本出版物所报道的研究工作得到国家卫生研究院全国普通医学研究所的支持, R35GM124913、普渡大学 PI4D 奖励计划和 PVM 内部竞争基础研究基金项目。内容完全是作者的责任, 不一定代表供资代理人的官方意见。我们感谢高一川上刚为 Tol2 构造, 林为 ASAP1 构造, 和伦纳德 Zon为ubi 启动子构造通过 Addgene。

斑马鱼被安置在一个 AAALAC 批准的动物设施中, 所有的实验都是根据普渡动物保育和使用委员会 (PACUC) 批准的协议进行的。
1. Tol2 座子质粒结构制备
注: Tol2 是在鳉鱼中发现的座子, 广泛应用于斑马鱼研究社区8,9。它已成功地应用于网关站点特定的基于重组的克隆系统, 并被称为 Tol2 套件10。Tol2 套件允许创建自定义表达式构造的更方便的方法, 同时也提高了转基因的效率。因此, 这是一个简单的决定, 利用这个系统, 并创建一个无处不在的 ASAP1 表达斑马鱼线使用经过验证的泛素启动器来驱动 ASAP111。
<ol><li>创建中间条目 ASAP1 构造: pDONR221-ASAP1<ol>
<li>获得基因编码的电压传感器 ASAP1 构造, pcDNA3.1/普洛-ASAP1 (Plasmid#52519), 从 Addgene。要放大 ASAP1 编码区域, 请使用自定义的引物 (attB1-ASAP1F 和 attB2-ASAP1R) 在底漆的 5 ' 末端 (图 1) 两侧 attB 序列建立 PCR。Phusion DNA 聚合酶的高效性, PCR 条件根据先前发布的协议12进行了优化。</li>
		<li>将50µL PCR 产品装入1% 泰式凝胶中, 使用有200µL 提示的常规吸管, 并在水平凝胶罐中160伏进行电泳, 约30分钟。</li>
		<li>检查在 uv transilluminator (353 nm) 下的凝胶, 用刀片/手术刀在 uv transilluminator 下使用刮刀/刀, 如先前发布的13,14, 并将含有凝胶样品的 DNA 放入干净的1.5 毫升离心管。</li>
		<li>对 ASAP1 PCR 产品进行凝胶纯化。使用商业 DNA 凝胶纯化试剂盒, 在制造商的指导下, 恢复分离凝胶中的 dna, 并将 dna 洗脱到20µL 水中。以1µL 为样本, 用分光光度计测量 DNA 浓度。使用水作为空白控件15流软件指令。</li>
		<li>取100的纯化 PCR 产物, 并将其与 150 pDONR221 质粒在10µL 的 TE 缓冲器 (10 毫米三, 1 毫米 EDTA pH 8.0)16混合。添加2µL 的 BP Clonase II 反应, 并在室温下孵化反应在夜间。</li>
		<li>第二天, 将蛋白酶 K 的1µL 加入反应中, 并在37摄氏度上孵化出30分钟的反应。</li>
		<li>执行转换。转移反应, 并与50µL Top10 能力的大肠杆菌细胞混合, 并在冰上孵化细胞30分钟。然后, 将反应管转移到42°c 水浴中1分钟, 立即取出管并在冰上孵化2分钟。接下来, 把管子放入一个37摄氏度的振动筛中, 孵化1小时。</li>
		<li>取出管子, 将细胞板到卡那霉素 LB 板上。接下来, 在37摄氏度的一夜之间孵化出盘子 (16-18 小时)。</li>
		<li>选择单一和分离良好的殖民地, 并接种到14毫升细胞培养管与3毫升的 LB 培养基。文化他们隔夜在37°c 在一个振动筛以自转速度 250 rpm (自转每分钟)。</li>
		<li>在其指令手册17之后, 使用商业 miniprep 工具包执行 miniprep。</li>
		<li>序列3-4 质粒与 pDONR221-ASAP1 测序, 以识别阳性克隆使用 M13F 和 M13R 序引物。</li>
	</ol>
</li>
	<li>创建用于显微注射的 Tol2 构造: pDestTol2-ubi-ASAP1<ol>
<li>选择已验证 pDONR221-ASAP1 克隆的测序, 并使用水作为空白控件15的软件指令后, 用分光光度计测量其 DNA 浓度。</li>
		<li>采取100µg 的 pDONR221-ASAP1 和混合它与100µg Tol2 套件5端质粒 (pENTR5'_ubi, Addgene #27320), µg p3E-polyA (Tol2 套件 #302) 和150µg pDestTol2pA2 (Tol2 套件 #394)。使用 TE 缓冲器 (10 毫米三, 1 毫米 EDTA, pH 8.0) 在1.5 毫升离心管中调整总容积到8µL, 并由 2-5 s 短涡混合。然后, 添加2µL 的 LR Clonase II 加, 并孵化在室温下的反应过夜。</li>
		<li>第二天, 用10µL 吸管将蛋白酶 K 的1µL 加入反应中, 并在37摄氏度上孵化出30分钟的反应。</li>
		<li>执行转换并标识正克隆, 如上文所述 (步骤 1.1. 7-11)。</li>
		<li>使用分光光度计15测量测序验证的 pDestTol2-ubi-ASAP1 克隆 (图 1B) 的 DNA 浓度。通常集中是大约 200 ng 或µL。</li>
	</ol>
</li>
</ol>2. 制备 Tol2 Transposase mRNA 和注射液
<ol><li>将大肠杆菌甘油 pCS2FA-transposase 质粒 (Tol2 试剂盒 #396) 的股票用灭菌的接种循环放到 LB 板上 (用100µg/毫升氨苄西林)。一夜之间孵化37摄氏度的盘子。第二天, 选择一个单一的殖民地, 并接种它成3毫升磅 (100 µg/毫升氨苄西林) 使用消毒10µL 吸管提示。文化它隔夜在37°c 在一个振动筛以 250 rpm 的自转速度。</li>
	<li>在大肠杆菌区域性上使用商业 miniprep 工具包在其指导手册之后执行 miniprep。用1分钟离心机在 1.4万 rpm 洗脱质粒 dna 30-50 µL, 用分光光度计测定其 dna 浓度。</li>
	<li>进行线性化1-2 µg 质粒与不我限制性和纯化 dna 与 dna 清洁套件后, 它的指导手册后, 我不消化。洗脱将 DNA 转化为5µL 水, 离心1.4万转1分钟。预期浓度约为200-300 µL。</li>
	<li>执行体外转录没有我线性化 pCS2FA-transposase 作为 DNA 模板使用商业 SP6 转录试剂盒。</li>
	<li>一旦反应完成, 纯化 Tol2 transposase mRNA 使用商业 RNA 清洁套件后, 制造商的指示。最后, 将洗脱 mRNA 转化为20µL RNAase 水, 并测定分光光度计中的 RNA 浓度。预期浓度约为1-3 µg/µL. 如果需要, 样品可以储存在-80 摄氏度的冰箱里。</li>
	<li>用 Tol2 在 transposase 管中混合20µL pDestTol2-ubi-ASAP1 和µL 离心 mRNA (100 ng/吹打), 制备微注射溶液。为了防止重复解冻和冷冻引起的核酸降解, 整除6µL 每管, 并将其存储在-80 °c 冷藏库中供将来使用。</li>
</ol>3. 显微注射
<ol><li>在注射前的下午, 设置4-6 个繁殖槽, 至少有2名男性和2名女性。这些鱼不能在下午喂饱。这一步骤将减少鱼的数量和节省时间, 以清洁他们第二天早上, 同时也有助于诱导育种的反应。</li>
	<li>第二天早上, 从-80 °c 冰箱中取出准备好的注射液 (pDestTol2ubi-ASAP1 构造和 Tol2 mRNA), 并将其放在冰上。</li>
	<li>拉在鱼饲养池的分隔, 让鱼交配。一般来说, 鱼在拉出分隔线后20-30 分钟内产卵。如果没有, 再等1-2 小时。有些鱼可能根本不下蛋。在这种情况下, 重复这个实验的鱼胚收集。</li>
	<li>在等待, 确保有针准备与尖端打破了一个角度创建一个斜面边缘与镊子, 或打破一个微妙的任务雨刷。<ol>
<li>使用以下参数在微拉拔上从毛细管玻璃上拔出针头: 热 545;拉 60;速度 80;时间 250;压力500。用镊子在解剖范围 (用眼睛片断标尺为直径估计) 打破针在一个角度近似45°。根据 microinjector 的设置, 所需的针径可以是可变的, 但更小的直径是降低胚胎死亡率的首选。</li>
	</ol>
</li>
	<li>一旦鱼产卵, 收集他们在一个10厘米直径培养皿, 并把他们带到解剖范围。清除所有异常的胚胎和鱼废料。</li>
	<li>将受精的胚胎吸管成3% 琼脂糖注射模。去除多余的水分, 以帮助保持胚胎的到位。</li>
	<li>一旦所有的行都充满了可行的胚胎, 安排它们, 使单个细胞都朝同一个方向向针, 这是关于45°角度水平。这将使注入更容易后。</li>
	<li>戴上手套时, 用20µL 装吸管尖, 从冰管上取出5µL。</li>
	<li>小心地将尖端插入到断毛管的后端, 直到它开始圆锥, 以使试剂接近尖端。如果还有气泡, 摇动针头, 确保不折断笔尖。</li>
	<li>将针头直接插入微注射针夹中, 仔细拧紧, 直到针固定到位。将角度调整为关于45°。</li>
	<li>一旦针准备好并附上, 打开显微镜和气体压力罐。商业 CO2坦克通常是好的为此目的。注射容积由保持和弹射压力调整: 大约 0.5 psi 为持有和 30 psi 为弹射。请务必检查解决方案时, 按下踏板。</li>
	<li>使用一滴矿物油的舞台千分尺, 调整溶液的体积和流量, 以150µm 直径 (约 2 nL)。确保背部压力会让少量滴出针。如果没有足够的背部压力, 毛细管作用将导致液体进入针头和破坏 mRNA。</li>
	<li>一旦针被校准, 开始注射的结构进入受精胚胎的单细胞。 注意: 这需要大量的练习, 耐心和技巧, 因为细胞膜很难刺穿。重要的是, 把溶液注入细胞, 而不是蛋黄, 以生成转基因斑马鱼。这与吗啉代注射液不同。只要结构进入细胞, 针进入细胞的哪一侧就无关紧要了。如果注入蛋黄而不是细胞, 转基因的成功率会很低。单细胞阶段注射也很重要, 或体细胞嵌合体鱼将被创造。这将减少转基因进入生殖细胞的几率。</li>
	<li>使用凝胶缺口的边缘提供一个支持, 保持胚胎到位, 并允许针施加压力, 而不移动胚胎。一旦针的尖端是在单一的细胞, 按下踏板释放所需的解决方案量。对所有的胚胎重复这个过程。</li>
	<li>完成后, 将注入的胚胎通过用鱼系统水和一次性的3.4 毫升转移吸管从琼脂糖槽中冲洗出来, 将注射的胚转移到标记皿中。将胚胎储存在28.5 摄氏度的孵化器中, 让它们发育。在一天内检查清除死鱼胚胎, 并在鱼水中用0.1% 亚甲基蓝代替水。</li>
	<li>注射后大约6-8 小时, 取10个单独注射的鱼胚, 并使用最厉害的方法18制备基因组 DNA。</li>
	<li>第二天早上, 用一个荧光光源解剖显微镜来整理在非蛋黄组织中显示 GFP 的胚胎。这些鱼类胚胎应包含注入结构。</li>
	<li>执行 Tol2 检验以检查座子活动, 如前所述19。如果能检测到切除的质粒, 则保留注入的鱼类胚胎并将其饲养。如果不能检测到切除的质粒, 重复 Tol2 mRNA 的合成和显微注射过程, 直到达到 Tol2 的阳性结果。</li>
</ol>4. 建立转基因 ASAP1 鱼, Tg (ubi: ASAP1)
<ol><li>将注入的鱼 (F0代) 提高到成年, 如斑马鱼书20中所述。这通常需要4月左右。</li>
	<li>采取一个单一的成人 F0鱼和交叉它与单一的性别 wildtype 鱼。在后天繁殖后收集鱼类胚胎。将收集到的鱼类胚胎保存在28.5 摄氏度的鱼水孵化箱中, 0.1% 亚甲基蓝。</li>
	<li>第三天, 用 GFP 过滤器检查荧光解剖显微镜下的鱼胚。分类出绿色鱼类胚胎, 如果有的话, 并提高他们成年为 F1代转基因鱼, Tg (ubi: ASAP1)。 注: 由于大多数父母 F0鱼都是胚芽线遗传合体, 因此不需要孟德尔比。</li>
	<li>交叉单 F1成年鱼与 wildtype 鱼和收集鱼类胚胎。将绿色鱼类的胚胎分类, 并将它们提高到成年时为 F2代鱼。 注: 如果有单一的转基因, 绿色和非绿色鱼类胚胎应接近1:1。</li>
	<li>要查看肿瘤像恶性周围神经鞘瘤 (MPNST) 的电位变化, 交叉 F2代 Tg (ubi: ASAP1) 鱼与rpl35hi258/wt 鱼。众所周知, 可见肿瘤开始在6-8 月大的成人21,22中找到。</li>
</ol>5. 成像
<ol><li>为了图像斑马鱼胚胎, 采取多 F2世代创建者鱼并且横渡他们与 wildtype 鱼在各自的对。根据斑马鱼的分期指南23, 在不同的理想发育阶段收集鱼类胚胎。</li>
	<li>在鱼胚的早期阶段, 在10厘米直径的培养皿中, 用鱼系统水, 在解剖范围内仔细地剥除胚胎的 chorions。</li>
	<li>用3.4 毫升一次性转移吸管将一些鱼类胚胎转移到凹玻璃滑块上, 使用3% 纤维素。将胚胎调整到所需位置, 使用荧光解剖范围下的针来查看蜂窝 GFP 活动。</li>
	<li>对于鱼类胚胎的移动阶段 (较早于 12 somite 阶段), 使用 0.05% tricaine 甲磺酸麻醉鱼胚, 然后将其转移到滑梯上。简单地, 鱼胚在鱼水中被发现成 0.05% tricaine 甲磺酸, 直到它们停止游泳和失去身体平衡。此外, 添加一滴 0.05% tricaine 甲磺酸与纤维素在幻灯片上。</li>
	<li>对于不到 12 somite 阶段的鱼类胚胎, 使用一种免疫荧光复合显微镜与兼容的相机和软件进行成像。对于12岁以上的 somite 阶段鱼类胚胎, 使用荧光解剖显微镜。</li>
	<li>为了图像肿瘤细胞的电压, 首先发现鱼与 MPNST 肿瘤。然后, 麻醉鱼与 0.05% tricaine 甲磺酸。对于整个安装成像, 把鱼变成一个10厘米直径培养皿。为了查看肿瘤细胞的电活动, 在全装成像后可能会解剖出鱼肿瘤。</li>
</ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerase+chain+reaction:+basic+protocol+plus+troubleshooting+and+optimization+strategies.">Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies.</a> Journal of Visualized Experiments. (63), e3998 (2012).</li><li>Ordovas, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+small-size+DNA+fragments+using+agarose+gel+electrophoresis.">Separation of small-size DNA fragments using agarose gel electrophoresis.</a> Methods in Molecular Biology. 110, 35-42 (1998).</li><li>Downey, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+DNA+from+agarose+gels.">Extraction of DNA from agarose gels.</a> Methods Mol Biol. 235, 137-139 (2003).</li><li>Desjardins, P., Conklin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NanoDrop+microvolume+quantitation+of+nucleic+acids.">NanoDrop microvolume quantitation of nucleic acids.</a> Journal of Visualized Experiments. 45 (45), (2010).</li><li>Green, M. R., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning : a laboratory manual. , (2012).</li><li>Zhang, S., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purifying+plasmid+DNA+from+bacterial+colonies+using+the+QIAGEN+Miniprep+Kit.">Purifying plasmid DNA from bacterial colonies using the QIAGEN Miniprep Kit.</a> Journal of Visualized Experiments. (6), 247 (2007).</li><li>Meeker, N. D., Hutchinson, S. A., Ho, L., Trede, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+isolation+of+PCR-ready+genomic+DNA+from+zebrafish+tissues.">Method for isolation of PCR-ready genomic DNA from zebrafish tissues.</a> Biotechniques. 43 (5), (2007).</li><li>Kawakami, K., Koga, A., Hori, H., Shima, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excision+of+the+tol2+transposable+element+of+the+medaka+fish,+Oryzias+latipes,+in+zebrafish,+Danio+rerio.">Excision of the tol2 transposable element of the medaka fish, Oryzias latipes, in zebrafish, Danio rerio.</a> Gene. 225 (1-2), 17-22 (1998).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>Amsterdam, A., 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=Many+ribosomal+protein+genes+are+cancer+genes+in+zebrafish.">Many ribosomal protein genes are cancer genes in zebrafish.</a> PLoS Biology. 2 (5), E139 (2004).</li><li>Lai, K., 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=Many+ribosomal+protein+mutations+are+associated+with+growth+impairment+and+tumor+predisposition+in+zebrafish.">Many ribosomal protein mutations are associated with growth impairment and tumor predisposition in zebrafish.</a> Developmental Dynamics. 238 (1), 76-85 (2009).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Developmental Dynamics. 203 (3), 253-310 (1995).</li><li>Zhang, G., 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=Comparative+oncogenomic+analysis+of+copy+number+alterations+in+human+and+zebrafish+tumors+enables+cancer+driver+discovery.">Comparative oncogenomic analysis of copy number alterations in human and zebrafish tumors enables cancer driver discovery.</a> PLoS Genetics. 9 (8), e1003734 (2013).</li><li>Zhang, G., 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=Highly+aneuploid+zebrafish+malignant+peripheral+nerve+sheath+tumors+have+genetic+alterations+similar+to+human+cancers.">Highly aneuploid zebrafish malignant peripheral nerve sheath tumors have genetic alterations similar to human cancers.</a> Proceedings of the National Academy of Science USA. 107 (39), 16940-16945 (2010).</li><li>Urrego, D., Tomczak, A. P., Zahed, F., Stuhmer, W., Pardo, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potassium+channels+in+cell+cycle+and+cell+proliferation.">Potassium channels in cell cycle and cell proliferation.</a> Philosophical Transactions of the Royal Society of London Series B. 369 (1638), 20130094 (2014).</li><li>Yang, H. H., 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=Subcellular+Imaging+of+Voltage+and+Calcium+Signals+Reveals+Neural+Processing+In+Vivo.">Subcellular Imaging of Voltage and Calcium Signals Reveals Neural Processing In Vivo.</a> Cell. 166 (1), 245-257 (2016).</li><li>Chamberland, S., 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=Fast+two-photon+imaging+of+subcellular+voltage+dynamics+in+neuronal+tissue+with+genetically+encoded+indicators.">Fast two-photon imaging of subcellular voltage dynamics in neuronal tissue with genetically encoded indicators.</a> Elife. 6, (2017).</li><li>Hochbaum, D. 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作者没有什么可透露的。

虽然早在胚胎发育和人类疾病中发现了细胞和组织水平的电活动, 但体内动态电变化及其生物学作用仍然很大程度上是未知的。其中一个主要的挑战是可视化和量化的电气变化。膜片钳技术是跟踪单细胞的一种突破, 但是它在脊椎动物胚胎中的应用是有限的, 因为它们是由许多细胞组成的。由于敏感度、特异度和毒性, 目前的化学电压染料也不理想。最近在 GEVIs 发明方面的努力为我们提供了?...

生物电是指由离子通道和位于细胞膜1上的泵所介导的内源电子信号。细胞膜上的离子交换, 以及耦合电位和电流的变化, 对于兴奋性神经细胞和肌肉细胞的信号过程是必不可少的。此外, 生物电和离子梯度也有多种其他重要的生物学功能, 包括能量储存, 生物合成和代谢物运输。生物电信号也被发现作为胚胎模式形成的调节器, 如体轴, 细胞周期和细胞分化的1。因此, 这是至关重要的了解许多人类先天性疾病的结果, 这类信号的错误监管。虽然膜片钳已被广泛用于记录单个细胞, 但它仍然远未理想的同时监测在胚胎发育过程中的多个细胞在体内。此外, 由于其特异性、敏感性和毒性, 电压敏感小分子对于体内应用也不理想。
创建各种基因编码的荧光电压指示器 (GEVIs) 提供了一个新的机制来克服这一问题, 并允许易于应用研究胚胎发育, 即使他们最初是为了监测神经单元格2,3。当前可用的 GEVIs 之一是动作电位 1 (ASAP1)4的加速传感器。它由电压敏感磷酸酶的电压传感域和循环排列绿色荧光蛋白组成。因此, ASAP1 允许可视化的细胞电位变化 (极化: 明亮的绿色; 退极化: 深绿色)。ASAP1 有2毫秒的动力学, 可以跟踪阈的潜在变化4。因此, 这一遗传工具允许在实时生物监测活细胞的新水平的有效性。进一步了解生物电在胚胎发育和许多人类疾病 (如癌症) 中的作用, 将为疾病治疗和预防的关键机制提供新的基础。
斑马鱼已被证明是一个强大的动物模型, 研究发育生物学和人类疾病, 包括癌症5,6。他们分享70% 同源基因与人, 并且他们有相似的脊椎动物生物7。斑马鱼提供相对容易的护理, 大的离合器大小的鸡蛋, 易于处理的遗传学, 容易转基因, 和透明的外部胚胎发育, 这使他们成为一个优越的系统在体内成像5,6。随着大量的突变鱼线来源已经存在和一个完全有序的基因组, 斑马鱼将提供一个相对无限的科学发现范围。
为了研究细胞的体内实时电活动, 我们利用斑马鱼模型系统和 ASAP1。本文介绍了如何利用 Tol2 座子转基因将荧光电压生物传感器 ASAP1 到斑马鱼基因组中, 并在胚胎发育、鱼类幼虫运动和活肿瘤中可视化细胞电活动.

<table>
	<tbody>
		<tr>
			<td>14mL cell culture tubes</td>
			<td>VWR</td>
			<td>60818-725</td>
			<td>E.Coli culture</td>
		</tr>
		<tr>
			<td>Agarose electrophoresis tank</td>
			<td>Thermo Scientific</td>
			<td>Owl B2</td>
			<td>DNA eletrophoresis</td>
		</tr>
		<tr>
			<td>Agarose RA</td>
			<td>Amresco</td>
			<td>N605-500G</td>
			<td>For making the injection gels</td>
		</tr>
		<tr>
			<td>Attb1-ASAP1-F primer</td>
			<td>IDT DNA</td>
			<td>GGGGACAAGTTTGTACAAAAAAGCAGGCTTCACCATGGAGACGACTGTGAGGTATGAACA</td>
			<td>ASAP1 coding region amplification for subcloning</td>
		</tr>
		<tr>
			<td>Attb2-ASAP1-R primer</td>
			<td>IDT DNA</td>
			<td>GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAGGTTACCACTTCAAGTTGTTTCTTCTGTGAAGCCA</td>
			<td>ASAP1 coding region amplification for subcloning</td>
		</tr>
		<tr>
			<td>Bright field dissection scope</td>
			<td>Nikon</td>
			<td>SMZ 745</td>
			<td>Dechorionation, microinjection, mounting</td>
		</tr>
		<tr>
			<td>Color camera</td>
			<td>Zeiss</td>
			<td>AxioCam MRc</td>
			<td>Fish embryo image recording</td>
		</tr>
		<tr>
			<td>Concave slide</td>
			<td>VWR</td>
			<td>48336-001</td>
			<td>For holding fish embryos during imaging process</td>
		</tr>
		<tr>
			<td>Disposable transfer pipette 3.4 ml</td>
			<td>Thermo Scientific</td>
			<td>13-711-9AM</td>
			<td>Fish embryos and water transfer</td>
		</tr>
		<tr>
			<td>Endonuclease enzyme, Not I</td>
			<td>NEB</td>
			<td>R0189L</td>
			<td>For linearizing plasmid DNA</td>
		</tr>
		<tr>
			<td>Epifuorescent compound scope</td>
			<td>Zeiss</td>
			<td>Axio Imager.A2</td>
			<td>Fish embryo imaging</td>
		</tr>
		<tr>
			<td>Epifuorescent stereo dissection scope</td>
			<td>Zeiss</td>
			<td>Stereo Discovery.V12</td>
			<td>Fish embryo imaging</td>
		</tr>
		<tr>
			<td>Fluorescent light source</td>
			<td>Lumen dynamics</td>
			<td>X-cite seris 120</td>
			<td>Light source for fluorescence microscopes</td>
		</tr>
		<tr>
			<td>Forceps #5</td>
			<td>WPI</td>
			<td>500342</td>
			<td>Dechorionation and needle breaking</td>
		</tr>
		<tr>
			<td>Gateway BP Clonase II Enzyme mix</td>
			<td>Thermo Scientific</td>
			<td>11789020</td>
			<td>Gateway BP recombination cloning</td>
		</tr>
		<tr>
			<td>Gateway LR Clonase II Plus enzyme</td>
			<td>Thermo Scientific</td>
			<td>12538120</td>
			<td>Gateway LR recombination cloning</td>
		</tr>
		<tr>
			<td>Gel DNA Recovery Kit</td>
			<td>Zymo Research</td>
			<td>D4002</td>
			<td>DNA gel purification</td>
		</tr>
		<tr>
			<td>Loading tip</td>
			<td>Eppendorf</td>
			<td>930001007</td>
			<td>For loading injection solution into capilary needles</td>
		</tr>
		<tr>
			<td>Methylcellulose (1600cPs)</td>
			<td>Alfa Aesar</td>
			<td>43146</td>
			<td>Fish embryo mounting</td>
		</tr>
		<tr>
			<td>Methylene blue</td>
			<td>Sigma-Aldrich</td>
			<td>M9140</td>
			<td>Suppresses fungal outbreaks in Petri dishes</td>
		</tr>
		<tr>
			<td>Microinjection mold</td>
			<td>Adaptive Science Tools</td>
			<td>TU-1</td>
			<td>To prepare agaorse mold tray for holding fish embryos during injection</td>
		</tr>
		<tr>
			<td>Microinjector</td>
			<td>WPI</td>
			<td>Pneumatic Picopump PV820</td>
			<td>Microinjection injector</td>
		</tr>
		<tr>
			<td>Micro-manipulator</td>
			<td>WPI</td>
			<td>Microinjector mm33 rechts</td>
			<td>Microinjection operation</td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter instrument</td>
			<td>P-1000</td>
			<td>For preparing capillary needle</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Amresco</td>
			<td>J217-500ml</td>
			<td>For calibrating injection volume</td>
		</tr>
		<tr>
			<td>mMESSAGE mMACHINE SP6 Transcription Kit</td>
			<td>Thermo Scientific</td>
			<td>AM1340</td>
			<td>mRNA in vitro transcription</td>
		</tr>
		<tr>
			<td>Monocolor camera</td>
			<td>Zeiss</td>
			<td>AxioCam MRm</td>
			<td>Fish embryo image recording</td>
		</tr>
		<tr>
			<td>Plasmid Miniprep Kit</td>
			<td>Zymo Research</td>
			<td>D4020</td>
			<td>Prepare small amount of plasmid DNA</td>
		</tr>
		<tr>
			<td>Plastic Petri dishes</td>
			<td>VWR</td>
			<td>25384-088</td>
			<td>For holding fish or fish embryos during imaging process</td>
		</tr>
		<tr>
			<td>RNA Clean &#38; Concentrator-5</td>
			<td>Zymo Research</td>
			<td>R1015</td>
			<td>mRNA cleaning after in vitro transcription</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>NanoDrop 2000</td>
			<td>For measuring DNA and RNA concentrations</td>
		</tr>
		<tr>
			<td>Stage Micrometer</td>
			<td>Am Scope</td>
			<td>MR100</td>
			<td>Microinjection volume calibration</td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td>Bio-Rad</td>
			<td>T100</td>
			<td>DNA amplification for gene cloning</td>
		</tr>
		<tr>
			<td>Thin wall glass capillaries</td>
			<td>WPI</td>
			<td>TW100F-4</td>
			<td>Raw glass for making cappilary needle</td>
		</tr>
		<tr>
			<td>Tol2-exL1 primer</td>
			<td>IDT DNA</td>
			<td>GCACAACACCAGAAATGCCCTC</td>
			<td>Tol2 excise assay</td>
		</tr>
		<tr>
			<td>Tol2-exR primer</td>
			<td>IDT DNA</td>
			<td>ACCCTCACTAAAGGGAACAAAAG</td>
			<td>Tol2 excise assay</td>
		</tr>
		<tr>
			<td>TOP10 Chemically Competent E. coli</td>
			<td>Thermo Scientific</td>
			<td>C404006</td>
			<td>Used for transformation during gene cloning</td>
		</tr>
		<tr>
			<td>Tricaine mesylate</td>
			<td>Sigma-Aldrich</td>
			<td>A5040</td>
			<td>For anesthetizing fish or fish embryos</td>
		</tr>
		<tr>
			<td>UV trans-illuminator 302nm</td>
			<td>UVP</td>
			<td>M-20V</td>
			<td>DNA visualization</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Thermo Scientific</td>
			<td>2853</td>
			<td>For transformation process of gene cloning</td>
		</tr>
	</tbody>
</table>

visualization of cellular electrical activity in zebrafish early embryos and tumors

生物电, 由离子通道和细胞膜上的泵介导的内源电信号, 在兴奋性神经细胞和肌肉细胞的信号过程和其他许多生物学过程中起着重要作用, 如胚胎发展模式。但是, 在脊椎动物胚胎发生中需要进行体内电活动监测。基因编码的荧光电压指示器 (GEVIs) 的发展, 使我们有可能为这一挑战提供解决方案。在这里, 我们描述如何创建一个转基因的电压指示器斑马鱼使用已建立的电压指示器, ASAP1 (动作电位加速传感器 1), 作为一个例子。本研究选择了 Tol2 套件和无处不在的斑马鱼启动子ubi 。我们还解释了网关站点特定克隆的过程, Tol2 座子斑马鱼转基因, 以及早期的鱼类胚胎和鱼类肿瘤的成像过程, 使用常规 epifluorescent 显微镜。利用这条鱼线, 我们发现在斑马鱼胚胎发生和鱼类幼虫运动过程中存在着细胞电压的变化。此外, 据观察, 在一些斑马鱼恶性周围神经鞘肿瘤, 肿瘤细胞一般极化相比, 周围正常组织。

在一个成功的注射, 超过50% 注入鱼类胚胎将显示一定程度的绿色荧光在体细胞, 其中大部分将是积极的 Tol2 座子的消费检测 (图 2)。经过2-4 代与 wildtype 鱼的杂交 (直到荧光鱼达到 50%, 预期的孟德尔比), 转基因鱼被用于成像实验, 以跟踪细胞膜电位在胚胎发育过程中。首先, 在斑马鱼早期胚胎发育阶段, 在整个细胞周期内检查膜电位的变化。据观察, 细胞...

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Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

斑马鱼早期胚胎和肿瘤细胞电活动的可视化研究

在这里, 我们展示了创建一个细胞电压报告斑马鱼线的过程, 以可视化胚胎发育, 运动和鱼类肿瘤细胞在体内。

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

visualization-cellular-electrical-activity-zebrafish-early-embryos

Research

JoVE Journal

Developmental Biology

8.7K 次观看.  Purdue University. 在这里, 我们展示了创建一个细胞电压报告斑马鱼线的过程, 以可视化胚胎发育, 运动和鱼类肿瘤细胞在体内。

视频: Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological systems. In FDAP experiments, the clearance of proteins within living organisms can be measured. A protein of interest is tagged with a photoconvertible fluorescent protein, expressed in vivo and photoconverted, and the decrease in the photoconverted signal over time is monitored. The data is then fitted with an appropriate clearance model to determine the protein half-life. Importantly, the clearance kinetics of protein populations in different compartments of the organism can be examined separately by applying compartmental masks. This approach has been used to determine the intra- and extracellular half-lives of secreted signaling proteins during zebrafish development. Here, we describe a protocol for FDAP experiments in zebrafish embryos. It should be possible to use FDAP to determine the clearance kinetics of any taggable protein in any optically accessible organism.

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Protein levels in cells and tissues are often tightly regulated by the balance of protein production and clearance. Using Fluorescence Decay After Photoconversion (FDAP), the clearance kinetics of proteins can be experimentally measured in vivo.

测量生活中的斑马鱼胚胎蛋白质稳定性使用荧光衰减光转化后（FDAP）

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

科研

发育生物学

Mechanical Vessel Injury in Zebrafish Embryos

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.

This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy.

在斑马鱼胚胎的机械血管损伤

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

转录组分析]<em&gt;活体</em&gt;生产牛胚胎植入前胚胎采用双色微阵列平台

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

分离和斑马鱼胚胎单细胞的表征

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

生物传感运动神经元膜电位在活斑马鱼胚胎

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

诱导生活青蛙和斑马鱼胚胎缺氧

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

多光子时间推移成像实时可视化发展: 迁移神经嵴细胞在斑马鱼胚胎中的可视化

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

斑马鱼胚胎和幼虫的整个山免疫性化学

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

为早期小鼠胚胎测序准备小型RNA库

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...