我们感谢詹妮弗Emtage博士的手稿的批判性阅读，瑞安吉尔技术援助和与试剂斑马鱼的照顾和斯蒂芬C Ekker博士（梅奥诊所，Rochster，明尼苏达州）的帮助。我们的工作已经由坦普尔大学科学技术学院和美国国立卫生研究院（R01-HD061749）的支持。

1。生产的F0代的基因陷阱的显微注射。 我们发现，不同遗传背景的胚胎显示不同的公差，以此过程中，与宠物店内为基础的野生型和图宾根 - 长翅（TLF）是最宽容的，而AB和图宾根株有较差的存活率。 <ol><li>制备转座子的DNA。准备GBT-B1（pDB783，Balciuniene 等人在准备中）使用标准QIAprep小量制备试剂盒（Qiagen 27106）质粒DNA。 关键是要包括在PB洗涤步骤（这是可选的QIAprep程序），否则小量制备的DNA将被RNA酶A被污染，导致转座的mRNA的降解。注射之前，稀释的DNA在无RNA酶的水（Ambion公司AM9937）至10纳克/微升。 </li><li>制备转座基因的。线性pT3TS/Tol2（pDB600）13使用XbaI和使用mMessage机T3转录线性化的DNA（Ambion公司AM1348） 在体外转录试剂盒。我们已经通过加入0.5微升的RiboLock RNA酶抑制剂（赛默飞世Fermentas公司EO0381）改性的体外转录反应。在DNA酶处理步骤后，使用RNeasy MINELUTE试剂盒（Qiagen 74204）纯化的mRNA。评估在体外转录的mRNA的琼脂糖凝胶电泳的质量。稀释的mRNA在无RNA酶的水至40纳克/微升，并存储为2微升等分试样在-80℃下</li><li>制备显微注射组合。注射前，加入8微升稀释的DNA转座子转座到mRNA的2微升等分。 </li><li>显微注射。注3 NL的DNA / RNA混合物到使用标准显微注射技术1单元斑马鱼胚胎的卵黄，旨在为蛋黄/卵裂球的接口。收集胚胎每隔20分钟，确保注射1细胞阶段。根据我们的经验，一个熟练的人可以很容易地注入1,000胚胎在1-1.5小时。 </li><li>注射后，运行5微升的左过在1％琼脂糖凝胶上进行质量控制，以确保该转座的mRNA没有被降解注射液。 </li><li>注射胚胎护理。在下午，去除未受精和胚胎畸形和分发胚胎60-80每100毫米的培养皿。异常和死亡的胚胎在第1天受精后（DPF），DPF 2和3 DPF去除。 </li><li>筛选GFP的表达。屏幕无胚胎的GFP荧光在3 DPF（ 图3）明显的缺陷。筛查可以做到无论是在配备有荧光体视显微镜（我们使用尼康一SMZ1500）或直立显微镜（我们使用了蔡司AxioImager用5X Fluar目标）。低GFP的表达表示可能不成功注入或转座子RNA降解由于RNase污染（ 图3A）。选择约30％最亮的胚胎，任何一方有绿色荧光蛋白表达在多种组织或在特定组织的细胞高比例的（ 比较 figures 3B和3C）。从注射1,000胚胎，我们通常有多达100个发育正常，高表达GFP的胚胎。 </li><li>提高使用标准的斑马鱼饲养过程中选择F0的胚胎。这是合理的预期，选择饲养注入胚胎的20-30％能够活到成年。 </li></ol> 2。维修的无人机系统:MRFP测试仪线。 我们已经产生了14xUAS:MRFP行标通过镜头专用γCry:绿色荧光蛋白。由于无人机须通过DNA甲基化沉默，它用沉默来传播股票的低水平选择鱼是很重要的。 <ol><li>设置独立的UAS:MRFP纯合子与我们最可靠的Gal4基因诱捕线之一交配，NSF tpl6（Balciuniene等人在准备中。）。 </li><li>在第二天，转的UAS:MRFP鱼这给胚胎个别静态储罐，而胚胎收集和清洗。 </li><li>屏幕胚胎GFP和RFP的荧光在1 DPF和3 DPF。 </li><li>这给胚胎具有高MRFP表达建立下一代无人机的互交鱼:MRFP线。 </li></ol> 3。筛选F0鱼。 <ol><li>交叉个体F0与纯合子无人机:MRFP鱼（ 图4）。我们通常注入我们GBTS与野生型或豹色素沉着图案线，并且我们的纯合子的UAS:MRFP线是在黄铜的背景。因此大成注入F0鱼可以是从UAS容易辨别:MRFP鱼，省去了记录被筛选的F0是否是男性还是女性，以及从在记录和/或确定的性别失误造成潜在的错误每条鱼。因此，人才与经验非常有限，比如我们的本科学生，都能够设置和分解鱼交配。这种方法的缺点是两个不同的遗传背景有正在使用，潜在复杂功能的表型丢失的解释。 </li><li>第二天，返回黄铜无人机:MRFP鱼他们的坦克。将这些都给胚胎成单个静态储罐（我们使用哈根出兵马俑Faunarium小，但任何小坦克可以用于此目的），而胚胎的收​​集和筛选了F0的鱼。 </li><li>清洁和转移胚胎收集到新的培养皿（&lt;100每盘）在第0天的下午。 </li><li>屏幕胚胎GFP和RFP的荧光在1数码相框，数码相框2和3 DPF。拉胚积极为GFP和RFP出来，通过GFP / RFP的表达模式对它们进行排序（如果超过一个模式是在离合器观察），提高他们树立F1代。 </li><li>安乐死而只产生消极的胚胎（出50-100胚胎的总数）的F0鱼。 </li><li>越过这又产生GFP / RFP阳性后代获得第二批阳性的F0鱼。 </li></ol> 4。筛选F1的鱼。 F1的鱼进行筛选，建立F2​​家庭，记录​​基因陷阱的表达模式和5&#39;RACE和反向PCR冷冻胚胎批次分子鉴定的基因位点的陷阱。 <ol><li> MRFP鱼（ 图4）:与纯合子无人机穿越各个F1鱼。 </li><li>第二天，将F1的鱼，给了胚胎成单个静态储罐，而胚胎的收​​集和筛选。 </li><li>屏幕胚胎GFP和RFP的荧光在1数码相框，数码相框2和3 DPF。独立和照片胚胎阳性GFP和RFP 24。 </li><li>在5 DPF，冻结20 GFP / RFP阳性和20 GFP / RFP阴性胚胎鉴定insertionally突变基因通过反向PCR和5&#39;RACE 9,10批次。提高剩余GFP / RFP阳性的胚胎建立F2代。 </li></ol>

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这些作者都没有透露。

这里所描述的基因陷阱载体和方法已经被用于一些独立的实验室。根据我们的经验，也有在这个过程中的两个关键步骤。第一个关键步骤是实现基因陷阱整合注入F0胚胎率高。失败在这一步常常可以归因于RNA酶污染的注射剂，特别是小量制备的DNA。这也是非常重要的，在1阶段的细胞用于基因陷阱实验注入胚。在我们的经验，TOL2介导的基因转移是非常有效的，从而有可能以较低质量的DNA不迟于1细胞阶段或注射即使当获得的转基因株系。不合格的注射是更成问题开展基因突变的陷阱的时候，因为向量集成的只有一小部分产生有效的基因陷阱事件。第二个关键步骤是由穿越到我们的无人机，以确定基因陷阱事件:MRFP线。 MRFP表达的缺失是几乎总是指示一个增强子陷阱事件。但是，有时缺乏MRFP表达可指示14×UAS的沉默。因此，重要的是要保持在UAS:MRFP线在非沉默的状态通过使用具有高的RFP的表达的个体时越过NSF tpl6传播下一代。 我们可以预见做出了一些改进我们的基因陷阱向量和协议。第一是使用较少的重复的UAS的基因中的阱结构。它已经表明，一个5×UAS足以达到完全活化在细胞培养实验中，并且重复少的UAS序列是不容易的甲基化和沉默6,25。它也可以是可能的设计基因捕获载体为充分条件诱变，类似于使用的国际小鼠基因陷阱财团和最近适于斑马鱼2,26,27的载体。另一个改进是使用不同吨ransposon系统中，例如睡美人 28，以产生一个UAS:MRFP记者线。这将使注射TOL2基于基因陷阱直接进入记者线和F0s由incrossing筛选。此外，这将消除使用两种不同的遗传背景，使得功能表型损失的解释更简单。即使有了这些改进，这里介绍的一般Gal4的基因陷阱协议仍然是完全适用。

插入突变已被证明是一个有效的方法来剖析各种模型系统基因功能从单细胞生物如细菌和酵母多细胞生物如老鼠和植物。任何外源DNA（病毒，转座子或质粒）可以用作诱变剂通过中断基因，例如外显子或启动子的基本要素。这种简单的方法有效的目标是非常小的大的复杂的基因组中，由于外显子只占1-3％的典型脊椎动物基因组中。小的有效目标的大小可以通过非常高的矢量积分率被克服，其实例逆转录病毒插入突变的斑马鱼11,12的巨大成功。相比之下，内含子包括约20-30％的脊椎动物的基因组。在斑马鱼中，基于转座子的插入突变的载体，有效地引入空或整合后严重减效突变成内含子被命名为"基因突破transposoNS"（GBTS）8-10。为了实现高效的基因整合的陷阱，他们使用一个最小TOL2转13,14。GBTS的致突变性依赖于鱼源性剪接受体和转录终止/多聚腺苷酸化序列。负责大成生化诱变性的元素两侧通过直接loxP位点用于切除由Cre重组酶，因此，喷射的Cre表达的导致的大成诱发突变高效反转，即使一些转座子序列保持在整合基因座9,10。 最近公布的大成生化载体使用MRFP的基因陷阱记者9,10，导致两个潜在的缺点。首先，它不知道什么分数斑马鱼基因的表达在足够高的水平，通过直接荧光报告融合蛋白进行检测。第二，基因只有一小部分被预期具有重要功能。据估计，目前只有1,400-2,400所需的斑马鱼基因DEVELOPMENT 15,16。大多数基因突变陷阱预计不会显示明显的表型，因此将有限的效用。为了克服这两个限制，我们已经（在准备Balciuniene 等 ）改性GBT-R15 9,10与GAL4-VP16使用作为主要的基因陷阱记者。与AUG-MRFP少在大成R15，翻译起始位点是由GAL4-VP16删除。对于直接检测基因陷阱事件，我们的载体包含的14倍Gal4的UAS 17,18的控制下的EGFP报告。 几个附加的特性设计到我们的两分基因陷阱载体。无人机:绿色荧光蛋白盒被直接FRT位点（ 图1中灰色V形）两侧。注射Flp重组酶的mRNA导致切除的无人机系统:绿色荧光蛋白卡带，而使基因陷阱突变绿色荧光蛋白的荧光痕迹。然后，它可以在转基因线，标志着通过GFP荧光特定组织或发育事件中使用。如在其他GBTS，整个基因陷阱盒被直接loxP位点（开放五边形图1）两侧。这使得基因陷阱事件可逆由Cre重组酶的表达，很容易建立特定基因陷阱一体化之间因果关系的证明和观察到的表型（ 图2）。最后，基因诱捕盒被倒置的I-SceI的大范围核酸酶位点（黑三角图1）两侧。在果蝇中，转座子整合通常由P元素的不精确切除转化为缺失（缺失）。目前没有证据表明TOL2转座子（或任何其他转座子活跃在脊椎动物如睡美人的PiggyBac或AC / DS）的切除可导致缺失。因此，我们包括I-SceI的网站作为诱导缺失的一个潜在的替代方法，即使没有证据，我-SceI位可以在斑马鱼诱发缺失。应当指出的是，虽然I-SceⅠ位兆核酸酶可用于促进转基因在斑马鱼中，由I-SceI的大范围核酸酶切割DNA被用于研究DNA修复机制，包括易出错的非同源末端连接，在酵母和哺乳动物细胞19-22。 我们的基因陷阱载体的集成，可以通过两种不同的机制导致EGFP表达。第一个是真正的基因陷阱事件（ 图1C）:向量集成到一个基因（IMG为Insertionally突变基因），内源性IMG成绩单和GAL4-VP16的5&#39;之间的融合转录制成，并翻译成融合蛋白包含由IMG和GAL4-VP16编码的蛋白质的N-末端。该融合蛋白结合到14倍UAS和激活的eGFP的转录。第二，不太理想的情况下是增强子陷阱（ 图1D）:在eGFP的前面的最小启动子落在靠近整合位点的增强子的控制之下，从而导致产量的eGFP在abSENCE GAL4-VP16生产。我们估计，EGFP表达事件30-50％是由于增强子陷阱和50-70％是由于基因陷阱23（Balciuniene 等人在准备中）。这两个类的事件之间的区别，我们已经取得了14xUAS:MRFP转基因株系（ 图1B），为了方便标记镜头专用γCry:（。Balciuniene等人筹建中）的GFP。基因捕获事件由共表达的GFP和RFP（ 图2中比较C和D）进行验证。 在我们的试点屏幕，我们筛选270 F0鱼注射的DNA转座子转座及mRNA的混合物，后痊愈超过40基因陷阱事件。我们的两个基因陷阱集成时有发生到基因与先前公布的化学诱导突变体:美国国家科学基金会和FLR。我们的插入突变体NSF tpl6和FLR tpl24的表型是由表型的表面上难以区分S中的相应的化学诱导的突变体。我们还注意到，基因陷阱事件出现偏向附近基因的5&#39;端内含子，从而增加无效等位基因的概率。我们观察到在我们所有的基因陷阱线一定程度的无人机沉默（杂色），有的位点显然更容易受到斑斓色彩比其他人。我们不相信无人机沉默是一个显著问题的基因陷阱线传播，因为我们已经能够通过至少五个世代单独选择对GFP的表达很容易地传播我们的所有基因陷阱线。

<table border="1">
<tbody>
 <tr>
 <td>Name of the Reagent/Material</td>
 <td>Company</td>
 <td>Catalog #</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Miniprep Kit</td>
 <td>Qiagen</td>
 <td>27104</td>
 <td>Optional PB wash step is a must</td>
 </tr>
 <tr>
 <td>XbaI restriction enzyme</td>
 <td>ThermoFisher</td>
 <td>ER0681</td>
 <td></td>
 </tr>
 <tr>
 <td>mMessage Machine T3</td>
 <td>Ambion</td>
 <td>AM1348</td>
 <td></td>
 </tr>
 <tr>
 <td>RiboLock RNase Inhibitor</td>
 <td>ThermoFisher</td>
 <td>EO0381</td>
 <td></td>
 </tr>
 <tr>
 <td>RNeasy MinElute</td>
 <td>Qiagen</td>
 <td>74204</td>
 <td>Can be replaced by phenol extraction 			and precipitation. Do not use LiCl!</td>
 </tr>
 <tr>
 <td>Nuclease-free water</td>
 <td>Ambion</td>
 <td>AM9937</td>
 <td>Has to be non-DEPC treated</td>
 </tr>
 <tr>
 <td>Borosilicate glass capiliaries for 			microinjection needles</td>
 <td>World Precision Instruments</td>
 <td>1B100F-4</td>
 <td></td>
 </tr>
 <tr>
 <td>Microcapiliaries for calibration</td>
 <td>Drummond Scientific </td>
 <td>1-000-0010</td>
 <td></td>
 </tr>
 <tr>
 <td>Microloader tips</td>
 <td>Eppendorf</td>
 <td>5242 956.003</td>
 <td></td>
 </tr>
 <tr>
 <td>Needle puller *</td>
 <td>Sutter Instruments</td>
 <td>P-87</td>
 <td></td>
 </tr>
 <tr>
 <td>Stereomicroscope 			for microinjection * </td>
 <td>Nikon </td>
 <td>SMZ1500</td>
 <td>We also use Wild 5M and Olympus SZ61 			with transmitted light stand</td>
 </tr>
 <tr>
 <td>Picoinjector *</td>
 <td>Harvard Instruments</td>
 <td>Pli-90</td>
 <td>We also use Pli-100</td>
 </tr>
 <tr>
 <td>Screening microscope with GFP/RFP 			fluorescence *</td>
 <td>Zeiss</td>
 <td>Zeiss AxioImager</td>
 <td>5X Fluar objective has sufficient 			working distance</td>
 </tr>
 <tr>
 <td>Screening microscope with GFP/RFP 			fluorescence *</td>
 <td>Nikon </td>
 <td>SMZ1500</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>* We suggest this equipment only because we successfully use it in our laboratory. In each category, several comparable options from multiple manufacturers are available.</td>
 </tr>
 </tbody>
</table>

gene trapping using gal4 in zebrafish

大窝卵数和光学透明胚胎的外部发展使斑马鱼使用荧光记者标记突变基因的表达在体内插入突变的特殊脊椎动物模型系统。几个实验室已在斑马鱼建造及测试增强子和基因陷阱载体，用荧光蛋白，Gal4的和LEXA为基础的转录激活因子作为记者1-7。这些载体有两个潜在的缺点:不理想的严谨性（ 如缺乏的增强子和基因陷阱事件之间的区分能力）和低诱变性（ 如整合到基因很少产生无效等位基因）。基因断裂座子（GBTS）被开发来解决这些缺点8-10。我们已经修改了一个第一大成生化载体，GBT-R15，与GAL4-VP16使用作为主要基因陷阱记者和补充无人机:绿色荧光蛋白作为直接检测基因陷阱事件次要记者。一GAL4-VP16的应用的研究作为主要的基因陷阱记者提供了两个主要的优点。首先，它提高灵敏度表示在低表达水平的基因。其次，它使研究人员能够利用基因捕获线作为Gal4的司机在非常特殊的组织其他转基因的直接表达。这对于基因与非必要的或多余的功能，其中基因陷阱的整合可能不会导致明显的表型上尤为重要。利用GAL4-VP16作为主基因陷阱记者的缺点在于编码基因与N-末端信号序列的蛋白质是不适合于捕获，因为所得到的GAL4-VP16融合蛋白不大可能能够进入细胞核并激活转录。重要的是，利用GAL4-VP16的不预先选择的核蛋白质:我们恢复基因陷阱的突变基因编码的核，细胞质和细胞膜而发挥作用的蛋白质。

在一个成功的注射，至少80％的胚胎注射该基因捕获DNA / TOL2转座基因组合显示某种程度的GFP荧光（ 图3）。当各路GFP阳性胚胎（ 图3C）被选中建立F0池，大约十分之一的筛选F0鱼会产生基因陷阱后代。其中F0鱼的基因陷阱事件被回收，大部分传输除了几个非表达转座子集成一个单一的基因陷阱事件。然而，我们已经建立了从单一F0个人4基因诱捕线。在基因捕获线的GFP / RFP的表达模式的范围从非常具体的组织（例如，在嗅球），以相当普遍。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/50113/50113fig1highres.jpg" src="/files/ftp_upload/50113/50113fig1.jpg" /> 图1。 Ŧ含GAL4 - 他两分基因陷阱载体GBT-B1及其应用。该含Gal4的基因陷阱载体A.成分:TOL2 5&#39;和TOL2 3&#39;，最小的TOL2转座子结束13; CSA，鲤鱼β-肌动蛋白剪接受体8 ^ GAL4-VP16，AUG-少GAL4-VP16; ZP（ A），斑马鱼的β-肌动蛋白3&#39;非编码区和poly（A）从GBT-R15 9,10元素。 14XUAS，EGFP和SV40 P（A）是无人机系统的组成部分:绿色荧光蛋白表达盒17,18。箭头表示激活无人机系统:绿色荧光蛋白由GAL4-VP16的基因陷阱产生B的14XUAS:MRFP转基因标有镜头的具体γCry:GFP的卡带。箭头表示激活UAS的:MRFP通过GAL4-VP16 反式 C.基因陷阱事件。蓝框是个外显子。剪接是由虚线。D.增强陷阱事件表示。附近的增强子（棕色盒）的载体的整合可能放置的最小启动子的eGFP这增强子的控制下（棕色箭头）。这将导致的eGFP的组织特异性表达，但由于GAL4-VP16不言，MRFP表达不会被激活。 <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/50113/50113fig2highres.jpg" src="/files/ftp_upload/50113/50113fig2.jpg" /> 图2。逆转的使用Cre重组酶基因陷阱事件。基因突变陷阱（A）和酶Cre-收归基因陷阱等位基因（B）的A，B。图。C，D共表达绿色荧光蛋白（C）和RFP（D）在NSF tpl6基因的神经系统陷阱线，E，F。注射Cre重组酶的RNA进入NSF tpl6胚胎造成损失的GFP（E）和RFP（F）的表达。需要注意的是在透镜不降低的GFP表达的，正如所料。 图3。胚胎注射了GBT-B1（pDB783）基因陷阱。 :A.胚胎注射pGBT-B1（pDB783）身体的B独显小GFP荧光，C.胚胎注射pGBT-B1和TOL2转座基因:一个随机组胚胎（B）和选择饲养高表达细胞（C）。 <img alt="图4" fo:content-width="6in" fo:src="/files/ftp_upload/50113/50113fig4highres.jpg" src="/files/ftp_upload/50113/50113fig4.jpg" /> 图4。为建立Gal4的基因陷阱线实验大纲。部分重叠的红/绿结构表明共表达GFP和RFP的，而独自绿色表示嵌合基因陷阱或增强子陷阱事件。

Watch this Scientific Journal Video about Gene Trapping Using Gal4 in Zebrafish at JoVE.com

Gene Trapping Using Gal4 in Zebrafish

本协议描述的使用GAL4-VP16为主要记者和GFP / RFP在斑马鱼次要记者基因陷阱插入突变的方法。约于一体的高表达F0鱼产量的基因陷阱后代共同表达GFP和RFP十名。筛选过程可以容易地缩放，以适应执行插入诱变屏幕实验室的规模。

基因捕获斑马鱼中使用的Gal4

Large clutch size and external development of optically transparent embryos make zebrafish an exceptional vertebrate model system for in vivo insertional ...

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

18.2K 次观看.  Temple University. 本协议描述的使用GAL4-VP16为主要记者和GFP / RFP在斑马鱼次要记者基因陷阱插入突变的方法。约于一体的高表达F0鱼产量的基因陷阱后代共同表达GFP和RFP十名。筛选过程可以容易地缩放，以适应执行插入诱变屏幕实验室的规模。 

视频: Gene Trapping Using Gal4 in Zebrafish

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

斑马鱼的胚胎显微注射来分析基因的功能

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

科研

生物学

Global Gene Expression Analysis Using a Zebrafish Oligonucleotide Microarray Platform

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray technology is used in numerous biological disciplines in a variety of applications including global gene expression analysis in relation to developmental stage, to a disease state, and in toxic responses. Herein, we include a demonstration of global gene expression analysis using a comprehensive zebrafish-specific oligonucleotide microarray platform. The zebrafish expression microarray platform contains 385,000 probes, 60 base pairs in length, interrogating 37,157 targets with up to 12 probes per target. For this platform, all cDNA and genomic information available for the zebrafish was collected from various genomic databases including Ensembl (http://www.ensembl.org), VEGA (http://vega.sanger.ac.uk), UCSC (http://genome.ucsc.edu), and ZFIN (http://www.zfin.org). As a result this expression array provides complete coverage of the current zebrafish transcriptome. The zebrafish expression microarray was printed by Roche NimbleGen (Madison, WI). This technical demonstration includes the fluorescent labeling of a cDNA product, hybridization of the labeled cDNA product to the microarray platform, and array scanning for signal acquisition using the one color analysis strategy.  

Gene microarrays are powerful tools in gene expression profiling at a genome-wide level. This technology has application in a variety of biological disciplines including developmental biology and toxicology. In this video, we detail a protocol for global gene expression analysis using a comprehensive oligonucleotide microarray platform for the zebrafish.

全球基因表达分析，使用斑马鱼的寡核苷酸芯片平台

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

在细胞培养中缺氧诱导和测试

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

在体内电穿孔Morpholinos到成年斑马鱼视网膜

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Gavaging成年斑马鱼

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

使用4D共聚焦显微镜分析颅面形态的斑马鱼

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...