之久，厚的角膜缘条纹。 有趣的是，颜色的多样性，有时最小（图2D）。在我们手中，偶尔，主要是红细胞在整个角膜鉴定，表明颜色多样性可以朝特定荧光团被偏置。这个不希望的偏差可以通过剂量反应测试，在Br1.0L 31和 Br2.1 21以前报道进行校准。 LES / ftp_upload / 53370 / 53370fig1.jpg"/&gt; 在 R26R-五彩纸屑 结构 图1.示意图 。（A） 该R26R-纸屑构造包含CAG启动子高表达水平，一个loxP位（黑三角）-flanked 新霉素抗性-roadblock和Brainbow2.1录像带在ROSA26轨迹16,21。该Brainbow2.1磁带包括两个头 - 尾loxP的两侧二聚体。一个二聚体包括核定位绿色荧光蛋白（GFP）和反向导向胞质黄色荧光蛋白（YFP，的反转方向被指定 - PFY）的。第二二聚体包含胞质RFP和反向取向膜-拴系的青色荧光蛋白（CFP，的反转方向被指定- PFC）16。 （B - E）当Cre重组酶的活化，可能会出现不同的切除和反转事件;可能的结果是exemplifiED B中-电子。华创活动引起，导致基因移除和/或倒置，从而导致随机表达四种荧光蛋白之一的盒随机重排。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53370/53370fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53370/53370fig2.jpg" /> 图2 角膜谱系下稳态和损伤跟踪。R26R-纸屑/ K14-的Cre小鼠注射4毫克三苯氧胺在协议中详述。在指定的时间点后感应扁平角膜成像（A - B）。角膜伤人加上Cre重组酶的诱导是由他莫昔芬的局部应用溶于DMSO实现（C - D）。获得的马赛克图像通过多通道光谱平铺共聚焦显微镜显示（A - D）。在稳态，慢速迁移细胞的放射状条纹缘角膜缘和角膜中心之间扩展。这些条纹发展缓慢，达到4-5个月（b）在角膜中心。角膜细胞簇是明显的，以及（白色箭头，B）。与此相反，大角膜缘条纹出现一个星期内伤人条件（C）下。一个例子对红细胞（D）颜色多样性的偏见。角膜缘角膜边界由虚线注解。比例尺为500微米。缩写:CFP，青色荧光蛋白;绿色荧光蛋白，绿色荧光蛋白; RFP，红色荧光蛋白; YFP，黄色荧光蛋白。这个数字已经被修改24。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53370/53370fig2large.jpg" target="_blank">请点击此处查看大图</a>这个数字。

Watch this Scientific Journal Video about A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice at JoVE.com

A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

In this article we describe the principles for designing and performing a multi-color lineage tracing experiment using R26R-Confetti mice. We provide a specific protocol for tracking corneal epithelial cells which can be modified for other tissues of interest.

方法对角膜细胞的谱系追踪使用多色荧光记者小鼠

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied ...

a-method-for-lineage-tracing-corneal-cells-using-multi-color

Universidad de Cartagena UDEC

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Developmental Biology

17.3K 次观看.  Israel Institute of Technology. In this article we describe the principles for designing and performing a multi-color lineage tracing experiment using R26R-Confetti mice. We provide a specific protocol for tracking corneal epithelial cells which can be modified for other tissues of interest.

视频: A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System

Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human cells has been low, which prevents the generation of human cell lines at a desired rate. The past two years have witnessed a rapid progression on improving efficiency of genetic manipulation by genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system. This manuscript describes a protocol for generating lineage specific human induced pluripotent stem cell (hiPSC) reporters using CRISPR/Cas system assisted homologous recombination. Procedures for obtaining necessary components for making neural lineage reporter lines using the CRISPR/Cas system, focusing on construction of targeting vectors and single guide RNAs, are described. This protocol can be extended to platform establishment and mutation correction in hiPSCs.

Genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system have greatly improved gene targeting efficiency in human induced pluripotent stem cells (hiPSCs). This manuscript describes a protocol for generating lineage specific hiPSC reporter using CRISPR/Cas system assisted homologous recombination.

的hiPSC神经细胞系特异性敲开记者代高效使用CRISPR / Cas9和Cas9双切口酶系统

Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human ...

科研

发育生物学

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

多能干细胞的海藻酸钠封装使用同轴喷嘴

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

Method of Studying Palatal Fusion using Static Organ Culture

Cleft lip and palate are among the most common of all birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium that adheres to form the midline epithelial seam (MES). The theories suggest that MES cells follow an epithelial to mesenchymal transition (EMT), apoptosis and migration, making a fused palate 1. Complete disintegration of the MES is the final essential phase of palatal confluence with surrounding mesenchymal cells. We provide a method for palate organ culture. The developed in vitro protocol allows the study of the biological and molecular processes during fusion. The applications of this technique are numerous, including evaluating responses to exogenous chemical agents, effects of regulatory and growth factors and specific proteins. Palatal organ culture has a number of advantages including manipulation at different stages of development that is not possible using in vivo studies.

Studies of palate development are motivated by the incidence of cleft palate, a birth defect that imposes a tremendous health burden and can leave lasting disfigurement. We demonstrate here a technique to culture palatal shelves that can be used to study different signaling pathways involved in palatal development and fusion.

使用静态器官培养研究腭融合的方法

Cleft lip and palate are among the most common of all birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium that ...

Co-localization of Cell Lineage Markers and the Tomato Signal

The cell lineage tracing system has been used predominantly in developmental biology studies. The use of Cre recombinase allows for the activation of the reporter in a specific cell line and all progeny. Here, we used the cell lineage tracing technique to demonstrate that chondrocytes directly transform into osteoblasts and osteocytes during long bone and mandibular condyle development using two kinds of Cre, Col10a1-Cre and Aggrecan-CreERT2 (Agg-CreERT2), crossed with Rosa26tdTomato. Both Col10 and aggrecan are well-recognized markers for chondrocytes.
On this basis, we developed a new method-cell lineage tracing in conjunction with fluorescent immunohistochemistry-to define cell fate by analyzing the expression of specific cell markers. Runx2 (a marker for early-stage osteogenic cells) and Dentin matrix protein1 (DMP1; a marker for late-stage osteogenic cells) were used to identify chondrocyte-derived bone cells and their differentiation status. This combination not only broadens the application of cell lineage tracing, but also simplifies the generation of compound mice. More importantly, the number, location, and differentiation statuses of parent cell progeny are displayed simultaneously, providing more information than cell lineage tracing alone. In conclusion, the co-application of cell lineage tracing techniques and immunofluorescence is a powerful tool for investigating cell biology in vivo.

We developed two sets of tracing combinations in Rosa26tdtomato (ubiquitously expressed in all cells)/Cre (specifically expressed in chondrocytes) mice: one with 2.3Col1a1-GFP (specific to osteoblasts) and one with immunofluorescence (specific to bone cells). The data demonstrate the direct transformation of chondrocytes into bone cells.

细胞系标记的共定位和番茄信号

The cell lineage tracing system has been used predominantly in developmental biology studies. The use of Cre recombinase allows for the activation of the ...

Cell Lineage Analyses and Gene Function Studies Using Twin-spot MARCM

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological studies to depict intricate morphologies and to manipulate the function of genes in subsets of neurons within otherwise unmarked and unperturbed organisms. Genetic mosaics generated in the MARCM system are mediated through site-specific recombination between homologous chromosomes within dividing precursor cells to produce both marked (MARCM clones) and unmarked daughter cells during mitosis. An extension of the MARCM method, called twin-spot MARCM (tsMARCM), labels both of the twin cells derived from a common progenitor with two distinct colors. This technique was developed to enable the retrieval of useful information from both hemi-lineages. By comprehensively analyzing different pairs of tsMARCM clones, the tsMARCM system permits high-resolution neural lineage mapping to reveal the exact birth-order of the labeled neurons produced from common progenitor cells. Furthermore, the tsMARCM system also extends gene function studies by permitting the phenotypic analysis of identical neurons of different animals. Here, we describe how to apply the tsMARCM system to facilitate studies of neural development in Drosophila.

Here, we present a protocol for a mosaic labeling technique that permits the visualization of neurons derived from a common progenitor cell in two distinct colors. This facilitates neural lineage analysis with the capability of birth-dating individual neurons and studying gene function in the same neurons of different individuals.

细胞谱系分析和基因功能研究使用双点MARCM

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

水平整体安装:厚三维组织横截面皮肤的新型加工和成像方案

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

活完好斑马鱼的单细胞 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Tracing Gene Expression Through Detection of &#946;-galactosidase Activity in Whole Mouse Embryos

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. The classical histochemical reaction is based on the hydrolysis of the substrate X-gal in combination with ferric and ferrous ions, which produces an insoluble blue precipitate that is easy to visualize. Therefore, &#946;-galactosidase activity serves as a marker for the expression pattern of the gene of interest as the development proceeds. Here we describe the standard protocol for the detection of &#946;-galactosidase activity in early whole mouse embryos and the subsequent method for paraffin sectioning and counterstaining. Additionally, a procedure for clarifying whole embryos is provided to better visualize X-gal staining in deeper regions of the embryo. Consistent results are obtained by performing this procedure, although optimization of reaction conditions is needed to minimize background activity. Limitations in the assay should be also considered, particularly regarding the size of the embryo in whole mount staining. Our protocol provides a sensitive and a reliable method for &#946;-galactosidase detection during the mouse development that can be further applied to the cryostat sections as well as whole organs. Thus, the dynamic gene expression patterns throughout development can be easily analyzed by using this protocol in whole embryos, but also detailed expression at the cellular level can be assessed after paraffin sectioning.

Here we describe the standard protocol for the detection of &#946;-galactosidase activity in early whole mouse embryos and the method for paraffin sectioning and counterstaining. This is an easy and quick procedure to monitor gene expression during development that can also be applied to tissue sections, organs or cultured cells.

通过检测全小鼠胚胎β乳糖酶活性追踪基因表达

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

使用由内皮结肠形成细胞和间质干细胞形成的共培养球体的三维血管生成测定系统

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...