Name: quantitative measurement of relative retinoic acid levels in e8.5 embryos and neurosphere cultures using the f9 rare-lacz cell-based reporter assay
Uploaded: 2016-09-06T14:00:00.000+00:00
Duration: 9 min 49 s
Description: Watch this Scientific Journal Video about Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay at JoVE.co

We would like to acknowledge Dr. Michael Wagner (SUNY Downstate Medical Center) and Ben Thiede (Dr. Jeff Corwin lab, University of Virginia) for providing us with the F9 RARE-LacZ cells and technical support. This study is funded by the New Jersey Commission on Spinal Cord Research (NJCSCR) (Grantnumber:10-3092-SCR-E-0) and the New Jersey Commission on Brain Injury Research (NJCBIR) (Grant number: CBIR13FEL006).

所有的动物工作是按照批准罗格斯-RWJMS IACUC方法进行。 1.解决方案和媒体准备<ol><li>准备库存和缓冲溶液按表1。按表2准备工作的解决方案。 </li></ol> 2.文化和F9稀土的LacZ细胞的维持<ol><li> 解冻冷冻细胞 <ol><li>如表2中详述的制备F9稀土LacZ的细胞培养基。 </li><li>外套10厘米培养皿（处理过的细胞培养物）用10毫升的0.2％明胶（参照表2）进行30分钟，在室温。洗去多余的明胶用10毫升蒸馏水水两漂洗。立即加入9毫升F9稀土的LacZ细胞培养基到烧瓶中，以防止干燥的明胶。 </li><li> 3分钟-在37℃水浴中2解冻F9稀土的LacZ细胞14的小瓶。板的细胞（约1×10 6个细胞/ ml）到涂覆菜Containing9毫升培养基。在37℃下培养，5％ 的 CO 2。改变介质的第二天。 </li></ol></li><li> F9稀土的LacZ细胞的维持 <ol><li>以1:10的比为3天 - 保持有规律的通路，每2。 注意:不要让细胞长成汇合，因为这会导致其分化。 </li><li>分裂培养物在80-90％铺满。删除媒体并用10ml 1×磷酸盐缓冲盐水（PBS）洗涤细胞。移除PBS并加入胰蛋白酶1毫升，孵育5分钟。再加9毫升培养介质中，吸移管的上下和分裂细胞1:10进行传代。 </li></ol></li><li> 冷冻F9稀土LacZ的细胞 <ol><li> Trypsinize含有F9稀土的LacZ细胞在80 10 cm培养皿 - 90％汇合如在步骤2.2.2详述。转移离解的细胞入15ml离心管中并旋转，在200×g离心5分钟。 </li><li>在10毫升F9稀土的LacZ的F取出上清，悬浮细胞沉淀reezing培养基（ 见表2）。分装1毫升到标有冷冻管并转移到小瓶冷冻容器。将第二天-80℃冰箱O / N和转让小瓶液氮罐中冷冻集装箱。 </li></ol></li></ol> 3. E8.5胚胎解剖<ol><li> 笼交配设置和检查插头 <ol><li>设置含有一种交配对配合笼。第二天上午，检查阴道塞的存在和指定日的插头被发现为0.523天 。在E8.5天，收获的胚胎。 </li></ol></li><li> 胚胎解剖 <ol><li>牺牲通过颈脱位水坝和喷雾用70％乙醇腹部区域（ 见表 2）。使削减用手术剪在腹部上方的皮肤和横向拉开两片（前部和后部），露出腹部。作出类似的削减在腹膜以露出腹部。 </li><li>找到子宫和输卵管子宫释放，并使用剪刀23系膜及。放置在用10ml 6cm皿（处理过的非细胞培养物）的子宫1×PBS中洗掉多余的脂肪和血。转移到含有新鲜10毫升1X PBS另外6 cm培养皿。 </li><li>单独通过削减其关闭用手术剪刀包含一个胚胎中提取一个子宫。切除子宫及蜕膜和释放含有使用两对无胚胎卵黄囊。 5镊子23。 </li><li>从胚胎中分离卵黄囊和卵黄囊转移到1.5ml微量离心管中用于基因组DNA提取将用于基因分型。使用P20吸管用大口径尖，整个胚胎移植到含有10微升胰蛋白酶的1.5 ml离心管。放置在冰上的管中。 </li><li>重复步骤3.2.3 - 3.2.4，直到所有的胚胎都被解剖。 </li></ol></li><li> 胚胎离解<ol><li>孵育含有胰蛋白酶的胚胎1.5毫升管在37℃下5分钟，以促进酶解。 <ol><li>磨碎轻轻使用用于胚胎机械解离一个P20移液器，和板含有一个解离的胚胎在F9稀土的LacZ细胞的一个孔在96孔板在整个10微升体积（见步骤5.1电镀F9稀土的LacZ细胞在一个96孔板）。培养O / N在37℃和5％的CO 2。 </li></ol></li></ol></li></ol> 4.椎板切除术，成人的解剖腰脊髓神经球和文化<ol><li> 椎板切除术 <ol><li>通过颈椎脱臼牺牲一个成人（P60）鼠标。 </li><li>喷雾用70％乙醇的背侧，并用手术剪一个横向切割。拉活板（前部和后部）分开，露出背部和脊柱。 </li><li>用手术剪刀，剪掉覆盖脊椎的肌肉。定位脊髓的腰部区域，肋的下方。使用剪刀，做一个深深的伤口，以切断脊椎在这一点上暴露脊髓。 </li><li>拉起脊柱的腰部区没有。 5钳子使得脊髓的露出横截面朝向实验者。用细剪刀提示，剪断在露出端的3点钟和9点钟位置的脊椎和肌肉。抓住用钳子切断脊椎的背部皮瓣。直到脊髓腰段的长度露出尾部继续这些削减。 </li><li>不使用从椎骨释放脊髓。 5以及钝端钳。脊髓转移到装有3毫升的DMEM / F12培养基的15毫升管。保持冰，直到所有的腰椎脊髓已被隔离。 </li></ol></li><li> 脊髓解剖 <ol><li>倾与脊髓培养基成6cm皿（无细胞培养物处理的）。 </li><li>下一个立体显微镜，通过抓住使用两对无神经节和血管移除脊髓周围段背根神经节和血管。 5镊子轻轻地从脊髓拉着他们离开。转移脊髓段到一个直径6cm培养皿没有介质和细使用手术刀刀片剁碎的组织。 </li><li>转移组织糜到含有1ml的神经球的解离介质15毫升管（ 见表2）。把管冰，并重复步骤4.2.1 - 4.2.2，直到所有脊髓已被处理。 </li><li>孵育含离解培养基组织糜管在37℃下进行10分钟，轻弹的一侧的管进行混合，然后在37℃孵育另外10分钟。经过酶消化，用磨碎的直径减小机械解离火抛光移液器。 注:准备递减之前进行实验直径火抛光的吸管。以一个玻璃巴斯德吸管和插头广角端用干净的棉花。使用酒精灯，火抛光在火焰中移液器的提示，以创建不同直径:"常规"，"精"和"超等"。对于"常规"抛光移液器，捻在火焰向圆边缘的移液管的端部不降低直径（约1毫米）。旋转中的火焰要略微较长的吸移管的端部以创建"精"（〜&gt;在1mm和&gt;直径5mm）和"超细"（〜0.5mm）的抛光移液器。不要用直径小于0.5mm使用吸管。 </li></ol></li><li> 神经干文化 <ol><li>含有降速管解离脊髓组织在200×g离心10分钟。在1ml神经球培养基除去上清，重悬细胞沉淀（ 见表2）。用P1,000吸管后跟P200移液管，以促进分解磨碎。 注意:避免产生气泡，这会降低细胞活力。 </li><li>转移10微升解离的细胞的成血球和获得细胞计数。板的细胞在用10毫升神经球培养基的T25烧瓶中的1×10 5/10 ml的密度。在37℃和5％CO 2的7天，直到神经球出现24。 </li></ol></li><li> 神经球离解 <ol><li>收集神经球文化和转移到15 mL管。旋转，在200×g离心10分钟。去除大部分上清留下1毫升后面。用P1,000吸管磨碎到重悬细胞沉淀并分离神经球成单个细胞。转移10微升解离的细胞的成血球并获得细胞计数。 </li><li>板1×10 5解离的神经球细胞过度F9 RARE-LacZ的的一个孔在96孔板（见5.1中96孔板电镀F9 RARE-LacZ的）。板3口井的重复。培养O / N在37℃和5％的CO 2。</li></ol></li></ol> 5. E8.5胚胎脊髓神经球的RA含量<ol><li> 电镀F9稀土LacZ的在96孔板 <ol><li>前一天收获E8.5胚或神经球的解离，涂布96孔板用100μl每孔的0.2％的明胶在室温30分钟。吸出明胶，并用200微升蒸馏水淋洗两次。 </li><li> Trypsinize F9稀土LacZ的细胞保持10 cm的培养皿如步骤2.2.2详细说明。板1×10 5细胞/孔的F9-RARE LacZ的细胞，但使用培养基不含G418这个时候。 </li><li>准备足够的井胚胎共培养（〜12 - 20，取决于产仔），神经球（3孔/基因型），以及用于在一式三份的标准曲线（27孔）。 </li></ol></li><li> F9稀土的LacZ和E8.5胚胎或脊髓神经球共培养 <ol><li>详见3.2节和板块上的F9顶部收获胚胎稀土的LacZ从5.1节的96孔板。以类似的方式，解离于在步骤4.4作为详细的96孔板F9 RARE-LacZ的顶部神经球和盘。培养O / N在37℃和5％的CO 2。 </li></ol></li><li> 生成标准曲线 <ol><li> 100纳米，10纳米，1纳米，100点，10点，下午1点，100 FM，由AT-的连续稀释:要生成一个标准曲线，具有以下浓度的准备全反式RA（AT-RA）解决方案RA股票（ 见表1）F9稀土LacZ的培养基。 </li><li> AT-RA浓度从5.1节加入100μl这些不同的到F9稀土LacZ的细胞。指定一个包含未处理F9稀土LacZ的细胞作为阴性对照井。制备各RA浓度和阴性对照孔的一式三份。培养O / N在37℃和5％的CO 2。 注意:F9 RARE-LacZ的细胞以剂量依赖的线性方式不同浓度的全 - 反式RA的响应。 </li></ol></li><li> RA含量 <ol><li>含有标准曲线和共培养的96孔板的O / N培养后，取出介质，并通过加入100微升2.5％的戊二醛固定的培养物（ 见表2）15分钟，室温。取出固定液，并用200微升1×PBS，每洗10分钟，洗两次澡。 </li><li>除去PBS中，并用200微升的LacZ洗涤溶液洗涤三次（ 见表2），每次洗涤10分钟。在第三次洗涤，准备在一个15毫升管包裹在箔的X-gal染色溶液（ 见表2）。计算多少染色液需要并准备适当的量（200微升/孔）。 注意:X-gal的染色液是光敏感。制备后用15分钟内的染色溶液。 </li><li>通过将用蒸馏水蘸纸巾移入塑料容器大到足以容纳一个96孔板制备的潮湿室中。加入200μl在X的每96孔板的半乳糖苷酶染色溶液并将板在湿润的腔室中。 </li><li>孵育加湿室含有96孔板在37℃，以允许的蓝色的沉淀物的发展。对于E8.5胚胎，孵育板O / N（12 - 16小时）。对于神经球文化，孵育4板 - 8小时。 </li></ol></li><li> 在OD 610和成像读取吸光度 <ol><li>显色反应后，取出的LacZ染色溶液和用200μl1×PBS中进行更换。使用标准ELISA板读数器来量化记者响应于RA的测量在610纳米的吸光度。图像单个孔使用装备有相机的标准立体显微镜。 </li></ol></li></ol> 6.亚克隆F9稀土的LacZ细胞<ol><li> 检查F9稀土的LacZ rResponse到RA <ol><li>在开始任何实验之前，先测试F9稀土LacZ的细胞对RA的响应。板F9稀土的LacZÇ在96孔板厄尔（参见5.1节），并添加1纳米全反式维甲酸（见步骤5.3）。在37℃CO / N孵育在湿润的腔室中。 </li><li>可视化使用显微镜蓝色沉淀的发展。如果颜色发展不贯穿孔中的细胞均匀（参照图1B，左），执行下面亚克隆详述。 </li></ol></li><li> F9稀土的LacZ细胞的亚克隆 <ol><li>斯普利特10 cm的培养皿中培养的F9稀土LacZ的细胞作为步骤2.2.2详细说明。代替1:10的比例的，板1×10 5个细胞/ 10毫升的低接种密度在10cm皿以允许分离的菌落的生长，使得每个集落产生从单个F9稀土的LacZ细胞。 </li><li>两天后，涂布24孔板用500μl每孔的0.2％明胶的30分钟。除去明胶溶液，并用500微升蒸馏水冲洗孔两次。用500μlF9稀土LacZ的细胞培养基的更换水。 </li><li>挑选使用无菌P10的枪头和转让到一个很好的殖民地;做24次，得到24个独立的殖民地。培养在37℃，3，5％CO 2 - 4天。 </li><li>拆分单个菌落成两个24孔板。后在培养2天后，添加的RA为1nm至之一24孔板和培养O / N各孔中。按照第5.4节详述，以测试高反应进行的LacZ染色。每个可视化的显色反应以及在显微镜下才能确定强和均匀的响应。 </li><li>一旦一个强亚克隆响应被确定，从另一24孔板展开相应的培养。进一步扩大在10cm皿的殖民地，冻结下来几个小瓶在2.3节详述，并放弃所有其他殖民地。 注意:只有关于原始菌落的四分之一导致高​​反应者。典型地，第一轮亚克隆不导致均匀的LacZ染色，并需要随后的轮亚克隆需做达到均匀强烈反应。 </li></ol></li></ol>

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I., 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+orphan+GPCR,+Gpr161,+regulates+the+retinoic+acid+and+canonical+Wnt+pathways+during+neurulation.">The orphan GPCR, Gpr161, regulates the retinoic acid and canonical Wnt pathways during neurulation.</a> Dev Biol. 402 (1), 17-31 (2015).</li><li>Hogan, B., Constantini, F., Lacy, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the Mouse Embryo: A Laboratory Manual. , (1986).</li><li>Deleyrolle, L. P., Reynolds, B. 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The authors have nothing to disclose.

所述F9稀土LacZ的细胞系14是一个功能强大的系统，该系统可用于通过组织外植12,14,17,18产生的检测的RA。它是对RA的浓度低至100 FM敏感，与在未处理的RA 12,14,18细胞没有检测到的非特异性反应。此外，报道细胞向全反式RA响应以线性方式浓度的特定范围内，从而使LacZ的反应进行量化，并用于确定RA水平12,14,18的幅度。这里介绍的快速和容易的RA记者测定法利用对胚胎和培养的成体神经干细胞的可视化（ 图 2）和相对定量测量（ 图3）的F9 RARE-LacZ报道细胞系。记者系统允许单个样品之间的内源性的RA的水平，如在图2中所示的比较，它具有足够的灵敏度，以便能够检测的RA由培养的神经球（ 图2B）产生个别E8.5胚胎（ 图2A）以及RA产生。该细胞系的不同的RA浓度的剂量依赖性响应允许对RA的水平（ 图3A）在样品中的样本（ 图3B）之间的量化以及相对的RA水平的比较。值得注意的是，F9 RARE报道细胞系测定的RA由组织样品在培养时产生的总量是很重要的。这相当于RA合成和分解代谢的净平衡。 虽然F9稀土LacZ的细胞报告系统是高度敏感的，多功能的，有我们为了保证这个系统的可靠性已经把几个关键的故障排除步骤。首先， 如图1中 ，以维持这些细胞通过定期通道的健康没有让他们g是重要排至汇合。第二，我们观察到后面的通道趋向于导致不一致RA响应，即使该培养物都在不断下选择用G418保持。要解决此，建议经过20代放弃文化和解冻新瓶。此外，必须进行定期检查，以确保记者细胞依然强劲反应到RA在媒体的存在。第三，如果早期传代显示到RA（ 图1B，左侧 ），一个或多个轮亚克隆的差或不均匀反应，直到获得（ 图1B，右击 ）强烈反应可能是必要的。最后，共培养之前，建议组织样品如胚胎和神经球解离为单细胞。我们已观察到的样品的结果中更一致的色度测量的该离解。这可能是由于一个在培养孔更均匀分布的RA，作为离解的细胞是在单与记者的细胞形式的接触。 若干修改也向原始协议14进行。第一，F9 RARE-LacZ的细胞的RA响应被直接固定细胞，并在610nm处测定吸光度，而不是收集单元并分析β-半乳糖苷酶活性14裂解物进行定量。另一种修饰是与F9稀土LacZ的离解样品的直接共培养之前RA响应的定量。以前的方法分别汇报培养样品，并从这些文化只增加媒体F9稀土的LacZ 16,18。 尽管它是一个强大的系统中，F9稀土LacZ报告分析有几个限制。这个报道细胞系中的一个限制是，虽然它强烈响应全 - 反式RA的存在，它也响应于其他维甲酸的异构体，包括9-顺​​式RA和13-顺式-RA;然而，它们是100倍更敏感的全反式RA COMPA红色的其它异构体18。另一个限制该系统是线性的剂量依赖性反应仅在10 -13中号下降到10 -7 M在-RA 14,18;因此，如果这些范围之外的比较RA的水平，这可能是必要执行样品的系列稀释液，直到测量值落在线性范围内。最后，由于这是主要的比色测定法，发色的端点将样品和实验之间变化。因此，一个标准曲线始终平行，以便镀为每一个实验中使用它的报道细胞应答的定量是重要的。 总之，我们已经报道了使用F9稀土的LacZ RA记者细胞系的定量测量RA水平E8.5胚胎和，以前未在神经球。此报告系统的多功能性可允许在几乎任何细胞或组织类型的RA水平的定量，以及mAY导致在未来变化的应用的开发。

视黄酸（RA）是视黄醇或维生素A的代谢物衍生，并通过几个蜂窝脱氢酶1的连续活动产生的。因为它的两亲化学性质的，它很容易地跨越脂质膜，并且可以作为可溶性形态形成因子1行动。这是通过作为核视黄酸受体配体和激活的基因表达2-10调控众多发育和成年细胞过程的基本分子。如果没有RA，这些RA受体（维甲酸受体）的DNA，并作为阻遏绑定拉雷什; RA结合这些受体把他们变成导致靶基因的表达1,5,6转录激活。 虽然RA信号通路是很好的特点，小尺寸（〜300 DA）和RA的两亲性使得直接组织可视化和RA的测量目前在技术上是不可行的11。对于目录的唯一方法RA水平等的测量是通过使用高效液相色谱（HPLC）11组织样品的RA隔离。这种方法通常需要大量的组织，通过集中不同的样品12便利。因此，该技术是适合于在单个胚胎或少量样品/组织的RA水平的测定不良。 为了研究RA的细胞功能，几种方法已被开发来间接推断的RA的存在。这些方法利用含有高度响应RAR-βRARE驱动的β-半乳糖苷酶或萤光素酶11,13,14的表达记者系统。通过Rossant 等人 13产生的转基因的稀土LacZ报告鼠标是用于原位 11,13,15,16可视RA信号的时空区域的理想的系统中，但不适合于定量测量。该F9稀土LacZ的细胞系14，在T他另一方面，为在组织外植体11,12,14,17,18检测的RA和RA水平的定量测量是有用的。 F9畸胎瘤细胞表达内源性α-，β-和γ-维甲酸受体19，和暴露于RA 19-21之后发起分化成顶叶内胚层。 F9细胞已长期被确立为RA诱导的细胞分化的模型，这就是为什么它被选定为一随机存取报告测定的理想细胞系。该F9衍生SIL-RA 15报告细胞瓦格纳等人产生14行包含一个E. coli&#39;LacZ基因的64 bp的维甲酸反应元件人β-维甲酸受体（RAR-β）基因（RARE）的一个副本下游。为了选择并维持稳定的克隆，该构建体也包含氨基糖苷磷酸转移酶（ 新霉素抗性 ）基因作为在G418存在的选择标记。这种结构合作nfers在RA的存在，这可以通过的LacZ染色进行可视化β-半乳糖苷的诱导表达和这个响应可以是随后使用比色法11,12,14,18定量。 这个非常通用报道细胞系已被广泛地穿过组织样本的共培养与记者细胞，如耳蜗17和胚胎底板植14用于检测内源性的RA生产。此外，记者线已通过培养分别胚胎脊髓的汇集部分和从这些培养至F9稀土的LacZ细胞18加入条件培养基用于在显影脊髓RA水平的定量。定量分析的LacZ染色后通过比色读数使用标准ELISA板读数器11,12,18进行。最后，此报道细胞系已经在检测由monitori存在RA代谢酶的使用NG改变RA水平12,18。 此处我们报告，此报道细胞系的敏感性也允许从单独共培养E8.5胚胎产生的RA水平的测量。这使得不同基因型的胚胎个体之间的比较。作为一个具体的例子，Gpr161是孤儿GPCR穿过RA信号通路22调节神经胚部分，和我们报告使用此报道细胞系来调查隐性突变在Gpr161（Gpr161 VL）上的整体的RA水平的影响胚胎。除了这一点，通用性和易于使用该报告系统的允许测量和不同组织样品中，比较，即使在从成年脊髓干细胞获得的神经球培养RA水平的自由。在这里，我们描述了通过直接共培养胚胎和神经球文化相对RA水平的定量测定与F9稀土的LacZ RA代表orter细胞系。

<table><tbody><tr><td>C3H/HeSn-Gpr161vl/J mouse strain</td><td>Jackson Laboratory</td><td>000316</td><td></td></tr><tr><td>F9 RARE-LacZ reporter cell line</td><td></td><td></td><td>from Dr. Michael Wagner (SUNY Downstate Medical Center) and Ben Thiede (Dr. Jeff Corwin lab, University of Virginia)</td></tr><tr><td>DMEM/F-12 (with L-glutamine and HEPES)</td><td>ThermoFisher Scientific</td><td>11330-057</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS), qualified</td><td>ThermoFisher Scientific</td><td>26140-079</td><td>Store at -20 &amp;deg;C in 10-ml aliquots to avoid repeated freeze-thaw; warm in 37 &amp;deg;C for media preparation</td></tr><tr><td>Penicillin/Streptomycin</td><td>ThermoFisher Scientific</td><td>15140-122</td><td>Store at -20 &amp;deg;C in 10-ml aliquots to avoid repeated freeze-thaw; light-sensitive</td></tr><tr><td>G418</td><td>ThermoFisher Scientific</td><td>10131-027</td><td>Store at 4 &amp;deg;C; light-sensitive</td></tr><tr><td>2% Gelatin</td><td>Sigma Aldrich</td><td>G1393</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>B-27 supplement (2 % stock solution)</td><td>ThermoFisher Scientific</td><td>17504-044</td><td>Store at -20 &amp;deg;C in 1-ml aliquots to avoid repeated freeze-thaw; warm at room temperature for media preparation</td></tr><tr><td>Murine Epidermal Growth Factor (EGF)</td><td>PeproTech</td><td>315-09</td><td>Prepare 100 &amp;mu;g/ml stock solution in 0.1% bovine serum albumin (BSA) in 1xPBS; prepare working concentrations by diluting with 1x PBS; store at -20 &amp;deg;C</td></tr><tr><td>Murine Fibroblast Growth Factor (FGF)-basic</td><td>PeproTech</td><td>450-33</td><td>Prepare 100 &amp;mu;g/ml stock solution in 0.1% bovine serum albumin (BSA) in 1x PBS; prepare working concentrations by diluting with 1x PBS; store at -20 &amp;deg;C</td></tr><tr><td>50% glutaraldehyde</td><td>Amresco</td><td>M155</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Sodium deoxycholate monohydrate (C24H39NaO4 &amp;middot; H2O)</td><td>Sigma Aldrich</td><td>D5670</td><td>Store at RT</td></tr><tr><td>X-gal (5-bromo-4-chloro-3-indolyl-&amp;beta;-d-galactopyranoside)</td><td>Promega</td><td>V3941</td><td>Store stock at -20 &amp;deg;C&amp;nbsp;</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma Aldrich</td><td>41639</td><td>Store at RT</td></tr><tr><td>all-trans Retinoic Acid (at-RA)</td><td>Sigma Aldrich</td><td>R-2625</td><td>Store 1 ml aliquots of stock in -80 &amp;deg;C; extremely sensitive to light</td></tr><tr><td>TrypLE Express</td><td>ThermoFisher Scientific</td><td>12605-010</td><td>Store at RT</td></tr><tr><td>0.25% trypsin-EDTA</td><td>ThermoFisher Scientific</td><td>25200-056</td><td>alternative to TrypLE Express; aliquot and store at -20 &amp;deg;C</td></tr><tr><td>Hank&#039;s Balanced Salt Solution (HBSS)</td><td>ThermoFisher Scientific</td><td>14170-112</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>10x phosphate buffered solution (PBS)</td><td>ThermoFisher Scientific</td><td>10010-023</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Papain</td><td>Worthington</td><td>LK003176</td><td>Store at 4 &amp;deg;C.&amp;nbsp;</td></tr><tr><td>Trypsin</td><td>Worthington</td><td>LS003703</td><td>Store in 1 ml aliquots at -20 &amp;deg;C.</td></tr><tr><td>Hyaluronic acid&amp;nbsp;</td><td>Calbiochem</td><td>385908</td><td>Store 200 &amp;mu;l aliquots at -20 &amp;deg;C.&amp;nbsp;</td></tr><tr><td>DNaseI</td><td>Worthington</td><td>LS002139</td><td>Store in 200 &amp;mu;l aliquots at -20 &amp;deg;C</td></tr><tr><td>Kynurenic acid</td><td>Sigma Aldrich</td><td>K3375</td><td>Store in 200 &amp;mu;l aliquots at -20 &amp;deg;C</td></tr><tr><td>95% ethanol</td><td></td><td></td><td></td></tr><tr><td>Mr. Frosty Freezing container</td><td>ThermoFisher Scientific</td><td>5100-0001</td><td></td></tr><tr><td>10 cm culture dish (non-cell culture treated)</td><td></td><td></td><td></td></tr><tr><td>10 cm culture dish (cell culture treated)</td><td></td><td></td><td></td></tr><tr><td>6 cm culture dish (non-cell culture treated)</td><td></td><td></td><td></td></tr><tr><td>96-well cell culture plates</td><td></td><td></td><td></td></tr><tr><td>1.5 ml microcentrifuge tubes</td><td></td><td></td><td></td></tr><tr><td>1.5 ml amber microcentrifuge tubes</td><td></td><td></td><td></td></tr><tr><td>1.5 ml cryovials</td><td></td><td></td><td></td></tr><tr><td>37 &amp;deg;C water bath</td><td></td><td></td><td></td></tr><tr><td>surgical scissors</td><td></td><td></td><td></td></tr><tr><td>fine surgical scissors</td><td></td><td></td><td></td></tr><tr><td>no. 5 forceps</td><td></td><td></td><td></td></tr><tr><td>blunt-ended forceps</td><td></td><td></td><td></td></tr><tr><td>scalpel blade</td><td></td><td></td><td></td></tr><tr><td>glass pasteur pipettes</td><td></td><td></td><td></td></tr><tr><td>cotton</td><td></td><td></td><td></td></tr><tr><td>alcohol lamp</td><td></td><td></td><td></td></tr><tr><td>ELISA plate reader</td><td></td><td></td><td></td></tr><tr><td>stereomicroscope</td><td></td><td></td><td></td></tr></tbody></table>

quantitative measurement of relative retinoic acid levels in e8.5 embryos and neurosphere cultures using the f9 rare-lacz cell-based reporter assay

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, neurogenesis, patterning of the hindbrain and spinal cord, and the development of multiple organ systems. Due to its chemical nature as a small amphipathic lipid, direct detection and visualization of RA histologically remains technically impossible. Currently, methods used to infer the presence and localization of RA make use of reporter systems that detect the biological activity of RA. Most established reporter systems, both transgenic mice and cell lines, make use of the highly potent RA response element (RARE) upstream of the RAR-beta gene to drive RA-inducible expression of reporter genes, such as beta-galactosidase or luciferase. The transgenic RARE-LacZ mouse is useful in visualizing spatiotemporal changes in RA signaling especially during embryonic development. However, it does not directly measure overall RA levels. As a reporter system, the F9 RARE-LacZ cell line can be used in a variety of ways, from simple detection of RA to quantitative measurements of RA levels in tissue explants. Here we describe the quantitative determination of relative RA levels generated in embryos and neurosphere cultures using the F9 RARE-LacZ reporter cell line.

所述F9稀土LacZ报道是生长在明胶包被的表面的粘附胚胎癌细胞系。的报道构建允许细胞与β-半乳糖苷酶。11,12,14,18的比例感应定量响应的RA。这些细胞显示上皮形态（ 图1A）。因为它们的高倍增率的，则建议这些细胞每2分割 - 以1:10的比为3天。在我们的与此报道细胞系的工作，我们已经观察到，即使在加入G418到培养基中，这些细胞对RA的响应减弱经过多次传代（ 图1B， 左 ），而这可能会影响的记者容量该细胞系。响应这种损失可能是由于在后面的段落转基因的部分损失。这样，周期性亚克隆有必要确保强有力和均匀的响应者RA（ 图1B，右）。 虽然该细胞系的各种用途，已经出版12,14-18，报道这里是使用该细胞系来衡量个别E8.5胚胎不同基因型的内源性RA水平。在Gpr161 VL / VL突变导致胚胎降低RA信号22。如由解离胚胎与F9稀土的LacZ细胞共培养中，这些Gpr161 VL / VL胚胎已相对于野生型同窝（ 图2A）降低内源性的RA。因为此报告细胞系，在这里也报道的通用性的是一种新型的用这些细胞以检测由从成人Gpr161 重量/重量的小鼠（ 图2B）中得到的神经球培养物产生的RA水平。记者细胞系能够证明从成年脊髓干细胞神经球文化产生内源性RA。伟大的差异erence在染色强度指示相对于E8.5胚胎脊髓神经球更大记者细胞反应。这可能是由于由神经球随时间产生的大得多的RA水平相比E8.5胚胎，这可能是由于这样的事实，神经球文化比E8.5胚胎更均匀，因此含有较多的RA生产细胞。 此外在报道细胞的剂量依赖性线性响应不同浓度在-RA的结果的。这个反应可以用比色法通过在610nm处读取吸光度来测量。然后所产生的标准曲线可用于定量样品中的RA的水平，如在野生型神经球培养物（ 图3A）或E8.5胚胎（ 图3B）。 <img alt="图1" src="/files/ftp_upload/54443/54443fig1.jpg" /> 图1. MAINTenance和F9稀土的LacZ细胞的亚克隆。 （A）F9稀土的LacZ细胞的相衬图像显示。这些细胞具有〜10小时的倍增时间，而且必须每3天通过。 -以1:10的比例（约70 80％汇合）（B）和加入1纳米的在-RA和随后的LacZ文化的周期检测染色必须完成，以确保文化保持强劲而均匀的响应者RA（右）。在贫穷和非均匀反应（左）的情况下，必须进行亚克隆。比例尺为100μm。 <a href="https://www.jove.com/files/ftp_upload/54443/54443fig1large.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/54443/54443fig2.jpg" /> 图2 中的LacZ染色共培养样品（游离E8.5胚胎/神经球）和F9稀土LacZ的细胞。（A）E8.5小鼠胚胎<html>不同基因型第解离并接种在F9稀土的LacZ细胞的顶部。 LacZ的染色24小时后可通过共培养样品产生的F9稀土LacZ的细胞内源性RA响应的可视化。所述Gpr161 VL突变导致降低内源性的RA作为可视化的报道细胞的降低的反应。（B）的左。从成人脊髓的神经干细胞培养的神经球的相衬图像被示出。对。从成年Gpr161 重量/重量脊髓衍生神经球解离并接种在F9稀土的LacZ细胞的顶部。蓝色沉淀之后的LacZ染色的存在表明内源性RA在这些神经球的存在。在神经球和胚胎E8.5共培养之间染色强度的差异表明神经球比E8.5胚胎目前RA的更高水平。比例尺为100μm。目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/54443/54443fig3.jpg" /> 图3. 标准曲线和RA楼层比色定量。在F9稀土的LacZ报道细胞系的响应可以比色在610nm处使用标准ELISA板读数器来测量。（A）中使用的量化的相对的RA水平代表标准曲线野生型神经球文化秒。从共培养E8.5胚胎相对RA水平（B）的定量。 N = 3; * P &lt;0.05，学生t检验。误差棒SEM。 <a href="https://www.jove.com/files/ftp_upload/54443/54443fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 申通CK缓冲器/解决方案</td><td> 组件 </td><td> 储存条件 </td></tr><tr><td> 10％的脱氧胆酸钠水合物（C 24 H 39的NaO 4·H 2 O） </td><td> 10毫升100毫升卫生署2 O </td><td> 4ºC </td></tr><tr><td>的1M氯化镁（MgCl 2的） </td><td></td><td> RT </td></tr><tr><td> 100X亚铁氰化钾（K 4 [铁（CN）6]·3H 6 O） </td><td> 2.12克在10毫升卫生署2 O </td><td>室温在黑暗中</td></tr><tr><td> 100×铁氰化钾（K 3的[Fe（CN）6]） </td><td> 1.64克在10毫升卫生署2 O </td><td>室温在黑暗中</td></tr><tr><td> 20毫克/毫升X-gal的（5-溴-4-氯-3-吲哚基β-D-吡喃半乳糖苷） </td><td>加入5毫升二甲基甲酰胺，使20毫克/毫升储液</td><td> -20ºC </td></tr><tr><td> 10毫米（3毫克/毫升）的所有-TRANS维甲酸</td><td> 16.67毫升无水乙醇溶解50毫克。制备1ml等份。 </td><td> -80ºC在黑暗中，在1.5ml琥珀色微量离心管</td></tr><tr><td>木瓜蛋白酶</td><td>溶解一小瓶7毫升HBSS </td><td> 4ºC </td></tr></tbody></table> 表1.缓冲器和解决方案组成。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 工作缓冲器/解决方案 </td><td> 组件 </td><td> 储存条件 </td></tr><tr><td> F9稀土LacZ的细胞培养基</td><td> 50毫升胎牛血清（FBS）（终浓度10％） </td><td>过滤消毒。 </td></tr><tr><td></td><td> 5毫升100X青霉素链霉素（终浓度1×） </td><td>保存在4ºC。 </td></tr><tr><td></td><td> 4毫升50毫克/毫升G418（终浓度0.4毫克/毫升） </td><td></td></tr><tr><td></td><td>至500ml DMEM / F-12（有L-谷氨酰胺和HEPES） </td><td></td></tr><tr><td> 0.2％的明胶</td><td> 25毫升2.0％明胶</td><td>保存在4ºC。 </td></tr><tr><td></td><td> 80毫升蒸馏水</td><td></td></tr><tr><td></td><td>拌匀</td><td></td></tr><tr><td> 70％的乙醇</td><td> 30毫升95％乙醇</td><td>在喷雾瓶中放置。 </td></tr><tr><td></td><td> 70毫升蒸馏水</td><td>存储在室温</td></tr><tr><td>神经干分离介质</td><td> 1毫升20U木瓜蛋白酶</td><td>准备新鲜的每一次</td></tr><tr><td></td><td> 100微升13毫克/毫升胰蛋白酶</td><td></td></tr><tr><td></td><td> 100微升7毫克/毫升透明质酸</td><td></td></tr><tr><td></td><td> 28微升10毫克/毫升DNA酶I </td><td></td></tr><tr><td></td><td>50微升4毫克/毫升犬尿酸</td><td></td></tr><tr><td>神经球培养基</td><td> DMEM / F-12（有L-谷氨酰胺和HEPES） </td><td>准备新鲜的每一次</td></tr><tr><td></td><td> 2％B-27的补充</td><td></td></tr><tr><td></td><td> 20纳克/ ml成纤维细胞生长因子（FGF） </td><td></td></tr><tr><td></td><td> 20ng的表皮生长因子（EGF） </td><td></td></tr><tr><td> 2.5％戊二醛</td><td> 250微升50％的戊二醛</td><td>准备新鲜的每一次</td></tr><tr><td></td><td> 4750微升1X PBS </td><td></td></tr><tr><td> LacZ的洗涤液</td><td> 0.5毫升10％脱氧胆酸盐（终浓度0.1％） </td><td>保存在4ºC。 </td></tr><tr><td></td><td> 1.0毫升100％NP-40（终浓度0.2％） </td><td></td></tr><tr><td></td><td> 50毫升10X PBS </td><td></td></tr><tr><td></td><td>蒸馏水至500ml </td><td></td></tr><tr><td>的X-gal染色溶液</td><td> 1个亚铁氰化钾</td><td>准备新鲜的每一次</td></tr><tr><td></td><td> 1X铁氰化钾</td><td></td></tr><tr><td></td><td>为1mg / ml的X-gal </td><td></td></tr><tr><td></td><td>以适当的音量使用LacZ的洗涤液</td><td></td></tr><tr><td> F9稀土LacZ的冷冻介质</td><td> F9稀土LacZ的培养基+ 5％DMSO </td><td>准备新鲜的每一次</td></tr></tbody></table> 表2.工作缓冲器和解决方案组成。

Watch this Scientific Journal Video about Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay at JoVE.co

Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay

Methods to accurately measure retinoic acid (RA) levels in small amounts of tissue do not exist. This protocol describes the easy, quantitative measurement of relative RA levels in E8.5 embryos and neurospheres using an RA reporter cell line.

使用F9稀土LACZ基于Cell的报告基因检测在E8.5胚胎和神经球文化相对维甲酸水平的定量测量

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, ...

quantitative-measurement-relative-retinoic-acid-levels-e85-embryos

Nanyang Technological University

Research

JoVE Journal

Neuroscience

7.8K 次观看.  Rutgers University. Methods to accurately measure retinoic acid (RA) levels in small amounts of tissue do not exist. This protocol describes the easy, quantitative measurement of relative RA levels in E8.5 embryos and neurospheres using an RA reporter cell line. 

视频: Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay

Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to tissue and cell-type specific regulation of gene expression during the cellular differentiation. Thus, their identification and further investigation is important in order to understand how cell fate is determined. Integration of gene expression data (e.g., microarray or RNA-seq) and results of chromatin immunoprecipitation (ChIP)-based genome-wide studies (ChIP-seq) allows large-scale identification of these regulatory regions. However, functional validation of cell-type specific enhancers requires further in vitro and in vivo experimental procedures. Here we describe how active enhancers can be identified and validated experimentally. This protocol provides a step-by-step workflow that includes: 1) identification of regulatory regions by ChIP-seq data analysis, 2) cloning and experimental validation of putative regulatory potential of the identified genomic sequences in a reporter assay, and 3) determination of enhancer activity in vivo by measuring enhancer RNA transcript level. The presented protocol is detailed enough to help anyone to set up this workflow in the lab. Importantly, the protocol can be easily adapted to and used in any cellular model system.

In this work we provide an experimental workflow of how active enhancers can be identified and experimentally validated.

预测与基因调控元件的验证激活过程中维甲酸诱导胚胎干细胞分化

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to ...

科研

发育生物学

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to cis-regulatory elements (CREs), short (&tilde;300-600 bp) stretches of genomic DNA which can lie upstream, downstream, or within the introns of the genes they control. CREs (i.e., enhancers/promoters) typically consist of multiple clustered binding sites for both transcriptional activators and repressors1-3. They serve as logical integrators of transcriptional input giving a unitary output in the form of spatiotemporally precise and quantitatively exact promoter activity. Most studies of mammalian cis-regulation to date have relied on mouse transgenesis as a means of assaying the enhancer function of CREs4-5. This technique is time-consuming, costly and, on account of insertion site effects, largely non-quantitative. On the other hand, quantitative assays for mammalian CRE function have been developed in tissue culture systems (e.g., dual luciferase assays), but the in vivo relevance of these results is often uncertain. 
Electroporation offers an excellent alternative to traditional mouse transgenesis in that it permits both spatiotemporal and quantitative assessment of cis-regulatory activity in living mammalian tissue. This technique has been particularly useful in the analysis of cis-regulation in the central nervous system, especially in the cerebral cortex and the retina6-8. While mouse retinal electroporation, both in vivo and ex vivo, has been developed and extensively described by Matsuda and Cepko6-7,9, we have recently developed a simple approach to quantify the activity of photoreceptor-specific CREs in electroporated mouse retinas10. Given that the amount of DNA that is introduced into the retina by electroporation can vary from experiment to experiment, it is necessary to include a co-electroporated 'loading control' in all experiments. In this respect, the technique is very similar to the dual luciferase assay used to quantify promoter activity in cultured cells. 
When assaying photoreceptor cis-regulatory activity, electroporation is usually performed in newborn mice (postnatal day 0, P0) which is the time of peak rod production11-12. Once retinal cell types become post-mitotic, electroporation is much less efficient. Given the high rate of rod birth in newborn mice and the fact that rods constitute more than 70% of the cells in the adult mouse retina, the majority of cells that are electroporated at P0 are rods. For this reason, rod photoreceptors are the easiest retinal cell type to study via electroporation. The technique we describe here is primarily useful for quantifying the activity of photoreceptor CREs.

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

This protocol describes a simple and inexpensive way to quantify the activity of cis-regulatory elements (i.e., enhancer/promoters) in living mouse retinas via explant electroporation. DNA preparation, retinal dissection, electroporation, retinal explant culture, and post-fixation analysis and quantification are described.

量化的活动顺</em在小鼠视网膜>调控元件外植体电穿孔

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to ...

神经科学

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

二维和三维胚体小鼠胚胎干细胞的维甲酸诱导神经分化的分析

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

解剖，文化与分析<em&gt;非洲爪蟾</em&gt;胚胎视网膜组织

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

体外实时成像在小鼠神经上皮单细胞分裂

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

Neurogenesis Using P19 Embryonal Carcinoma Cells

The P19 cell line derived from a mouse embryo-derived teratocarcinoma has the ability to differentiate into the three germ layers. In the presence of retinoic acid (RA), the suspension cultured P19 cell line is induced to differentiate into neurons. This phenomenon is extensively investigated as a neurogenesis model in vitro. Therefore, the P19 cell line is very useful for molecular and cellular studies associated with neurogenesis. However, protocols for neuronal differentiation of P19 cell line described in the literature are very complex. The method developed in this study are simple and will play a part in elucidating the molecular mechanisms in neurodevelopmental abnormalities and neurodegenerative diseases.

The P19 mouse embryonic carcinoma cell line (P19 cell line) is widely used for studying the molecular mechanism of neurogenesis with great simplification compared to in vivo analysis. Here, we present a protocol for retinoic acid-induced neurogenesis in the P19 cell line.

使用P19胚胎癌细胞的神经发生

The P19 cell line derived from a mouse embryo-derived teratocarcinoma has the ability to differentiate into the three germ layers. In the presence of ...

Multiplexed Analysis of Retinal Gene Expression and Chromatin Accessibility Using scRNA-Seq and scATAC-Seq

Powerful next generation sequencing techniques offer robust and comprehensive analysis to investigate how retinal gene regulatory networks function during development and in disease states. Single-cell RNA sequencing allows us to comprehensively profile gene expression changes observed in retinal development and disease at a cellular level, while single-cell ATAC-Seq allows analysis of chromatin accessibility and transcription factor binding to be profiled at similar resolution. Here the use of these techniques in the developing retina is described, and MULTI-Seq is demonstrated, where individual samples are labeled with a modified oligonucleotide-lipid complex, enabling researchers to both increase the scope of individual experiments and substantially reduce costs.

Here, the authors showcase the utility of MULTI-seq for phenotyping and subsequent paired scRNA-seq and scATAC-seq in characterizing the transcriptomic and chromatin accessibility profiles in retina.

使用 scRNA-Seq 和 scATAC-Seq 对视网膜基因表达和染色质可及性进行多重分析

Powerful next generation sequencing techniques offer robust and comprehensive analysis to investigate how retinal gene regulatory networks function during ...

Measuring Retinoic Acid Levels in Neurospheres Using a Retinoic Acid Reporter Cell Line

Source: Ababon, M. R. et al., <a href="https://app.jove.com/v/54443">Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay.</a>&#160;J. Vis. Exp.&#160; (2016).
This video demonstrates the measurement of retinoic acid (RA) secretion from neurosphere cells using RA reporter cells. It outlines the steps involved in co-culturing neurosphere cells with RA reporter cells and performing a colorimetric RA assay to measure RA levels.

使用视黄酸报告细胞系测量神经球中的视黄酸水平

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Solution and Media ...

JoVE Encyclopedia of Experiments

Neuropathology

Developmental and Pediatric Disorders

Employing Reporter Cells in a Co-Culture to Study Retinoic Acid Production by Mouse Embryonic Cells

Source: Ababon, M. R., et al. <a href="https://app.jove.com/v/54443">Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay.</a> J. Vis. Exp. (2016).
This video demonstrates a technique for assessing retinoic acid activity in mouse embryos. Upon isolating cells from the mouse embryos, the cells are co-cultured with retinoic acid (RA) reporter cells. Embryonic cells produce RA, which induces beta-galactosidase expression in reporter cells, indicating RA activity in the embryonic cells.

在共培养物中使用报告细胞研究小鼠胚胎细胞产生视黄酸

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines
1. Solution and Media Preparation

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Neuronal Culture Techniques

Co-culture and Interaction Studies

Retinoic Acid-Induced Neurogenesis using Mouse Embryonic Carcinoma Cells

Source: Leszczy&#324;ski, P., et al.,&#160; <a href="https://app.jove.com/v/58225">Neurogenesis Using P19 Embryonal Carcinoma Cells</a> J. Vis. Exp. (2019)
This video demonstrates a simple technique for retinoic acid-induced neurogenesis in P19 mouse embryonic carcinoma cells. Retinoic acid activates transcription factors and triggers gene expression for neuronal differentiation.

使用F9稀土LACZ基于Cell的报告基因检测在E8.5胚胎和神经球文化相对维甲酸水平的定量测量

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