这项工作是由美国国立卫生研究院R01＃DE021475（CTM），美国国立卫生研究院博士前培训资助5T32GM007127（PAE）和美国国家科学基金会研究生研究奖学金（NAE）的部分资助。我们感谢凯文施瓦尔巴赫用于产生CRISPR Sanger测序数据进行BAC重组工程和注射，尼克东德和凯瑟琳利帕里关于注射方案有帮助的反馈。

所有的鱼的工作是经美国加州大学伯克利分校的机构动物护理和使用委员会（协议号为R330）。 1.准备核酸注射液<ol><li> TOL2质粒转基因（从费舍尔26改编）。 <ol><li>削减10微克转座质粒（PCS-TP）39 10单位的NotI在提供的缓冲区1小时，在37°C至线性化。 注:材料转让协定可能需要获得TOL2质粒。 </li><li>根据标准方案40用乙酸钠异戊醇及乙醇沉淀:提取用25切割的质粒:氯仿:24:苯酚的1:1混合物。悬浮的质粒在50μl无RNA酶的水。 注意:酚 - 氯仿应在通风橱中使用和废弃，必须根据机构方针进行适当设置。 </li><li>根据制造商的说明建立一个SP6启动转录反应。 </li><li>使用RNA ISOL通货膨胀试剂盒根据制造商的说明清理转录反应;在50微升不含RNA酶的水重悬的RNA。 </li><li>除去RNA的1微升等份。热至65℃5分钟以变性的二级结构，然后立即冷却在冰上。冷冻在-80℃，其余的转录反应。 </li><li>运行在运行缓冲液在一个车道的RNA大小标准在0.5×TAE 1％琼脂糖凝胶（Tris碱，乙酸，乙二胺四乙酸）的RNA的等分试样。预期产物为2200碱基对;如果总RNA&gt; 5％出现在涂抹小超过2200碱基对，这表明广泛降解（ 图1）丢弃。 </li><li>使用分光光度计在260nm定量RNA。稀释至350纳克/μl，以无RNase水和存储1微升等分在-80°C（好至少两年）。 </li><li>克隆TOL2报道质粒（例如，使用pT2HE 8或质粒从TOL2套件41）， 顺-利息调控元件。简言之，PCR扩增与含在质粒中发现的限制性位点的引物感兴趣的基因组DNA序列，消化的PCR产物和载体与酶（多个），结扎插入到载体中，并转化产生的质粒导入感受态E.大肠杆菌 40。隔离质粒与包括根据制造商的协议的内毒素冲洗的试剂盒。 </li><li>以根据制造商的协议的PCR纯化试剂盒进行TOL2质粒的第二次纯化。洗脱在30微升不含RNA酶的水。 注意:此步骤中的产率可能很低（输入质粒有时低于50％）。 </li><li>稀释质粒在无RNase水〜125毫微克/微升。 </li></ol></li></ol><img alt="图1" src="/files/ftp_upload/54055/54055fig1.jpg" /> 图1.转座基因凝胶纯化的转录反应亲管道（1微升）的混合物加热至65℃，在冰上冷却，并与0.5×TAE电泳缓冲液在100伏的RNA梯度的在千碱基的大小（kb的）在1％琼脂糖凝胶上运行指示向左。全长转座基因在2.2〜2kb的亮带。降级或不完整的mRNA的小，但可接受的量被认为低于2.2 kb的。 <a href="https://www.jove.com/files/ftp_upload/54055/54055fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="2"><li> BAC转基因（BAC为重组工程技术见Suster 32,33） <ol><li>从E.准备BAC 大肠杆菌根据制造商的协议使用的BAC纯化试剂盒。用乙醇沉淀以回收DNA，在无RNase水标准的醋酸钠-乙醇萃取40和重悬的DNA在〜250毫微克/微升。 </li><li>准备转座子基因在1.1节。 </li></ol></li><li>突变诱导TALENS（见塞马克<s达&gt; 35 TALENS设计） <ol><li>使用基于网络的应用程序设计TALENS用于感兴趣42的基因。如果可能的话，设计TALENS破坏限制性内切酶酶切位点，以促进分子分析。 </li><li>克隆TALENS并准备质粒转录以下发布的协议35。 </li><li>转录TALEN的mRNA具有根据制造商的说明一个SP6启动转录反应并如第1.1.4转座描述清理的mRNA。用分光光度计量化并稀释至核糖核酸酶自由水200纳克/微升。在凝胶上运行TALEN的mRNA，以确保它是正确的大小并且在步骤1.1.6描述不降解。 </li><li>设计一对PCR引物以扩增约100-200碱基对周围使用的引物设计工具和靶DNA序列43的TALEN靶序列。订购适当的限制酶来测试基于步骤1.3.1在靶部位的病变。 </li></ol> &lt;/ LI&gt; <li> CRISPR转基因（见塔尔博特和Amacher 38设计CRISPRs的制备）: <ol><li>设计并准备CRISPRs并根据协议38 Cas9的mRNA，并为了适当的核查引物和限制性内切酶步骤1.3.4所述。 </li></ol></li></ol> 2.准备注射试剂<ol><li>使用硼硅毛细血管准备针的介绍如下。 注意:这些毛细管并不用于斑马鱼显微注射标准毛细管，并且由一个较厚的和更强的玻璃是穿刺强硬刺绒毛膜关键的。 <ol><li>拔针头时，一定要戴上丁腈或乳胶手套，不要让针头接触皮肤或皮肤油脂。 </li><li>通过以下的微量拉马的制造商的说明坡道试验确定微量经验拉动参数。例如，用一个框丝，以下参数被阻止开采是最佳的:（热火= 515，拉= 60，速度= 60，延时= 85，压力= 500）。这些设置产生逐渐变细为〜2毫米急剧在约12°，然后一个长延伸逐渐变细以约2°为〜6毫米（ 图2）的针。 注意:合适的参数将由牵拉和长丝而变化，并且盲目使用程序而不先确定的参数通过斜坡测试可以永久地破坏牵拉的长丝，它是困难且替换昂贵的。 </li><li>按照制造商的说明从拉硼硅玻璃至少4针微量在2.1.2确定的设置。 </li><li>商店针垂直与尖端朝下毛细管储存罐。 </li><li>注射前，置于冰上毛细管储存罐寒意针。一张湿纸巾添加到罐子防止水分蒸发，一旦针填补。 </li></ol></li><li>卵子受精（全在室温下进行的步骤） <ol><li>轻轻挤压腹部和前部抚摸到后方向穿过泄殖腔和成35×10mm的2培养皿推鸡蛋出从妊娠雌性刺条蛋离合器。添加在培养皿的一侧（不接触蛋）湿润一块纸巾的创建湿度室中。将盖子培养皿使鸡蛋保持湿润。 </li><li>安乐死雄性刺在用0.1％碳酸氢钠缓冲0.025％三卡因-S。 </li><li>剖开腹腔，取出睾丸和250微升汉克的解决方案（见韦斯特44全协议汉克溶液的制备）浸渍。 </li><li>施肥最多100个蛋用50μl精子溶液，用枪头，轻轻搅动以确保所有卵受精。如果离合器&gt; 100鸡蛋，受精半卵后的，以确保所有的胚胎是在注射时的单细胞阶段。鸡蛋可以留未受精在RT长达一个小时，和精子通常在Hank溶液在4℃下持续1-7天。 </li><li>要盖上培养皿的盖子胚胎受精后，防止干燥。允许第一个单元20-25分钟出现，并（在此期间准备注射的材料）膨胀起来。 </li><li>填写150毫米×15毫米的培养皿用刺鱼水。 （为了使刺水，先制备溶于去离子水10％碳酸氢钠，然后添加3.5克人造海水混合物和每1升去离子水0.217毫升10％碳酸氢钠，和搅拌/剧烈振荡以溶解盐）。 </li></ol></li><li>准备注射液（虽然鸡蛋施肥） <ol><li>根据表1和存储在冰上制备注射溶液。 注意:一些核酸的浓度已经从这些发表了斑马鱼增加由于刺卵裂球的体积增加。 </li></ol></li></ol><table fo:keep-together.within页="1"HEIGHT ="301"WIDTH ="511"&gt; <tbody><tr><td> 试剂 </td><td> TOL2注入 </td><td> BAC注射 </td><td> TALEN注入 </td><td> CRISPR注入 </td></tr><tr><td> TOL2表达</td><td> 350纳克</td><td> 350纳克</td><td> - </td><td> - </td></tr><tr><td>脱氧核糖核酸</td><td> 150-200 NG质粒</td><td> 200-300 NG BAC </td><td> - </td><td> - </td></tr><tr><td> TALEN表达</td><td> - </td><td> - </td><td> 200纳克</td><td> - </td></tr><tr><td> CRISPR RNA指南</td><td> - </td><td> - </td><td> - </td><td> 200纳克</td></tr><tr><td> Cas9表达</td><td> - </td><td> - </td><td> - </td><td> 400纳克</td></tr><tr><td> 0.5％酚红inDulbecco的PBS </td><td> 0.5微升</td><td> 0.5微升</td><td> 0.5微升</td><td> 0.5微升</td></tr> &lt;TR&gt; <td>无RNA酶的水</td><td> 5微升</td><td> 5微升</td><td> 5微升</td><td> 5微升</td></tr></tbody></table> 表1:注射试剂所有的混合物，应准备在5微升的总体积并在冰上储存。 <ol start="4"><li>填充针（冰上;允许10分钟以上的针填充）。 <ol><li>吹打0.5微升注射溶液涂布而针中的毛细管储存罐垂直悬挂在针的钝顶端回填至少三个针。要小心，下降停留在顶部和不滴下来的一侧，避免气泡。 </li><li>红色的液体大多排放到针的尖端后，添加另外0.5微升到针的钝端，并允许它排出。 </li></ol></li></ol> 3.准备注入钻井及针显微注射注意:这些步骤c，施肥蛋后通常进行。 <ol><li>打开透光的解剖显微镜，并放置一〜13厘米×13厘米的玻璃板上的显微镜光基座15厘米石膏看到在玻璃板7的叶片顶部。定向锯垂直于朝向针保持器朝向凹槽的注射装置。 </li><li>打开空气供应和确保压力从调节器设置为〜200千帕。 </li><li>打开控制盒和调整设置。设置压力〜150-175千帕。设置喷射持续时间为180毫秒。 </li><li>松开持针器，插入一个装满针插入支架，直到可以感觉到橡胶支架的阻力，并拧紧，直到手指拧紧。 </li><li>调整针角度约45°。 </li><li>使用显微控制，以便该端部中的视场中心的调整针。放大到〜40X放大率和集中在针的末端，这不应该被触摸下面的玻璃。 </li><li>通过与钟表匠的镊子抓它轻轻地打破了针尖。理想的情况下，不垂直地断裂，而是在一〜60°的角度。打破接近尖端（未超过2-3镊子宽度从端部远离， 图2）。 </li></ol><img alt="图2" src="/files/ftp_upload/54055/54055fig2.jpg" /> 图2.未破裂和破碎显微注射针头。顶部针是完整和底针尖已被打破用钳子（箭头）。针填充用含有0.05％酚红溶液。比例尺= 1毫米。 <a href="https://www.jove.com/files/ftp_upload/54055/54055fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="8"><li>按下喷射脚踏几次测试针是否损坏。轻点几下后，小红滴应该开始走出来的t他结束。如果没有，尝试打破针略高。 </li><li>如果针头有气泡，增加背压机组并迅速按下踏板几次上班泡出来。 </li><li>调整背压: <ol><li>使用一次性移液管放置刺水几滴在玻璃板上。 </li><li>轻轻放下针入水。 </li><li>增加背部压力，直到粉色的液体流隐隐从针出现（表示正压）。 注意:如果没有足够的反压，中针会通过毛细作用绘制出细胞质中。如果有过多的回压，注射量可能无意中太大。 </li><li>缩回针尽可能使得当制备胚胎它不会被损坏（见下文）。 </li></ol></li><li>可替代地，调整背压到较高的压力设置，以便液体恒定强流离开针瓦特母鸡它被淹没在水中。然后，按脚踏注入变得不必要了;然而，必须迅速地进行注射，以避免过度注入。 注:当第一次学习注入不要尝试这种技术。 </li></ol> 4.显微注射<ol><li>约25分钟受精后，用两个10微升枪头去除离合器和转移到玻璃板5-10胚胎。 </li><li>仍然使用枪头，轻轻胚胎成锯条的个别凹槽分开。请小心不要刺破胚胎。 </li><li>使用具有端部的移液管切断所以刺胚胎将适合内，加足够刺水以覆盖胚胎，留下3-5秒，然后用吸管除去多余的水，留下水涂层的小体积的水每个胚胎。 注意:太多的水会导致绒毛膜硬化以及打破针，但水的小体积是需要解除通道猎户座从细胞和蛋黄离开（ 图3A - B）。 </li><li>与胚胎最远开始，滑动玻璃板和放大，使所述第一胚胎填充视野的约25％。 </li><li>降低针进入视野，然后使用10μl的移液管尖轻轻旋转胚胎识别卵裂球，粒状，微黄色上调细胞质对蛋黄（ 图3B）的顶部凸块，然后旋转，使得的卵裂球直接垂直于针的末端（同时保持胚胎在锯片， 图3C的压痕）。 注:蛋黄液滴可移动的胚胎旋转，所以不要用它们作为卵裂球的位置的参考框架。 </li><li>降低针进入细胞质，但不通过推到底层蛋黄。刺入绒毛膜时，以避免破坏针施加压力缓慢，均匀。如果针弯曲严重，收回，在一个稍微不同的位置再试一次。 </li><li>踩下脚踏板3-4次注射，使得一个红色的丸药略有漫边缘充满细胞质的直径约〜1/8（参见图3D）。 <ol><li>避免得到红色丸药与不开始扩散，这表明注射进入细胞质（ 图3E）下方的蛋黄锐边。 </li><li>如果一个明亮的粉红色，红色斑点迅速扩散，进一步插入针头穿刺卵裂球。 </li><li>如果注射小丸变黄瞬间，旋转胚胎，以确保胚胎细胞已经有针对性的再次注入（ 图3F）。 </li></ol></li></ol><img alt="图3" src="/files/ftp_upload/54055/54055fig3.jpg" /> 图3.刺胚胎外观前和注射后，所有胚从第绘制往下看通过显微镜（除C&#39;和G）电子视角。 （A）之前添加水，受精胚胎具有均匀外观油滴浮动靠近卵黄的顶部。 （B）中加入水，绒毛膜膨胀，揭示了从蛋黄突出，并且是在信息可见一个卵裂球后。 （C）的胚胎的旋转使针垂直地入射到绒毛膜和卵裂球。 （C&#39;）已经刺破绒毛膜与细胞质的尖端的针的侧视图。 （D）注入在一个红色的斑点与褪色随着时间的推移弥漫边缘细胞质结果。 （E）注入蛋黄的红点与定义的边缘细胞质结果根本。 （F）注入从红色到黄色的pH值引起的色彩偏移的细胞质结果相反的蛋黄。 （G）横向鉴于注射的结果，与X的过子注入的理想地点。 <a href="https://www.jove.com/files/ftp_upload/54055/54055fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="8"><li>使用显微控制，以收回该针头，使用10微升枪头，如果它坚持针按住胚胎。 </li><li>缩回针之后，滑动玻璃板对准与针的下胚。 </li><li>所有注射胚胎后，用移液管水150毫米的培养皿满刺水加入到胚胎，然后收集他们在移液管和地点。 </li><li>擦干用纸巾玻璃板，以避免积累太多的水。 </li><li>分发新鲜胚胎到锯并重复步骤4.4至4.10。 </li><li>保持〜胚胎作为未注射的对照的10％，以确保未注射的胚胎正常发育和野生型对照使用在t他的分子检测如下所述。 </li></ol> 5.注射后护理<ol><li>注射后在18℃孵育在培养皿中的胚胎直至孵化10。下面出雏，在水族箱后的幼虫所描述的10。 </li><li>轻轻倒出用水刺1天注射后，用新鲜水刺更换。新鲜的刺水至少在这之后每隔一天进行更换。 </li><li>每天检查死或畸形胚胎。除去这些胚，以防止污染的水腐烂。 </li></ol> 6.注射结果分析<ol><li>对的荧光报告注射，使用荧光解剖显微镜用GFP或RFP过滤（取决于荧光转基因）每日监测胚胎在黑暗的房间。基因表达的记录解剖模式与数字照片和/或书面描述和鱼的不同EXPRES数制表锡永域（ 图4和5）。 注:棘鱼有自发荧光，这下GFP过滤器是可见的后4天受精（DPF）开始星状色素细胞。 <ol><li>为了产生稳定的线条，保存荧光检测胚胎，并成长为描述10到成年。 </li><li>使用异型杂交按步骤描述6.1找荧光的后代，表示成功的转基因传递在2.2节和屏幕后代荧光所述体外受精程序注入的成鱼与野生型鱼。 </li><li>为了显现孵化，自由游动的幼虫荧光，500微升0.8％三卡因添加到150毫米的培养皿来麻醉鱼，等到鱼停止移动图像。立即冲洗数次用新鲜水刺观察之后和成像。 </li><li>或者，要保留进一步成像荧光，安乐死在幼虫0.025％（250毫克/升）三卡因缓冲用0.1％碳酸氢钠，并在4℃下固定在4％低聚甲醛在1×磷酸盐缓冲盐水（PBS）中4小时。存储在1×PBS中达两周。 注:随着时间的推移背景自体荧光增加。 </li></ol></li><li>诊断PCR /酶切基因分型突变诱导与CRISPRs或TALENS - 第2天受精后表现最好的。 <ol><li>使用移液管放置10注射的胚胎（2旦，还在绒毛膜）到第一10个孔的12孔PCR条管。将未注射控制鱼在过去的两个井。 </li><li>去除多余的水分刺鱼。 </li><li>加入50μl裂解缓冲液（10mM的Tris pH值8.3，50mM的氯化钾，1.5mM的MgCl 2的，0.3％吐温20和0.3％NP-40），以每孔中。 </li><li>上管地方帽和孵育在95℃下，在热循环20分钟。 注意:沸腾步骤后蛋黄就会变白和橡胶。 </li><li>移除盖子和使用不同的第一百一个1000微升枪头浸渍到每管胚胎。 注:白色碎屑收集在试管的底部。 </li><li>添加2.5微升10毫克/毫升的蛋白酶K至每个孔中。 </li><li>更换盖子和在55℃孵育1小时以消化蛋白质，随后95℃20分钟，在-20℃下灭活蛋白酶K储存以避免霉菌的生长。 </li><li>使用以下制造商的说明高保真聚合酶进行PCR。使用胚胎裂解液DNA模板。 注意:请小心从管DNA模板顶部去除液体，避免在试管底部任何可见的绒毛或蛋黄碎片。 </li><li>混合PCR产物在等体积与含有1x特定酶缓冲液和每采样0.25微升酶的限制性内切酶母液。始终节省一半的未切割的PCR产物法检测凝胶上，确认PCR产物的预期大小。孵育PCR检测为适当的条件与酶混合限制性内切酶。 注意:一些酶可能需要酶缓冲PCR产物的其他比例;调节缓冲液的浓度，如果未注射控件不显示完整的消化。 </li><li>以下消化，在1％琼脂糖凝胶上运行的产品旁的DNA大小梯子确认预期的产物大小。 注意:在注射胚胎足本频带指示分子的病变的存在（ 图6）和未注射的对照必须完全消化，以解释结果。 </li><li>要确认病灶，从琼脂糖凝胶切出未切割带，并用胶提取试剂盒纯化DNA。使用Sanger测序，理想地使用测序引物内部的PCR引物，以确认病灶。在f 0的注入胚胎，病变的组合将有可能出现，造成桑格质量读取目标站点附近降低。 </li><li>为了产生一个稳定的路线，节省从分子病变筛查阳性离合器所有注射胚胎。 G行了鱼，异型杂交，然后筛选通过6.2.11在步骤6.2.1记载的F- 1胚胎分子病变的子集。如果杂合子携带者被确定，成长其余˚F1胚胎到成年，并确定使用从尾鳍夹提取的DNA杂合子个体。 </li></ol></li></ol>

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注射单细胞胚胎刺为转基因或基因组编辑提出了三大挑战。首先，相对于斑马鱼胚胎中，胚胎刺蛋壳坚韧往往会打破针。这个问题可以通过使用较厚的和更强的玻璃微并垂直于绒毛膜注入部分地克服（参见协议， 图2）。确保尽可能少​​水尽可能加入到胚胎（刚好足以引起绒毛膜膨胀并解除从细胞以外）有助于减少绒毛膜硬度。绒毛膜变硬随着时间的推移，所以润燥胚胎后迅速的工...

了解生物多样性是如何产生是确定的在自然界进化表型变化的遗传和发育基础的一个基本组成部分。该三刺鱼，Gasterosteus叶树 ，先后涌现作为研究进化的遗传基础的优秀典范。刺鱼已经发生了许多适应性进化改变海鱼都围绕殖民地北半球无数的淡水环境，造成巨大的形态，生理和行为的变化1。个人从21刺种群的基因组已经被测序，组装，和高密度图谱已经产生，进一步提高了装配2,3。遗传作图实验已经确定的基因组区域4底层演变表型- 6，和在一些情况下，具体的候选基因的功能角色已经测试-7,8-。一些潜在的形态改变的基因组区域的已确定与希望的候选基因，但这些候选人尚未功能测试9 - 12。此外，刺鱼是群体遗传学/基因组13,14，形态15，行为1，内分泌学16，生态毒理学17，免疫18和19寄生虫研究常见型号。在这些领域未来的研究将受益于在刺鱼进行功能基因操作的能力。除了 ​​操纵他们的编码序列，候选基因的作用可以通过研究其顺 -regulatory序列和由在功能上增加，减少或消除了候选基因的表达进行评估。在刺鱼显微注射和转基因方法已经非常成熟7,8,20和最初开发利用大范围核酸酶介导方法21在青鳉22首先说明。这里提出的修改显微注射法已为TOL2介导的转基因进行了优化和最近开发的基因组编辑试剂，包括TALENS和CRISPRs。 到顺 -regulatory元件变化被认为是对形态演变关键，因为顺 -regulatory变化能够避免编码突变23的负多效性的后果。因此，检测和比较的顺 -regulatory序列已经成为越来越多的进化研究的中心目标。此外，大多数人疾病变体是调控变体24,25，和模型脊椎动物系统急需研究顺调控元件的功能和逻辑。鱼在外部受精及其胚胎大量提供强大的脊椎动物系统研究顺 -regulation。该TOL2座子系统，其中forei被整合在基因组中GN DNA通过TOL2转座结合位点和侧翼共注射与TOL2转座的mRNA，以高效率工作为成功整合质粒构建成鱼的基因组26 - 28。通常情况下，一个潜在增强剂克隆一个基本启动子（如hsp70l 29）和荧光报告基因，如绿色荧光蛋白（增强型绿色荧光蛋白）或mCherry在TOL2骨干的上游，并与转座基因26注入。的荧光报道的，无论是在注射的胚胎或具有稳定整合的转基因后代表达的观察，提供了关于由推定增强剂驱动的基因表达的时空管制信息。在进一步的实验中，验证了增强剂可以用来驱动目的基因的组织特异性表达。 对于较大的顺 -regulatory地区分析，高品质的大插入GENOM用细菌人工染色体（BAC）IC库已建成为海洋和淡水棘鱼30。这些BAC的可recombineered在一个大的（150-200 kb的）的环境中的荧光报道基因的基因组区31，以取代一个基因。的荧光报道然后在时空图案表示为通过BAC内调节序列来确定。在鱼的研究中，TOL2站点可以加入到BAC，促进基因组整合32,33。在发育的后期阶段，当在原位杂交在技术上是挑战性的，所述的BAC的荧光读出可用于研究基因表达的模式，如已经示出了刺骨形态发生蛋白6（BMP6）20。此外，在一个单独的荧光表达模式可随着时间的推移，它不能与原位杂交来实现跟踪。 BAC的还可以用来增加一个additiona一个基因组区l的副本，以增加的感兴趣的基因的剂量。 基因功能的研究，基因组编辑是一种爆发扩展字段可用于生产各种各样的生物体34的基因组序列的有针对性的变化。转录活化剂-样效应核酸（TALENS）是最初从能够精确设计以直接结合到所选择的基因组序列，并产生双链断裂35,36植物病原体分离模块化，序列特异性核酸酶。群集定期相互间隔短回文重复序列（CRISPR）/ CAS系统中的细菌最初发现并使用一个导的RNA和Cas9蛋白以生成到导向37的互补靶DNA序列中断。由两个TALENS和CRISPRs创建往往双链断裂的后续修复留下一个小的插入或缺失，其可以破坏靶序列的功能35-37。在刺鱼，TALENS已被用于针对增强20破坏基因的表达，TALENS和CRISPRs无论是在编码序列（未发表资料）已成功生产突变。对于CRISPRs在斑马鱼使用的生成详细的协议可以作为一个准则制定刺鱼38 CRISPRs。 转基因和基因组编辑实验需要引入核酸到一个新受精的单细胞胚胎。通过引入在发育早期的转基因或基因组编辑工具，在胚胎基因操纵子细胞的数目被最大化。注射胚胎，然后目测筛选荧光或分子筛选基因组修改。如果有助于生殖系细胞成功靶向，转基因或突变可以传递给后代的一个子集，即使当后喷射杀伤力是高的。马赛克鱼可还是远交互交和他们的后代中筛选到恢复突变等位基因或感兴趣的稳定整合的转基因。本协议描述引入转基因和基因组编辑试剂成单细胞胚胎刺和监测成功的基因修改方法。

<table border="0" cellpadding="0" cellspacing="0" width="647">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Stereomicroscope with transillumination</td>
			<td style="width:145px;">Leica</td>
			<td style="width:119px;">S6e/ KL300 LED</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Manual micromanipulator</td>
			<td style="width:145px;">Applied Scientific Instrumentation</td>
			<td style="width:119px;">MM33</td>
			<td style="width:167px;">Marzhauser M33 Micromanipulator</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pressure Injecion system</td>
			<td style="width:145px;">Applied Scientific Instrumentation</td>
			<td style="width:119px;">MPPI-3</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Back pressure unit</td>
			<td style="width:145px;">Applied Scientific Instrumentation</td>
			<td style="width:119px;">BPU</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Micropipette holder kit</td>
			<td style="width:145px;">Applied Scientific Instrumentation</td>
			<td style="width:119px;">MPIP</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Magnetic base holder</td>
			<td style="width:145px;">Applied Scientific Instrumentation</td>
			<td style="width:119px;">Magnetic base</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Foot switch</td>
			<td style="width:145px;">Applied Scientific Instrumentation</td>
			<td style="width:119px;">FSW</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Iron plate (magnetic base)</td>
			<td style="width:145px;">Narishige</td>
			<td style="width:119px;">IP</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Flaming/Brown Micropipette Puller</td>
			<td style="width:145px;">Sutter Instrument</td>
			<td style="width:119px;">P-97</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Disposable transfer pipettes</td>
			<td style="width:145px;">Fisher</td>
			<td style="width:119px;">13-711-7M</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.5% phenol red in DPBS</td>
			<td style="width:145px;">Sigma&#160;</td>
			<td style="width:119px;">P0290</td>
			<td style="width:167px;">injection tracer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">#5 forceps, biologie dumoxel&#160;</td>
			<td style="width:145px;">Fine Science Tools</td>
			<td style="width:119px;">11252-30</td>
			<td style="width:167px;">for needle breaking</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Micropipette Storage Jar</td>
			<td style="width:145px;">World Precision Instruments</td>
			<td style="width:119px;">E210</td>
			<td>holds needles</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">6&#34;, 6 teeth per inch plaster drywall saw</td>
			<td style="width:145px;">Lenox</td>
			<td>20571 (S636RP)</td>
			<td>hold eggs for injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">13 cm x 13 cm glass plate</td>
			<td style="width:145px;">any hardware store</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Borosilicate glass capillaries, 1.0 mm OD/0.58 mm ID&#160;</td>
			<td style="width:145px;">World Precision Instruments</td>
			<td style="width:119px;">1B100-F4</td>
			<td style="width:167px;">*harder glass than zebrafish injection capillaries</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">150 x 15mm petri dish</td>
			<td style="width:145px;">Fisher</td>
			<td style="width:119px;">FB0875714</td>
			<td>raise stickleback embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">35 x 10mm petri dish</td>
			<td style="width:145px;">Fisher</td>
			<td style="width:119px;">08-757-100A</td>
			<td>store eggs pre-injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Instant Ocean Salt</td>
			<td style="width:145px;">Instant Ocean</td>
			<td>SS15-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Bicarbonate</td>
			<td style="width:145px;">Sigma</td>
			<td>S5761-500G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tricaine methanesulfonate/MS-222</td>
			<td style="width:145px;">Western Chemical Inc</td>
			<td style="width:119px;">MS222</td>
			<td style="width:167px;">fish anaesthesia/euthanasia</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Sp6 transcription kit</td>
			<td style="width:145px;">Ambion</td>
			<td style="width:119px;">AM1340</td>
			<td style="width:167px;">For transcription of TALENs and transposase mRNA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">RNeasy cleanup kit</td>
			<td style="width:145px;">Qiagen</td>
			<td style="width:119px;">74104</td>
			<td style="width:167px;">purify transposase or TALEN RNA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">QiaQuick PCR cleanup kit</td>
			<td style="width:145px;">Qiagen</td>
			<td style="width:119px;">28104</td>
			<td style="width:167px;">clean up plasmids for injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Proteinase K 20 mg/ml</td>
			<td style="width:145px;">Ambion</td>
			<td style="width:119px;">AM2546</td>
			<td style="width:167px;">for DNA preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nucleobond BAC 100 kit</td>
			<td style="width:145px;">Clontech</td>
			<td style="width:119px;">740579</td>
			<td>for BAC DNA preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NotI</td>
			<td style="width:145px;">NEB</td>
			<td style="width:119px;">R0189L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phusion polymerase</td>
			<td style="width:145px;">Fisher</td>
			<td style="width:119px;">F-530L</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Qiagen PlasmidPlus Midi kit</td>
			<td style="width:145px;">Qiagen</td>
			<td style="width:119px;">12943</td>
			<td style="width:167px;">contains endotoxin rinse buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">QIAQuick Gel Extraction</td>
			<td style="width:145px;">Qiagen</td>
			<td style="width:119px;">28704&#160;</td>
			<td>for sequencing induced mutations</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phenol:chloroform:Isoamyl alcohol</td>
			<td style="width:145px;">Sigma</td>
			<td>P2069-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium acetate</td>
			<td style="width:145px;">Sigma</td>
			<td>S2889-250G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethanol (molecular biology grade)</td>
			<td style="width:145px;">Sigma</td>
			<td>E7023-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Agarose</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:119px;">A9539</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50X Tris-acetate-EDTA buffer</td>
			<td style="width:145px;">ThermoFisher</td>
			<td style="width:119px;">B49</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.5-10KbRNA ladder</td>
			<td style="width:145px;">ThermoFisher</td>
			<td style="width:119px;">15623-200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nanodrop&#160; Spectrophotometer</td>
			<td style="width:145px;">Thermo Scientific</td>
			<td style="width:119px;">Nanodrop 2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paraformaldehyde</td>
			<td style="width:145px;">Sigma</td>
			<td>158127-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10X PBS</td>
			<td style="width:145px;">ThermoFisher</td>
			<td>70011-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1kb Plus DNA Ladder</td>
			<td style="width:145px;">ThermoFisher</td>
			<td>10787-018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Potassium Chloride</td>
			<td style="width:145px;">Sigma</td>
			<td>P9541-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium Chloride</td>
			<td style="width:145px;">Sigma</td>
			<td>M8266-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NP-40</td>
			<td style="width:145px;">ThermoFisher</td>
			<td>28324</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tween 20</td>
			<td style="width:145px;">Sigma</td>
			<td>P1379-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris pH 8.3</td>
			<td style="width:145px;">Teknova</td>
			<td>T1083</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">12-strip PCR tube</td>
			<td style="width:145px;">Thermo Scientific</td>
			<td style="width:119px;">AB-1113</td>
			<td></td>
		</tr>
	</tbody>
</table>

microinjection for transgenesis and genome editing in threespine sticklebacks

该三刺鱼已成为一个强大的系统来研究各种各样的形态，生理的遗传基础和行为的表型。海洋种群适应无数淡水环境已经放出的非常多样化的表型，用交叉的海洋和淡水的形式的能力相结合，提供了难得脊椎动物系统，其中遗传学可用于映射的基因组区域控制演变特征。优秀的基因资源，现已有售，促进发展变化的分子遗传学解剖。而映射实验产生性的候选基因列表，官能基因操作都必须测试这些基因的作用。基因调控可以集成到使用TOL2转座系统的转基因报告质粒和BAC进行研究。具体的候选基因与顺 -regulatory元素功能可以通过诱导目标进行评估突变的TALEN和CRISPR / Cas9基因组编辑试剂。所有的方法都需要将核酸引入受精单细胞刺胚胎中，任务由刺胚胎的厚绒毛膜和相对小而薄的卵裂球制成具有挑战性。这里，一个详细的协议用于核酸显微注射到刺胚胎为转基因和基因组编辑应用程序来研究基因表达和功能，以及技术来评估转基因的成功和恢复稳定线说明。

对于具有增强子活性的报告基因的转基因，成功的注射会造成该转基因（ 图4A，4C）的特异性，细胞表达。然后注射鱼可以远交，以产生稳定的线（在图4B所示的BAC稳定线的例子）。注射DNA导入刺胚胎通常会导致比单独RNA远高于杀伤力。典型的是看到高达50％（有时甚至更多）杀伤力或畸形（ 见图4D - 女 ...

Watch this Scientific Journal Video about Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks at JoVE.com

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

显微注射转基因的基因组，并在编辑Threespine刺鱼

转基因操作和基因组编辑是功能测试基因和顺 -regulatory元素的作用是至关重要的。此处的基因组修饰（包括TOL2介导的荧光报道基因构建体，TALENS和CRISPRs）的生成的详细显微注射协议提出了紧急模型鱼的三刺。

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and ...

microinjection-for-transgenesis-genome-editing-threespine

Research

JoVE Journal

Developmental Biology

13.8K 次观看.  University of California, Berkeley. 转基因操作和基因组编辑是功能测试基因和顺 -regulatory元素的作用是至关重要的。此处的基因组修饰（包括TOL2介导的荧光报道基因构建体，TALENS和CRISPRs）的生成的详细显微注射协议提出了紧急模型鱼的三刺。 

视频: Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and organ formation during development. Determining the locations and levels of such chromatin features in vivo will provide valuable information about the spatio-temporal regulation of genomic elements, and will support aspirations to mimic embryonic tissue development in vitro. The most commonly used method for genome-wide and high-resolution profiling is chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq). This protocol outlines how yolk-rich embryos such as those of the frog Xenopus can be processed for ChIP-Seq experiments, and it offers simple command lines for post-sequencing analysis. Because of the high efficiency with which the protocol extracts nuclei from formaldehyde-fixed tissue, the method allows easy upscaling to obtain enough ChIP material for genome-wide profiling. Our protocol has been used successfully to map various DNA-binding proteins such as transcription factors, signaling mediators, components of the transcription machinery, chromatin modifiers and post-translational histone modifications, and for this to be done at various stages of embryogenesis. Lastly, this protocol should be widely applicable to other model and non-model organisms as more and more genome assemblies become available.

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

The question of how chromatin regulators and chromatin states affect the genome in vivo is key to our understanding of how early cell fate decisions are made in the developing embryo. ChIP-Seq&#8212;the most popular approach to investigate chromatin features at a global level&#8212;is outlined here for Xenopus embryos.

染色质监管机构和各国的全基因组快照<em&gt;爪</em&gt;通过胚胎的ChIP-SEQ

The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and ...

科研

发育生物学

Mosaic Zebrafish Transgenesis for Functional Genomic Analysis of Candidate Cooperative Genes in Tumor Pathogenesis

Comprehensive genomic analysis has uncovered surprisingly large numbers of genetic alterations in various types of cancers. To robustly and efficiently identify oncogenic &#8220;drivers&#8221; among these tumors and define their complex relationships with concurrent genetic alterations during tumor pathogenesis remains a daunting task. Recently, zebrafish have emerged as an important animal model for studying human diseases, largely because of their ease of maintenance, high fecundity, obvious advantages for in vivo imaging, high conservation of oncogenes and their molecular pathways, susceptibility to tumorigenesis and, most importantly, the availability of transgenic techniques suitable for use in the fish. Transgenic zebrafish models of cancer have been widely used to dissect oncogenic pathways in diverse tumor types. However, developing a stable transgenic fish model is both tedious and time-consuming, and it is even more difficult and more time-consuming to dissect the cooperation of multiple genes in disease pathogenesis using this approach, which requires the generation of multiple transgenic lines with overexpression of the individual genes of interest followed by complicated breeding of these stable transgenic lines. Hence, use of a mosaic transient transgenic approach in zebrafish offers unique advantages for functional genomic analysis in vivo. Briefly, candidate transgenes can be coinjected into one-cell-stage wild-type or transgenic zebrafish embryos and allowed to integrate together into each somatic cell in a mosaic pattern that leads to mixed genotypes in the same primarily injected animal. This permits one to investigate in a faster and less expensive manner whether and how the candidate genes can collaborate with each other to drive tumorigenesis. By transient overexpression of activated ALK in the transgenic fish overexpressing MYCN, we demonstrate here the cooperation of these two oncogenes in the pathogenesis of a pediatric cancer, neuroblastoma that has resisted most forms of contemporary treatment.

The goal of this study is to demonstrate how the mosaic transgenesis strategy can be used in zebrafish to rapidly and efficiently assess the relative contributions of multiple oncogenes in tumor initiation and progression in vivo.

马赛克斑马鱼转基因的候选基因合作在肿瘤发病机制的功能基因组分析

Comprehensive genomic analysis has uncovered surprisingly large numbers of genetic alterations in various types of cancers.  To robustly and efficiently ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

技术目标显微注射到显影<em&gt;爪</em&gt;肾

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Identification of Critical Conditions for Immunostaining in the Pea Aphid Embryos: Increasing Tissue Permeability and Decreasing Background Staining

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental studies. Like other aphids, A. pisum propagate rapidly via parthenogenetic viviparous reproduction, where the embryos develop within egg chambers in an assembly-line fashion in the ovariole. Previously we have established a robust platform of whole-mount in situ hybridization allowing detection of mRNA expression in the aphid embryos. For analyzing the expression of protein, though, established protocols for immunostaining the ovarioles of asexual viviparous aphids did not produce satisfactory results. Here we report conditions optimized for increasing tissue permeability and decreasing background staining, both of which were problems when applying established approaches. Optimizations include: (1) incubation of proteinase K (1 &#181;g/ml, 10 min), which was found essential for antibody penetration in mid- and late-stage aphid embryos; (2) replacement of normal goat serum/bovine serum albumin with a blocking reagent supplied by a Digoxigenin (DIG)-based buffer set and (3) application of methanol rather hydrogen peroxide (H2O2) for bleaching endogenous peroxidase; which significantly reduced the background staining in the aphid tissues. These critical conditions optimized for immunostaining will allow effective detection of gene products in the embryos of A. pisum and other aphids.

A protocol of whole-mount immunostaining on aphid embryos is presented, in which critical conditions for decreasing background staining and increasing tissue permeability are addressed. These conditions are specifically developed for effective detection of protein expression in the embryonic tissues of aphids.

临界条件在免疫染色豌豆蚜虫胚胎鉴定:增加组织通透性和降低背景染色

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental ...

Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning fundamental developmental biology through to large-scale genetic and chemical screens. However, many experiments, especially those related to infection and xenograft models, rely on microinjection and imaging of embryos and larvae, which are laborious techniques that require skill and expertise. To improve the precision and throughput of current microinjection techniques, we developed a series of microstructured devices to orient and stabilize zebrafish embryos at 2 days post fertilization (dpf) in ventral, dorsal, or lateral orientation prior to the procedure. To aid in the imaging of embryos, we also designed a simple device with channels that orient 4 zebrafish laterally in parallel against a glass cover slip. Together, the tools that we present here demonstrate the effectiveness of photolithographic approaches to generate useful devices for the optimization of zebrafish techniques.

Microinjection of zebrafish embryos and larvae is a crucial but challenging technique used in many zebrafish models. Here, we present a range of microscale tools to aid in the stabilization and orientation of zebrafish for both microinjection and imaging.

斑马鱼幼虫显微注射和成像的微装置

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning ...

An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing

Binary transcription systems are powerful genetic tools widely used for visualizing and manipulating cell fate and gene expression in specific groups of cells or tissues in model organisms. These systems contain two components as separate transgenic lines. A driver line expresses a transcriptional activator under the control of tissue-specific promoters/enhancers, and a reporter/effector line harbors a target gene placed downstream to the binding site of the transcription activator. Animals harboring both components induce tissue-specific transactivation of a target gene expression. Precise spatiotemporal expression of the gene in targeted tissues is critical for unbiased interpretation of cell/gene activity. Therefore, developing a method for generating exclusive cell/tissue-specific driver lines is essential. Here we present a method to generate highly tissue-specific targeted expression system by employing a &#34;Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-associated&#34; (CRISPR/Cas)-based genome editing technique. In this method, the endonuclease Cas9 is targeted by two chimeric guide RNAs (gRNA) to specific sites in the first coding exon of a gene in the Drosophila genome to create double-strand breaks (DSB). Subsequently, using an exogenous donor plasmid containing the transactivator sequence, the cell-autonomous repair machinery enables homology-directed repair (HDR) of the DSB, resulting in precise deletion and replacement of the exon with the transactivator sequence. The knocked-in transactivator is expressed exclusively in cells where the cis-regulatory elements of the replaced gene are functional. The detailed step-by-step protocol presented here for generating a binary transcriptional driver expressed in Drosophila fgf/branchless-producing epithelial/neuronal cells can be adopted for any gene- or tissue-specific expression.

An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing

Here, we present a method for generating tissue-specific binary transcription systems in Drosophila by replacing the first coding exon of genes with transcription drivers. The CRISPR/Cas9-based method places a transactivator sequence under the endogenous regulation of a replaced gene, and consequently facilitates transctivator expression exclusively in gene-specific spatiotemporal patterns.

基于基因组编辑的果蝇组织特异二进制转录系统的高效生成策略

Binary transcription systems are powerful genetic tools widely used for visualizing and manipulating cell fate and gene expression in specific groups of ...

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

基于微注射的免疫胚胎心肌细胞体内植入系统

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...

Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica

The firebrat Thermobia domestica is an ametabolous, wingless species that is suitable for studying the developmental mechanisms of insects that led to their successful evolutionary radiation on the earth. The application of genetic tools such as genome editing is the key to understanding genetic changes that are responsible for evolutionary transitions in an Evo-Devo approach. In this article, we describe our current protocol for generating and maintaining mutant strains of T. domestica. We report a dry injection method, as an alternative to the reported wet injection method, that allows us to obtain stably high survival rates in injected embryos. We also report an optimized environment setting to mate adults and obtain subsequent generations with high efficiency. Our method underlines the importance of taking each species&#8217; unique biology into account for the successful application of genome editing methods to non-traditional model organisms. We predict that these genome editing protocols will help in implementing T. domestica as a laboratory model and to further accelerate the development and application of useful genetic tools in this species.

Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica

We provide a detailed protocol for rearing, microinjection of eggs and for efficient mating of the firebrat Thermobia domestica to generate and maintain mutant strains after genome editing.

卵子微弹射和有效交配的基因组编辑在火布拉特 热比亚家

The firebrat Thermobia domestica is an ametabolous, wingless species that is suitable for studying the developmental mechanisms of insects that led to ...

Brain Organoid Generation from Induced Pluripotent Stem Cells in Home-Made Mini Bioreactors

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology has emerged recently. It is still in its infancy and has some limitations unsolved yet. The current protocols do not allow obtaining organoids to be consistent enough for drug discovery and preclinical studies. The maturation of organoids can take up to a year, pushing the researchers to launch multiple differentiation processes simultaneously. It imposes additional costs for the laboratory in terms of space and equipment. In addition, brain organoids often have a necrotic zone in the center, which suffers from nutrient and oxygen deficiency. Hence, most current protocols use a circulating system for culture medium to improve nutrition.
Meanwhile, there are no inexpensive dynamic systems or bioreactors for organoid cultivation. This paper describes a protocol for producing brain organoids in compact and inexpensive home-made mini bioreactors. This protocol allows obtaining high quality organoids in large quantities.

Here we describe a protocol for generating brain organoids from human induced pluripotent stem cells (iPSCs). To obtain brain organoids in large quantities and of high quality, we use home-made mini bioreactors.

在自制迷你生物反应器中从诱导多能干细胞中生成脑类器官

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology ...

显微注射转基因的基因组，并在编辑Threespine刺鱼

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