这项工作是由遗传，发育与细胞生物学和爱荷华州立大学的系和国立卫生研究院授予EY024941（WJ）。DR资助。杰弗里Essner提供稿件的意见。

所有的动物程序是按照美国国立卫生研究院的指导方针和机构动物护理和使用委员会在爱荷华州立大学和马里兰大学获得批准。 1. TALEN设计<ol><li>输入所需的靶序列到TALEN设计网站。 （例如: <a href="https://tale-nt.cac.cornell.edu/node/add/talen">https://tale-nt.cac.cornell.edu/node/add/talen</a> ）。输入选择间隔/复读数组的长度。 <ol><li>基因组序列拷贝到标有"序"框。 </li><li>内"提供定制间隔/ RVD长度"选项卡中选择间隔长度和数组的长度。 注:15个碱基对长度的间隔，重复15-17工作数组长度很好，使组装不太复杂。 </li></ol></li><li>选择TALEN对。围绕一个独特的限制性酶位点而设计TALEN对允许通过限制酶的基因分型消化一个PCR产物。 </li><li>设计引物扩增周围使用的网站，如:引物的30-32 TALEN目标站点的基因组区域。当用限制性酶进行基因分型，设计引物来扩增包含该限制酶位点只有一次的区域。因此建议该区域被放大和前TALEN施工，以确定存在于A.任何多态性测序要使用mexicanus实验室人口显微注射。 </li></ol> 2. TALEN大会（从TALEN包协议修改）33,34 有关其他详细信息和故障排除，请参阅该协议34。 <ol><li>备，并根据制造商的说明从TALEN试剂盒测序必要质粒。 </li><li>设立＃的反应，A和B. 1反应包括重复变量diresidues（RVDS）1-10和反应A.目的载体pFUS_A包括RVDS 11-（N-1）和第È目标向量pFUS_B在反应B（N-1），其中N是在TALEN RVDS的总数。 <ol><li>添加到每个反应:1微升含有各RVD（100毫微克/微升），目标向量（100毫微克/微升），1微升BSA限制性内切酶，1μl的牛血清白蛋白（BSA）（2毫克/毫升每种质粒的），1微升连接酶，2微升10×连接酶缓冲液和x的微升水为20微升的总体积。例如，参照表1。 注:半反应也可以用。 </li><li>将反应在热循环和运行周期:10倍（37°C / 5分钟+ 16°C / 10分钟）+ 50°C / 5分钟+ 80°C / 5分钟。 </li></ol></li><li>孵育核酸酶反应。各反应加1微升25毫摩尔ATP和1微升核酸。在37℃孵育反应1小时。 </li><li>变换反应。 <ol><li>变换2.5微升每个反应到25微升化学感受态细胞。 注:自制能力Ç厄尔可以使用。然而，随着低能力的细胞可能会导致缺乏殖民地。 <ol><li>混合2.5微升25微升化学感受态细胞的反应。在冰上孵育五分钟。孵育30秒将细胞在42℃。 </li><li>放置在冰上的管2分钟。加入125微升中超最佳肉汤代谢产物抑制（SOC）的。摇管在37℃下1小时。 </li></ol></li><li>板100微升转化细胞在LB平板上壮观霉素（50微克/毫升）中，X-gal和异丙基β-D-1-硫代半乳糖苷（IPTG）。成长O / N在37℃。 </li></ol></li><li>挑各反应2-3的白色菌落，并使用引物pCR8_F1和pCR8_R1（ 表2）由菌落PCR确认。 <ol><li>使试剂的主结构。例如，参照表3中 。 </li><li>挑取菌落并涂抹成PCR管的底部，然后把殖民地的遗体于2ml LB与壮观霉素（50微克/毫升）。将15微升主混合物与殖民地管。 </li><li>运行以下PCR程序（ 表4） </li><li>检查的PCR（运行整个卷）上通过电泳在1.5％琼脂糖凝胶。正确的克隆将具有预期大小的带，以及一涂抹和频带的梯形。为适当的涂抹的一个例子，见34,33。 </li><li>生长2毫升培养物在LB培养基O / N的正确的克隆于37℃在振荡培养箱中。 </li></ol></li><li>根据制造商的说明小量制备质粒。 </li><li>顺序检查TALEN序列以pCR8_F1和pCR8_R1。按照前述方法35。 </li><li>利用测序验证正确的克隆，建立了反应＃2（TALEN包），这将放置在A和B的载体和最终RVD的RVDS到目标向量。 <ol><li>制备用于反应2（ 表5）的组合。 注:哈LF反应都可以使用。 </li><li>发生在一个热循环反应，并运行下面的程序:37℃/ 10分钟+ 16°C / 15分钟+ 37°C / 15分钟+ 80°C / 5分钟。 </li></ol></li><li>变换反应。 <ol><li>变换2.5微升每个反应到25微升化学感受态细胞。 <ol><li>混合2.5微升25微升化学感受态细胞的反应。在冰上孵育5分钟。热休克细胞30秒，在42℃。 </li><li>放置管在冰上。加入125微升SOC的。放置在振荡培养箱管在37℃下1小时。 </li></ol></li><li>板100微升转化细胞在LB平板上的氨苄青霉素（100微克/毫升）中，X-gal和IPTG的。成长O / N，37°C。 </li></ol></li><li>挑1-3每个反应的白色菌落，并通过使用引物TAL_F1和TAL_R2（ 表2）菌落PCR检查。 <ol><li>使聚合酶mastermix，水和引物作为溶液在表3中，选择一个殖民地，它涂抹到PCR管的底部，然后把殖民地的残骸分成2毫升LB氨苄青霉素（100微克/毫升）。放置15微升溶液与集落的管。 </li><li>运行PCR程序（ 表6）。 </li><li>检查通过电泳在1.5％琼脂糖凝胶对PCR。正确的克隆将有拖尾和乐队的阶梯。为适当的涂抹的一个例子，见34,33。 </li><li>生长2毫升培养物在LB培养基O / N的正确的克隆于37℃在振荡培养箱中。 </li></ol></li><li>根据制造商的说明小量制备质粒。 </li><li>顺序检查TALEN序列以TAL_F1和TAL_R2以下先前描述的方法35。 </li></ol> 3. TALENS的转录表达<ol><li>消化序列验证模板的4微克与2微升萨克我2小时，在37℃。 </LI&gt; <li>上通过电泳在1.5％琼脂糖凝胶上运行2微升萨克消化的质粒。被正确地消化质粒将显示单一条带。 </li><li>净化后的PCR纯化试剂盒协议其余囊消化的质粒。与之前洗脱洗涤溶液洗涤两次。洗脱到的无核酸酶水30微升。 </li><li>遵循T3的mRNA生产的标准协议。 <ol><li>设置使用线性模板的0.5微克半反应（上述制备）。孵育在37℃下2小时。 </li><li>添加脱氧核糖核酸酶（包含在试剂盒）0.5微升，在37℃下孵育15分钟。 </li></ol></li><li>净化的mRNA，按照制造商的说明进行操作。洗脱到30μl的无核酸酶水。 </li><li>通过电泳跑1.5％琼脂糖凝胶来检查的mRNA。 <ol><li>清洁凝胶设备与产品，以消除RNase污染，并准备一个1.2％的凝胶。 </li><li>混合料1微升mRNA的+4＃181:L不含核酸酶的水+ 5微升乙醛酰样染料。在50℃孵育样品30分钟。离心机在运行前凝胶管短暂地置于冰上。 </li></ol></li><li>检查用定量核酸浓度的方法，该浓度并存储在等分试样的RNA在-80℃。选择的等分试样尺寸，使得RNA的不冻结/解冻一次以上。 注:500-1000纳克的浓度/通常获得NL。 （由带，而不是在凝胶上的涂抹评定），为RNA的完整性被保持具有较低浓度的RNA可以用作长。 </li></ol> 4.注入Astyanax mexicanus胚胎TALEN表达<ol><li>准备工具注入。 <ol><li>通过在鱼水注入1.2％琼脂糖（水用碳酸氢钠和海盐至pH 7.4，电导率700μS空调）到一个培养皿倾注射板。将模具（与预测塑料片，使鱼井鸡蛋）的菜里面。除去当琼脂糖硬化的模具和储存在4℃。有关模具详见36。 </li><li>拉使用根据制造商的说明的针拔出器的注射针。 注意:适当的针的长度是注射重要。针太长会打针太灵活。拉针的协议将用来拉针设备的不同而不同。合适的针的一个例子示于图1。对于我们的设备，我们拉是5-6厘米长针，以及破碎时在约0.011毫米尖端的外径。然而，针头应校准（步骤4.3.4），以确定开口宽度。拉针的示例程序可以在表7中找到。 </li><li>通过打破吸管使开口足够大的鸡蛋通过准备胚胎移植玻璃吸管。火焰破月底到它不再尖锐。 注意:使用玻璃吸管和玻璃碗重要的是与A.时mexicanus卵子和胚胎，因为他们是粘，将坚持以塑料。 </li></ol></li><li>收集1-细胞阶段的鸡蛋。 <ol><li> A.品种mexicanus以下标准协议37。 注:例如，如果鱼保持在14光:10暗周期和和ZT14使用Zeitgerber时间（ZT）与ZT0作为灯的灯关，我们的ZT15和ZT19的表面鱼卵。确切的产卵时间必须为每个单独的实验室来确定。 </li><li>通过交配之前过度喂食的鱼3-4天，并把鱼放入清水催产。提高温度2°F。注:我们最初的水温为74左右°F。 </li><li>表面收集鱼卵在黑暗中，检查每15分钟在1细胞阶段获得鸡蛋。数百蛋可以从一个单一的一对表面的鱼获得。 </li><li>搜集鸡蛋在玻璃碗防止粘到塑料表面和排序，以在1-细胞期胚胎注射前通过在显微镜下观察卵和收集卵是单个细胞分离。保持蛋在新鲜的系统中的水（水箱，其中成鱼被容纳，这已为pH和电导率处理的）。 </li></ol></li><li>注射TALEN表达。 <ol><li>注入不同量的总mRNA（在一对等量各TALEN的），以确定注射的最佳浓度为毒性和效率由TALEN对而异。通过注入总mRNA的浓度是400-800皮克开始。稀释并结合基因到所需的浓度为1.5注射NL。 </li><li>负载稀释的mRNA进针的背面和针附着到微注射器。 </li><li>使用镊子断针。 </li><li>校准针。例如，喷射10次，并收集在一个微毛细管所得降。 10×100一次性1.081; L，32毫米微毛细管，下降应填写微毛细管至0.5毫米，1.5升/注射1次。根据需要调整注射时间和压力。 </li><li>针插入的单电池和注入的mRNA。直接注入​​基因进入细胞内，没入蛋黄。 </li><li>收集在玻璃碗注射胚胎。胚胎保持在23-25​​℃。除去死胚定期注射后的头几天（即变得浑浊和不规则形胚）。从控制（未注射）和注射板死亡，畸形胚胎创纪录的数字。 注:增加mRNA的浓度会导致增加毒性和胚胎畸形/死亡。因此，相对于毒性效率必须平衡，以确定mRNA的最佳浓度注入。 </li></ol></li></ol> 5.表型方正鱼和评估TALEN效率<ol><li>根据机构的动物祭祀协议胚胎。 注意:我们安乐死胚胎通过快速冷却冰。 </li><li>收集胚胎成用移液0.8微升PCR联管。 注:基因分型可在单个胚胎或胚胎的池来进行。 </li><li>提取DNA。 <ol><li>地方胚胎入100微升50氢氧毫氢氧化钠（NaOH）和孵育在95℃下进行30分钟，然后冷却至4℃。 </li><li>添加的1M的Tris-HCl pH为8的1/10体积（10微升）。 </li></ol></li><li>使用在步骤1.3所设计的引物（ 见表8和9样品的协议）的区域执行PCR。个别胚1微升的DNA是足够的PCR反应。 </li><li>消化用合适的限制性酶将所得的PCR产物，并通过电泳运行1.5％琼脂糖凝胶。 <ol><li>例如，对于在注射的胚胎基因分型的眼皮肤白化病2（OCA2）位点用限制酶BSR我通过加入0消化PCR产物0.5微升BSR我直接的12.5微升完成PCR反应，在65℃下2小时温育。运行未消化和在凝胶中消化产物。限制性内切酶抗性带（ 即 ，不易消化频带）表明TALEN诱发突变都存在（ 图2）。 </li></ol></li><li> （可选的）通过使用凝胶分析工具如先前29描述的来计算每个频带的强度值在斐济38分析的凝胶的图像确定未切割产物的百分比计算的百分比的突变率。 </li><li>由TA克隆凝胶纯化限制酶抗性突变体带和测序克隆确定突变等位基因的序列。 <ol><li>凝胶纯化限制按照制造商的说明书酶抗性突变体带。 TA克隆带按照制造商的说明进行操作。挑选殖民地和在摇动生长在1.5ml LB O / N在37℃孵化器。 </li><li>小量制备培养物按照制造商的说明进行操作。发送该DNA测序。 </li><li>使用程序如猿，对准突变序列与野生型序列。 <ol><li>复制并粘贴两个序列成APE文件。选择"工具"菜单中的"对齐两个序列"的​​工具。 </li><li>指定使用下拉菜单两个DNA序列。 注意:克隆的序列的反向互补可能需要根据在PCR进入克隆载体的方向被使用。如果序列不对齐，重复步骤检查在"对齐DNA"中的"启-Com的"框。 </li></ol></li></ol></li><li>评价创始人鱼在适当阶段的表型和使用用于预期表型29适当的方法（例如， 图3）。表型的方法，将基于使用protoc用于基因预期的表型由研究人员的目标醇。 </li></ol> 6.屏幕种系传递注:A. mexicanus在4-8个月达到性成熟。 <ol><li>交叉性成熟创始人鱼类与野生型鱼。 </li><li>屏幕胚或利用在步骤5.1-5.7方法成鱼（ 图4）。对于成年鱼，一块尾巴可按照麻醉被裁剪。 <ol><li>通过在三卡因（3-氨基苯甲酸乙酯）的溶液中浸没鱼减少翅片削波期间的应力麻醉鱼。继鳍剪裁，让鱼在淡水中恢复。鱼快速恢复;观察直至病愈（通常是游泳）。 </li></ol></li></ol>

The authors have nothing to disclose.

大踏步近年来已取得一了解性状进化的遗传基础。虽然一些性状的进化底层候选基因已被确定，它由于缺乏最进化有趣物种遗传易处理的仍然是具有挑战性的，以测试在体内这些基因。这里，我们报告中的A.基因组编辑的方法mexicanus，使用一个物种，研究洞穴动物的进化。遗传图谱研究1,21,23和候选基因方法19,40确定了一些候选基因的性状在A.洞穴形式的演变mexicanus。近期cavefish基因组41出版物提供确定候选基因洞穴性状进化的附加 ​​功能强大的工具。许多测试这些候选基因的功能，需要技术来降低基因的表达。学习目前唯一的选择ING在A.降低基因表达mexicanus是通过使用吗啉的。然而，吗啉代基因敲除是短暂的，仅限于几天受精后，并且是不适合的成年动物，如像上学17，摄食过度19和振动吸引力行为42成人洞穴和表面鱼之间行为上的差异学习性状是有用的。基因，如那些可以使用TALENS制成的功能等位基因的损失的产生，将用于测试的候选基因的作用，这些性状是至关重要的。 方法已为TALEN对33的装配容易和用于该方法的详细协议被开发可用34。该协议是由比德尔等 7斑马鱼进行了优化，采用了不同的最终目的载体，pT3Ts-goldyTALEN（pT3TS-GT）。该载体允许用于注入单细胞斑马鱼胚胎TALEN mRNA的转录。我们已经用这种改良的装配方法，详细讲解了这里，组装和转录TALENS的注入A. mexicanus。我们发现，当注射到单细胞表面A. mexicanus胚胎，因为这个协议中所述，我们可能会变异A. mexicanus基因29。对于未来在该协议没有描述的候选基因的研究，序列可以在基因组cavefish 41被发现和用于识别TALEN目标站点。 关键成功注射是一种高品质TALEN表达。因此，通过对注射前的凝胶运行的RNA少量检查RNA的质量是很重要的（步骤3.6）。用于保持RNA完整性其他预防措施，如冻结等分以避免冻结解冻（步骤3.7），和通过注射（步骤4）中使用干净的水和无RNA酶的管和提示维持无菌条件下，应采取。为提高注入鱼额外关键步骤是清理注射后死胚，死胚能迅速影响水质。因此，我们早上取出死胚以下注射，并定期在未来数日内注射，以维持健康的活胚胎（步骤4.3.6）。 在Astyanax mexicanus TALEN突变能够高效;然而，效率的变化取决于TALEN对注入29。增加的mRNA的浓度会导致增加毒性和畸形和注射胚胎的死亡。因此，毒性与效率，必须进行测试，并平衡来确定mRNA的最佳浓度注入。对于高效TALEN对，表型可能在注射创始人鱼进行评估。例如，TALENS靶向OCA2的注射导致在表面鱼29白化补丁（ 图3）。对于其他性状或基因，然而，在创办人鱼的表型的评估可能是具有挑战性的，由于巧妙地表型或TALEN对注入效率低的。因此，对于许多应用，将是理想的，为的候选基因在非镶嵌动物中的作用的分析感兴趣的基因，以产生种系突变。在答 TALEN诱发突变的获得种系传递mexicanus是可能29（ 图4）。因此，这种技术可以适用于评估其它候选基因。 在Astyanax mexicanus进行遗传操作有一些限制存在在这个时候。面，在黑暗cavefish品种，在深夜。在实验室的地方是不可能扭转光暗循环，研究人员必须在深夜进来以执行注射，因为它是产卵后立即注入至关重要。此外，收集面鱼的胚胎在黑暗中是很重要的，因为光线会影响产卵。 其它技术，在particuLAR的CRISPR / CAS系统（43审查），存在基因组编辑，很可能会适用于A. mexicanus。实际上，存在导游RNA组装协议是快速和容易的44和协议报可以在这里进行修改CRISPR / CAS注入。此外，对于基因组编辑新的应用正在迅速发展，许多的这些可能被证明为A.有用mexicanus的研究人员。例如，在斑马鱼精确突变已在感兴趣的基因通过用含有突变7的单链寡coinjecting TALENS制成。这种技术可以用于评估洞穴等位基因中产生洞穴的表型，如在cavefish和表面鱼19之间观察到代谢的差异MC4R的某些洞穴等位基因的错义突变的角色的角色是有用的。 TALENS也被用来产生表达荧光标记我通过同源重组基因的等位基因ñ的模式类似于内源性基因座45。这些方法可以在A.使用mexicanus评价细胞或候选基因的表达的子集。的CRISPR / CAS系统已在斑马鱼中使用，以获得组织特异性基因敲除46。这些技术中，施加到A. mexicanus，可以评估诸如在cavefish眼损失过程的遗传基础是有用的。透镜起着眼损失的过程中的关键作用，在cavefish 47和组织特异性CRISPR / CAS系统可以用来评估特意在透镜与眼睛的其它组织的候选基因的眼损失的作用。这些和其他的基因组编辑技术可在A中被利用mexicanus在未来的研究回答关于溶洞特征的演变关键问题。

了解性状进化的遗传基础是进化生物学家的一个重要研究目标。相当大的进展在查明位点特征的演变和背后这些位点（例如1-3）内精确定位候选基因进行了。然而，在功能上测试这些基因的作用仍然具有挑战性用于研究性状的进化因为许多有机体当前不是遗传容易处理。基因组编辑技术问世已经大大增加了广泛的生物体的遗传操作性。转录活化剂-样效应核酸（TALENS）和群集定期相互间隔短回文重复序列（CRISPRs）已被用来产生在一个数生物体（例如4-11）在基因靶向的突变。这些工具，应用到进化相关制度，有潜力彻底改变进化生物学家研究进化的遗传基础。 Astyanax mexicanus是鱼类的物种存在两种形式:多穴居形式（cavefish）河居住的表面形状（表面鱼）和A. mexicanus cavefish从表面鱼类祖先（12综述）演变而来。 cavefish种群已经进化了许多特性，包括眼睛的损耗，减少或色素沉着的损失，增加了味蕾和颅神经丘和改变的号码行为，如办学行为的损失，增加侵略，在喂养姿势和亢进13的变化-19。 Cavefish和表面鱼的间，和遗传作图实验已经进行识别位点和候选基因性状的洞穴1,20-26。一些候选基因已经过测试，在细胞培养1,19促进洞穴性状的职能作用，在其他物种中的21模式生物或过表达27或短暂击倒ü唱歌吗啉28 A. mexicanus。然而，这些方法有局限性。生成这些基因在A的突变体等位基因的能力mexicanus对于理解它们的功能在cavefish的演变是至关重要的。因此，A。 mexicanus是基因编辑技术的应用的理想候选有机体。 在这里，我们概括为A.基因组编辑的方法mexicanus使用TALENS。这种方法可用于评估镶嵌注射创始人鱼的表型和用于分离在感兴趣29基因稳定突变鱼线。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Equipment</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Thermocycler</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Injection station</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gel apparatus</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Needle puller</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nanodrop</td>
			<td style="width:71px;"></td>
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			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
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			<td>Catalog Number</td>
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			<td height="21" style="height:21px;width:216px;">Supplies</td>
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		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Note: As far as we know, supplies from different companies can be used unless otherwise indicated</td>
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			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Golden Gate TALEN and TAL Effector Kit 2.0</td>
			<td style="width:71px;">Addgene</td>
			<td style="width:119px;">Kit #1000000024</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fisher BioReagents LB Agar, Miller (Granulated)</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">BP9724-500</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Fisher BioReagents Microbiology Media: LB Broth, Miller</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">BP1426-500</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Teknova TET-15 in 50% EtOH</td>
			<td style="width:71px;">Teknova (ordered through Fisher)</td>
			<td style="width:119px;">50-843-314</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Spectinomycin Dihydrochloride, Fisher BioReagents</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">BP2957-1</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Super Ampicillin (1000x solution)</td>
			<td style="width:71px;">DNA Technologies</td>
			<td style="width:119px;">6060-1</td>
			<td></td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">ThermoScientific X-Gal Solution, ready-to-use</td>
			<td style="width:71px;">Thermo Sci Fermentas Inc (Ordered through Fisher)</td>
			<td style="width:119px;">FERR0941</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">IPTG, Fisher BioReagents</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">BP1620-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petri dishes</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">08-757-13</td>
			<td></td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">BsaI</td>
			<td style="width:71px;">New England Biolabs (ordered through Fisher)</td>
			<td style="width:119px;">50-812-203</td>
			<td>Use BsaI, not BsaI-HF (as described in the Golden Gate TALEN and TAL Effector Kit protocol)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">BSA</td>
			<td style="width:71px;">New England Biolabs</td>
			<td style="width:119px;">provided with restriction enzymes</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">10x T4 ligase buffer</td>
			<td style="width:71px;">Promega (ordered through Fisher)</td>
			<td style="width:119px;">PR-C1263</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">GoTaq Green Master mix</td>
			<td style="width:71px;">Promega (ordered through Fisher)</td>
			<td style="width:119px;">PRM7123</td>
			<td>Other Taq can be used, but the reaction should be adjusted accordingly</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">Quick ligation kit</td>
			<td style="width:71px;">New England Biolabs (ordered through Fisher)</td>
			<td style="width:119px;">50-811-728</td>
			<td>We use Quick Ligase for all TALEN assembly reactions</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">One Shot TOP10 Chemically Competent E.coli</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">C4040-06</td>
			<td>Other chemically competent cells or homemade competent cells can be used</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">Esp 3I</td>
			<td style="width:71px;">Thermo Sci Fermentas Inc (Ordered through Fisher)</td>
			<td style="width:119px;">FERER0451</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Plasmid-Safe ATP-dependent DNase</td>
			<td style="width:71px;">Epicentre (Ordered through Fisher)</td>
			<td style="width:119px;">NC9046399</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QIAprep Spin Miniprep Kit</td>
			<td style="width:71px;">Qiagen</td>
			<td align="right" style="width:119px;">27106</td>
			<td>The Qiagen kit should be used for the initial plasmid preparation (as described in the Golden Gate TALEN and TAL Effector Kit protocol)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QIAquick PCR Purification Kit</td>
			<td style="width:71px;">Qiagen</td>
			<td align="right" style="width:119px;">28104</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">GeneMate LE Quick Dissolve Agaraose</td>
			<td style="width:71px;">BioExpress</td>
			<td style="width:119px;">E-3119-125</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Sac I</td>
			<td style="width:71px;">Promega (Ordered through Fisher)</td>
			<td style="width:119px;">PR-R6061</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">mMESSAGE mMACHINE T3 Transcription kit</td>
			<td style="width:71px;">Ambion</td>
			<td style="width:119px;">AM1348M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rneasy MinElute Cleanup Kit</td>
			<td style="width:71px;">Qiagen</td>
			<td align="right" style="width:119px;">74204</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">NorthernMax-Gly Sample Loading Dye&#160;</td>
			<td style="width:71px;">Ambion (ordered through Fisher)</td>
			<td style="width:119px;">AM8551</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Eliminase</td>
			<td style="width:71px;">Decon (ordered through Fisher)</td>
			<td style="width:119px;">04-355-32</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fisherbrand Disposable Soda-Lime Glass Pasteur Pipets</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">13-678-6B</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Standard Glass Capillaries</td>
			<td style="width:71px;">World Precision Instruments</td>
			<td style="width:119px;">1B100-4</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Microcaps</td>
			<td style="width:71px;">Drummond Scientific Company</td>
			<td style="width:119px;">1-000-0010</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Eppendorf Femtotips Microloader Tips for Femtojet Microinjector</td>
			<td style="width:71px;">Eppendorf (ordered through Fisher)</td>
			<td style="width:119px;">E5242956003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium hydroxide</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">S318-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris base</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">BP152-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

genome editing in astyanax mexicanus using transcription activator-like effector nucleases (talens)

Identifying alleles of genes underlying evolutionary change is essential to understanding how and why evolution occurs. Towards this end, much recent work has focused on identifying candidate genes for the evolution of traits in a variety of species. However, until recently it has been challenging to functionally validate interesting candidate genes. Recently developed tools for genetic engineering make it possible to manipulate specific genes in a wide range of organisms. Application of this technology in evolutionarily relevant organisms will allow for unprecedented insight into the role of candidate genes in evolution. Astyanax mexicanus (A. mexicanus) is a species of fish with both surface-dwelling and cave-dwelling forms. Multiple independent lines of cave-dwelling forms have evolved from ancestral surface fish, which are interfertile with one another and with surface fish, allowing elucidation of the genetic basis of cave traits. A. mexicanus has been used for a number of evolutionary studies, including linkage analysis to identify candidate genes responsible for a number of traits. Thus, A. mexicanus is an ideal system for the application of genome editing to test the role of candidate genes. Here we report a method for using transcription activator-like effector nucleases (TALENs) to mutate genes in surface A. mexicanus. Genome editing using TALENs in A. mexicanus has been utilized to generate mutations in pigmentation genes. This technique can also be utilized to evaluate the role of candidate genes for a number of other traits that have evolved in cave forms of A. mexicanus.

TALEN对注射导致的FokⅠ域的二聚的RVDS到特定的DNA核苷酸的结合，因此，产生双链断裂39可通过非同源末端连接（NHEJ）进行修复。 NHEJ常常引入导致在插入或缺失（插入缺失）错误。插入缺失可以通过扩增包围TALEN目标位点的区域和消化与TALEN间隔区之内的切割的限制性酶将所得的扩增子进行识别。没有一个插入缺失的等位基因，而含有等位基因改变限制性内切酶靶序列将不能消化插入缺失，产生一限制酶抗性带（ 图2）将消化。 TALEN注射可以很可能导致A.等位基因基因突变mexicanus 29。因此，一些表型可能在创始人鱼进行评估。对于<html>例如，我们评估与TALENS目标OCA2，假设是负责的cavefish 1,28多个白化人群白化病基因注入表面鱼色素沉着。我们发现在OCA2 TALEN注射鱼不存在于未注射的鱼29（ 图3）白化补丁。 对于许多实验，理想的是有鱼的突变系评估表型。方正鱼发送突变等位基因可以通过从创始人鱼杂交到野生型鱼（ 图4）的基因分型的子代来鉴定。 <img alt="图1" src="/files/ftp_upload/54113/54113fig1.jpg" /> 图1.针注射的表达 。被破碎用于注射TALEN的mRNA成单细胞胚胎之前的微量的照片。_upload / 54113 / 54113fig1large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/54113/54113fig2.jpg" /> 图2. TALEN效率从中Astyanax mexicanus OCA2的外显子9 OCA2。306 bp的PCR产物进行了检查的限制酶位点的损失时不同量TALEN的mRNA注射29。而扩增子的一部分在10 TALEN注射胚胎池是限制性消化抗性来自控制胚胎扩增子消化。限制性内切酶消化从具有TALEN的mRNA注射的胚胎耐频带靶向OCA2已被证明含有插入缺失29。注意，mRNA的增加浓度注射在增加TALEN效率（更未消化的DNA）的结果。 " - "用车道未消化，车道，"+"是diges泰德与限制性内切酶。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54113/54113fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54113/54113fig3.jpg" /> 图3.表型的创始人鱼色素沉着的变化。（A）控制未注射表面A. mexicanus。 （ 二 ）缺乏补丁与400皮克TALEN的mRNA注射创始人面鱼黑色素细胞靶向OCA2（箭头）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54113/54113fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54113/54113fig4.jpg" /> 图4. TALEN诱导mutatio的种系传递纳秒。从A. OCA2的外显子9 306 bp的PCR产物mexicanus检查从注入创始人鱼的10架F 1鱼池的限制性内切酶网站的损失。而扩增子的一部分在10架F 1胚胎池是限制性消化抗性来自控制胚胎扩增子消化。限制性内切酶消化从OCA2˚F1秒已被证明含有插入缺失29耐频带。与道" - "都未消化，车道，"+"与限制性内切酶消化。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54113/54113fig4large.jpg" target="_blank">请点击此处查看这个数字更大的版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 反应A </td><td></td><td></td><td> Reacti基于B </td><td></td></tr><tr><td> 量 </td><td> 试剂 </td><td></td><td> 量 </td><td> 试剂 </td></tr><tr><td> 4微升</td><td>水</td><td></td><td> 10微升</td><td>水</td></tr><tr><td> 1微升</td><td> pFUS_A </td><td></td><td> 1微升</td><td> pFUS_B4 </td></tr><tr><td> 1微升</td><td> BsaI </td><td></td><td> 1微升</td><td> BsaI </td></tr><tr><td> 1微升</td><td> BSA </td><td></td><td> 1微升</td><td> BSA </td></tr><tr><td> 1微升</td><td>连接酶</td><td></td><td> 1微升</td><td>连接酶</td></tr><tr><td> 2微升</td><td colspan="2"> 10X连接酶缓冲</td><td> 1微升</td><td> 10X连接酶缓冲</td></tr><tr><td> 1微升</td><td> pNH1 </td><td></td><td> 1微升</td><td> pNG1 </td></tr><tr><td> 1微升</td><td> pNH2 </td><td></td><td> 1微升</td><td> PHD2 </td></tr><tr><td> 1微升</td><td> pNG3 </td><td></td><td> 1微升</td><td> pNH3 </td></tr><tr><td> 1微升</td><td> pHD4 </td><td></td><td> 1微升</td><td> pNI4 </td></tr><tr><td> 1微升</td><td> pHD5 </td><td></td><td></td><td></td></tr><tr><td> 1微升</td><td> PHD6 </td><td></td><td></td><td></td></tr><tr><td> 1微升</td><td> pNG7 </td><td></td><td></td><td></td></tr><tr><td> 1微升</td><td> pHD8 </td><td></td><td></td><td></td></tr><tr><td> 1微升</td><td> pNG9 </td><td></td><td></td><td></td></tr><tr><td> 1微升</td><td> pHD10 </td><td></td><td></td><td></td></tr></tbody></table> 表1实施例反应组件A和B为TALEN RVDS含有NH-NH-NG-HD-HD高清-NG-HD-吴高清-NG-HD-NH-NI-NG。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 底漆名 </td><td> 序列（5&#39;-3&#39;） </td></tr><tr><td> pCR8_F1 </td><td> ttgatgcctggcagttccct </td></tr><tr><td> pCR8_R1 </td><td> cgaaccgaacaggcttatgt </td></tr><tr><td> TAL_F1 </td><td> ttggcgtcggcaaacagtgg </td></tr><tr><td> TAL_R2 </td><td> ggcgacgaggtggtcgttgg </td></tr></tbody></table> 表2的PCR引物用于菌落PCR，从34。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 试剂 </td><td> 量 </td></tr><tr><td> Taq酶mastermix，2倍</td><td> 50微升</td></tr><tr><td> pCR8_F1底漆，10嗯</td><td> 4微升</td></tr><tr><td> pCR8_R1底漆，10林M </td><td> 4微升</td></tr><tr><td>不含核酸酶的水</td><td> 42微升</td></tr><tr><td colspan="2"> *调整主混合物，如果使用不同的Taq </td></tr></tbody></table> 表3.预混100微升（微升15 /反应）为菌落PCR 1。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 步 </td><td> 温度（℃） </td><td> 时间（秒） </td></tr><tr><td> 1 </td><td> 95 </td><td> 120 </td></tr><tr><td> 2 </td><td> 95 </td><td>三十</td></tr><tr><td> 3 </td><td> 55 </td><td>三十</td></tr><tr><td> 4 </td><td> 72 </td><td> 105 </td></tr><tr><td>五</td><td colspan="2">转至步骤2 30个循环</td></tr><tr><td> 6 </td><td> 72 </td><td> 300 </td></tr></tbody></table> 表4. PCR程序为菌落PCR 1。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 量 </td><td> 试剂 </td><td> 浓度 </td></tr><tr><td> 12微升</td><td>水</td><td></td></tr><tr><td> 1微升</td><td>矢量A </td><td> 100毫微克/微升</td></tr><tr><td> 1微升</td><td>矢量B </td><td> 100毫微克/微升</td></tr><tr><td> 1微升</td><td>目的载体pT3Ts-GT </td><td> 50纳克/微升</td></tr><tr><td> 1微升</td><td>最后RVD（PLR-RVD） </td><td> 100毫微克/微升</td></tr><tr><td> 1微升</td><td> Esp3I </td><td></td></tr><tr><td> 1微升</td><td>连接酶</td><td></td></tr><tr><td> 2微升</td><td colspan="2"> 10X连接酶缓冲</td></tr></tbody></table> 表5. 协议第二组件的反应。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 步 </td><td> 温度（℃） </td><td> 时间（秒） </td></tr><tr><td> 1 </td><td> 95 </td><td> 120 </td></tr><tr><td> 2 </td><td> 95 </td><td>三十</td></tr><tr><td> 3 </td><td> 55 </td><td>三十</td></tr><tr><td> 4 </td><td> 72 </td><td> 180 </td></tr><tr><td>五</td><td colspan="2">转至步骤2 30个循环</td></tr><tr><td> 6 </td><td> 72 </td><td> 300 </td></tr></tbody></table> 表6. PCR程序菌落PCR 2。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 热 </td><td> 290 </td></tr><tr><td> 拉 </td><td> 150 </td></tr><tr><td> 速度 </td><td> 100 </td></tr><tr><td> 时间 </td><td> 150 </td></tr><tr><td colspan="2">这些参数对于一个火焰山/布朗微量拉马型号P-97使用低谷长丝</td></tr></tbody></table> 表7.样品针头拔程序。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 试剂 </td><td> 量 </td></tr><tr><td> Taq酶mastermix，2倍</td><td> 12.5微升</td></tr><tr><td>基因特异性正向引物，10μM </td><td> 1μ升</td></tr><tr><td>基因特异性反向引物，10μM </td><td> 1微升</td></tr><tr><td>不含核酸酶的水</td><td> 9.5微升</td></tr><tr><td>脱氧核糖核酸</td><td> 1微升</td></tr><tr><td colspan="2"> *调整主混合物，如果使用不同的Taq </td></tr></tbody></table> 表8. 样品协议进行基因特异性PCR。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 步 </td><td> 温度（℃） </td><td> 时间（秒） </td></tr><tr><td> 1 </td><td> 95 </td><td> 120 </td></tr><tr><td> 2 </td><td> 95 </td><td>三十</td></tr><tr><td> 3 </td><td> 56 </td><td>三十</td></tr><tr><td> 4 </td><td> 72 </td><td> 60 </td></tr><tr><td>五</td><td colspan="2">去送TEP 2为35个循环</td></tr><tr><td> 6 </td><td> 72 </td><td> 300 </td></tr><tr><td colspan="3"> *调节退火温度和延伸时间为特定的引物和PCR产物大小</td></tr></tbody></table> 基因特异性PCR 表9. 样品PCR程序。

Watch this Scientific Journal Video about Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases TALENs at JoVE.com

Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases TALENs

基因靶向诱变现在有可能在宽的范围内使用的基因组编辑技术生物体。在这里，我们演示了有针对性的基因突变的协议使用转录激活像Astyanax mexicanus，鱼品种，包括面鱼和cavefish效应核酸酶（TALENS）。

基因组编辑在<em&gt; Astyanax mexicanus</em&gt;使用转录因子样效应核酸酶（TALENS）

Identifying alleles of genes underlying evolutionary change is essential to understanding how and why evolution occurs.  Towards this end, much recent ...

genome-editing-astyanax-mexicanus-using-transcription-activator-like

Nanyang Technological University

Research

JoVE Journal

Biology

Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases (TALENs)

8.2K 次观看.  Iowa State University. 基因靶向诱变现在有可能在宽的范围内使用的基因组编辑技术生物体。在这里，我们演示了有针对性的基因突变的协议使用转录激活像Astyanax mexicanus，鱼品种，包括面鱼和cavefish效应核酸酶（TALENS）。 

视频: Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases TALENs

Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH

Delayed type hypersensitivity (DTH) is an immune reaction in which the main players are CCR7- effector / memory T lymphocytes. Here, we demonstrate a method for inducing and recording the progress of a DTH reaction in the rat ear. This is followed by a demonstration of the preparation of rat ear tissue for two-photon imaging of the CCR7- effector / memory T cell response.
An adoptive DTH is induced by the intraperitoneal injection of GFP-labeled Ova-specific CCR7- effector / memory T cell line (Beeton, C J. Visualized Experiments, Issue 8). Cells are then allowed to equilibrate in the rat for 48 hours before challenge by injecting one ear with saline (control ear) and the other with a 1:1 mix of Ova and Ova conjugated to Texas-Red (Ova-TR) to allow visualization of resident antigen-presenting cells.
We describe a method of tissue preparation useful for imaging the motility of cells within the deep dermal layer during an immune response, in conjunction with visualization of collagen fibers by second harmonic generation. Ear tissue is cut into 5 x 5 mm squares (slightly larger is better) and mounted onto plastic cover slips using Vetbond&trade;, which are then secured using silicone grease in an imaging chamber and superfused by oxygen-bubbled tissue culture medium at 37&deg;C.

Here we demonstrate a method for inducing and recording the progress of a delayed type-hypersensitivity (DTH) reaction in the rat ear. This is followed by a demonstration of the preparation of rat ear tissue for two-photon imaging of the effector / memory T cell response.

成像效应在耳朵上的记忆体T细胞诱导后收养的DTH

Delayed type hypersensitivity (DTH) is an immune reaction in which the main players are CCR7- effector / memory T lymphocytes. Here, we demonstrate a ...

科研

生物学

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.
Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

格罗斯和精细解剖内耳感觉上皮细胞在成年斑马鱼（斑马鱼）

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the ...

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, form a complex with Smad4 and translocate to the nucleus where they function as transcription factors to initiate BMP specific downstream effects 1. Traditional immuno-fluorescence techniques with antibodies against phospho-Smad peptides exhibit low sensitivity, high background and offer gross quantification as they rely on intensity of the antibody signal particularly if this is photosensitive fluorescent. In addition, phospho-Smads may not all be in complex with Smad4 and engaged in active transcription.
In situ PLA is a technology capable of detecting protein interactions with high specificity and sensitivity 2-4. This new technology couples antibody recognition with the amplification of DNA surrogate of the protein. It generates a localized, discrete signal in a form of spots revealing the exact position of the recognition event. The number of signals can be counted and compared providing a measurement. We applied in situ PLA, using the Duolink kit, with a combination of antibodies that allows the detection of the BMP signaling effectors phospho-Smad1/5/8 and Smad4 only when these are in proximity i.e. in a complex, which occurs only with signaling activation. This allowed for the first time, the visualization and measurement of endogenous BMP signaling with high specificity and sensitivity in a time course experiment under BMP4 stimulation.

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

Here we show how to use Proximity Ligation Assay (PLA), with a combination of antibodies to visualize Bone Morphogenetic Protein (BMP) signaling in fixed cells. This technique allowed us to follow the nuclear accumulation of endogenous BMP activated effector-complexes and quantify their levels over time under BMP4 stimulation.

检测信号效应复合物BMP4的下游使用原位解放军，接近结扎分析

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, ...

Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, genome-wide cDNA library cloned into the 3'UTR downstream from the dual-selection fusion protein, thymidine kinase-zeocin (TKzeo). The first round of selection is for stable transformants, followed with introduction of a miRNA of interest, and finally, selecting for cDNAs containing the miRNA's target. Selected cDNAs are identified by sequencing (see Figure 1-3 for Target ID Library Workflow and details).
To ensure broad coverage of the human transcriptome, Target ID Library cDNAs were generated via oligo-dT priming using a pool of total RNA prepared from multiple human tissues and cell lines. Resulting cDNA range from 0.5 to 4 kb, with an average size of 1.2 kb, and were cloned into the p3&#900;TKzeo dual-selection plasmid (see Figure 4 for plasmid map). The gene targets represented in the library can be found on the Sigma-Aldrich webpage. Results from Illumina sequencing (Table 3), show that the library includes 16,922 of the 21,518 unique genes in UCSC RefGene (79%), or 14,000 genes with 10 or more reads (66%).

The Target ID Library is a plasmid-based, genome-wide collection of cloned cDNA used to identify miRNA targets. Here we demonstrate its use and application.

miRNA的目标使用的任务目标ID库的全基因组屏幕

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, ...

Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such as chemical-based mutagenesis or random integration of DNA sequences confer indiscriminate genetic changes in an overall inefficient manner and require incorporation of undesirable synthetic sequences or use of aberrant culture conditions, potentially confusing biological study. By contrast, transient ZFN expression in a cell can facilitate precise, heritable gene editing in a highly efficient manner without the need for administration of chemicals or integration of synthetic transgenes.
Zinc finger nucleases (ZFNs) are enzymes which bind and cut distinct sequences of double-stranded DNA (dsDNA). A functional CompoZr ZFN unit consists of two individual monomeric proteins that bind a DNA "half-site" of approximately 15-18 nucleotides (see Figure 1). When two ZFN monomers "home" to their adjacent target sites the DNA-cleavage domains dimerize and create a double-strand break (DSB) in the DNA.1 Introduction of ZFN-mediated DSBs in the genome lays a foundation for highly efficient genome editing. Imperfect repair of DSBs in a cell via the non-homologous end-joining (NHEJ) DNA repair pathway can result in small insertions and deletions (indels). Creation of indels within the gene coding sequence of a cell can result in frameshift and subsequent functional knockout of a gene locus at high efficiency.2 While this protocol describes the use of ZFNs to create a gene knockout, integration of transgenes may also be conducted via homology-directed repair at the ZFN cut site.
The CompoZr Custom ZFN Service represents a systematic, comprehensive, and well-characterized approach to targeted gene editing for the scientific community with ZFN technology. Sigma scientists work closely with investigators to 1) perform due diligence analysis including analysis of relevant gene structure, biology, and model system pursuant to the project goals, 2) apply this knowledge to develop a sound targeting strategy, 3) then design, build, and functionally validate ZFNs for activity in a relevant cell line. The investigator receives positive control genomic DNA and primers, and ready-to-use ZFN reagents supplied in both plasmid DNA and in-vitro transcribed mRNA format. These reagents may then be delivered for transient expression in the investigator&#x2019;s cell line or cell type of choice. Samples are then tested for gene editing at the locus of interest by standard molecular biology techniques including PCR amplification, enzymatic digest, and electrophoresis. After positive signal for gene editing is detected in the initial population, cells are single-cell cloned and genotyped for identification of mutant clones/alleles.

Genome Editing with CompoZr Custom Zinc Finger Nucleases ZFNs

The CompoZr Custom Zinc-Finger Nuclease (ZFN) Service enables precise genome editing in any organism or cell line at any locus defined by the user. This article describes the process for the design, manufacture, validation and implementation of the CompoZr Custom ZFN Service.

基因组编辑与的CompoZr自锌指核酸酶（ZFNs）

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such ...

Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such architectures are not random and involve interactions between gene promoters and regulatory elements 13. The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination 1, 14. 
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures 5,6. Cells are fixed and interacting loci are captured by covalent DNA-protein cross-links. To minimize non-specific noise and reduce complexity, as well as to increase the specificity of the chromatin interaction analysis, chromatin immunoprecipitation (ChIP) is used against specific protein factors to enrich chromatin fragments of interest before proximity ligation. Ligation involving half-linkers subsequently forms covalent links between pairs of DNA fragments tethered together within individual chromatin complexes. The flanking MmeI restriction enzyme sites in the half-linkers allow extraction of paired end tag-linker-tag constructs (PETs) upon MmeI digestion. As the half-linkers are biotinylated, these PET constructs are purified using streptavidin-magnetic beads. The purified PETs are ligated with next-generation sequencing adaptors and a catalog of interacting fragments is generated via next-generation sequencers such as the Illumina Genome Analyzer. Mapping and bioinformatics analysis is then performed to identify ChIP-enriched binding sites and ChIP-enriched chromatin interactions 8. 
We have produced a video to demonstrate critical aspects of the ChIA-PET protocol, especially the preparation of ChIP as the quality of ChIP plays a major role in the outcome of a ChIA-PET library. As the protocols are very long, only the critical steps are shown in the video.

Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation

Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a method for de novo detection of chromatin interactions, for better understanding of transcriptional control.

配对末端标签测序（嘉PET）测绘染色质相互作用和了解转录调控染色质相互作用分析

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

在体内病态的人类基因组建模使用斑马鱼

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

Mouse Genome Engineering Using Designer Nucleases

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well as for modeling human diseases and testing therapeutic strategies. Recent advances in the use of designer nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system for site-specific genome engineering open the possibility to perform rapid targeted genome modification in virtually any laboratory species without the need to rely on embryonic stem (ES) cell technology. A genome editing experiment typically starts with identification of designer nuclease target sites within a gene of interest followed by construction of custom DNA-binding domains to direct nuclease activity to the investigator-defined genomic locus. Designer nuclease plasmids are in vitro transcribed to generate mRNA for microinjection of fertilized mouse oocytes. Here, we provide a protocol for achieving targeted genome modification by direct injection of TALEN mRNA into fertilized mouse oocytes.

Designer nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to modify the genome of mouse preimplantation embryos by triggering both the nonhomologous end joining (NHEJ) and homologous recombination (HR) pathways. These advances enable the rapid generation of mice with precise genetic modifications.

小鼠基因组工程使用Designer核酸酶

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well ...

Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to understand where and when transcription is taking place within nuclear space and to visualize the relationship between episomes infected within the same cell's nucleus. Here, both immunofluorescence (IFA) and RNA-FISH have been combinedto identify actively transcribing Kaposi's sarcoma-associated herpesvirus (KSHV) episomes. By staining KSHV latency-associated nuclear antigen (LANA), it is possible to locate where viral episomes exist within the nucleus. In addition, by designing RNA-FISH probes to target the intron region of a viral gene, which is expressed only during productive infection, nascent RNA transcripts can be located. Using this combination of molecular probes, it is possible to visualize the assembly of large viral transcription factories and analyze the spatial regulation of viral gene expression during KSHV reactivation. By including anti-RNA polymerase II antibody staining, one can also visualize the association between RNA polymerase II (RNAPII) aggregation and KSHV transcription during reactivation.

Viral transcriptional factories are discrete structures that are enriched with cellular RNA polymerase II to increase viral gene transcription during reactivation. Here, a method to locate sites of actively transcribing viral chromatin in 3D nuclear space by a combination of immunofluorescence staining and in situ RNA hybridization is described.

基于3D 荧光显微镜的病毒转录工厂功能成像

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to ...

Gamete Collection and In Vitro Fertilization of Astyanax mexicanus

Astyanax mexicanus is emerging as a model organism for a variety of research fields in biological science. Part of the recent success of this teleost fish species is that it possesses interfertile cave and river-dwelling populations. This enables the genetic mapping of heritable traits that were fixed during adaptation to the different environments of these populations. While this species can be maintained and bred in the lab, it is challenging to both obtain embryos during the daytime and create hybrid embryos between strains. In vitro fertilization (IVF) has been used with a variety of different model organisms to successfully and repeatedly breed animals in the lab. In this protocol, we show how, by acclimatizing A. mexicanus to different light cycles coupled with changes in water temperature, we can shift breeding cycles to a chosen time of the day. Subsequently, we show how to identify suitable parental fish, collect healthy gametes from males and females, and produce viable offspring using IVF. This enables related procedures such as the injection of genetic constructs or developmental analysis to occur during normal working hours. Furthermore, this technique can be used to create hybrids between the cave and surface-dwelling populations, and thereby enable the study of the genetic basis of phenotypic adaptations to different environments.

Gamete Collection and In Vitro Fertilization of Astyanax mexicanus

In vitro fertilization is a commonly used technique with a variety of model organisms to maintain lab populations and produce synchronized embryos for downstream applications. Here, we present a protocol that implements this technique for different populations of the Mexican tetra fish, Astyanax mexicanus.

阿斯蒂亚纳斯墨西哥的Gamete收集与体外受精

Astyanax mexicanus is emerging as a model organism for a variety of research fields in biological science. Part of the recent success of this teleost fish ...