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

13.9K 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

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene function. Gene knockout has been used to study these gene functions in vivo. The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system is a powerful tool used to edit genomic DNA sequences to alter gene function. While the traditional approach has been to introduce CRISPR/Cas9 mRNA into the single cell embryos through microinjection, this can be a slow and inefficient process in catfish. Here, a detailed protocol for microinjection of channel catfish embryos with CRISPR/Cas9 protein is described. Briefly, eggs and sperm were collected and then artificial fertilization performed. Fertilized eggs were transferred to a Petri dish containing Holtfreter's solution. Injection volume was calibrated and then guide RNAs/Cas9 targeting the toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) gene and rhamnose binding lectin (RBL) gene were microinjected into the yolk of one-cell embryos. The gene knockout was successful as indels were confirmed by DNA sequencing. The predicted protein sequence alterations due to these mutations included frameshift and truncated protein due to premature stop codons.

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

A simple and efficient microinjection protocol for gene editing in channel catfish embryos using the CRISPR/Cas9 system is presented. In this protocol, guide RNAs and Cas9 protein were microinjected into the yolk of one-cell embryos. This protocol has been validated by knocking out two channel catfish immune-related genes.

CRISPR/Cas9 蛋白注射到沟鲶、鮰松毛虫、基因编辑的胚胎

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene ...

科研

遗传学

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

Microinjection of Nasonia vitripennis embryos is an essential method for generating heritable genome modifications. Described here is a detailed procedure for microinjection and transplantation of Nasonia vitripennis embryos, which will greatly facilitate future genome manipulation in this organism.

金小蜂金小蜂基因组操作的胚胎显微注射和移植技术

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex ...

Generation of Genetically Modified Mice through the Microinjection of Oocytes

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear injection of DNA expression constructs into fertilized oocytes remains the most commonly used technique to generate transgenic mice for overexpression. With the introduction of CRISPR technology for gene targeting, pronuclear injection into fertilized oocytes has been extended to the generation of both knockout and knockin mice. This work describes the preparation of DNA for injection and the generation of CRISPR guides for gene targeting, with a particular emphasis on quality control. The genotyping procedures required for the identification of potential founders are critical. Innovative genotyping strategies that take advantage of the "multiplexing" capabilities of CRISPR are presented herein. Surgical procedures are also outlined. Together, the steps of the protocol will allow for the generation of genetically modified mice and for the subsequent establishment of mouse colonies for a plethora of research fields, including immunology, neuroscience, cancer, physiology, development, and others.

The microinjection of mouse oocytes is commonly used for both classic transgenesis (i.e., the random integration of transgenes) and CRISPR-mediated gene targeting. This protocol reviews the latest developments in microinjection, with a particular emphasis on quality control and genotyping strategies.

通过显微注射卵母细胞生成转基因小鼠

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear ...

发育生物学

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

显微注射mRNA和斑马鱼的胚胎吗啉反义寡核苷酸。

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

生物学

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

Manipulation of gene expression in tissues is required to perform functional studies. In this paper, we demonstrate the cerebroventricular microinjection (CVMI) technique as a means to modulate gene expression in the adult zebrafish brain. By using CVMI, substances can be administered into the cerebroventricular fluid and be thoroughly distributed along the rostrocaudal axis of the brain. We particularly focus on the use of antisense morpholino oligonucleotides, which are potent tools for knocking down gene expression in vivo. In our method, when applied, morpholino molecules are taken up by the cells lining the ventricular surface. These cells include the radial glial cells, which act as neurogenic progenitors. Therefore, knocking down gene expression in the radial glial cells is of utmost importance to analyze the widespread neurogenesis response in zebrafish, and also would provide insight into how vertebrates could sustain adult neurogenesis response. Such an understanding would also help the efforts for clinical applications in human neurodegenerative disorders and central nervous system regeneration. Thus, we present the cerebroventricular microinjection method as a quick and efficient way to alter gene expression and neurogenesis response in the adult zebrafish forebrain. We also provide troubleshooting tips and other useful information on how to carry out the CVMI procedure.

In this article, we demonstrate a method for manipulation of gene expression in the ventricular cells of the adult zebrafish telencephalon using antisense morpholino oligonucleotides. We present this method as an efficient and quick protocol that can be used for functional studies in the adult vertebrate brain.

显微吗啉代寡核苷酸脑室微量注射成年斑马鱼大脑中的基因表达

Manipulation of gene expression in tissues is  required to perform functional studies. In this paper, we demonstrate the  cerebroventricular ...

神经科学

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

将DNA微注射到异种胚胎的眼蕾中，以及GFP表达光学轴状轴向轴向的成像，活的Xenopus大白鼠

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...

Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily produced by electroporation of CRISPR factors (and single-stranded oligo DNA donors) into the zygote. In contrast, gene cassette (&#62;1 kb) knock-in and floxed mice are mainly generated by microinjection of CRISPR factors and double-stranded DNA donors into zygotes. Genome editing technologies have also increased the flexibility of genetically modified mice production. It is now possible to introduce the intended mutations in the target genomic regions in a number of beneficial inbred mouse strains. Our team has produced over 200 gene cassette knock-in mouse lines, and over 110 floxed mouse lines by zygote microinjection of CRISPR-Cas9 following requests from several countries, including Japan. Some of these genome editing used BALB/c, C3H/HeJ, and C57BL/6N inbred strains, however most used C57BL/6J. Unlike the electroporation method, genome editing by zygote microinjection in various inbred strains of mice is not that easy. However, gene cassette knock-in and floxed mice on single inbred genetic backgrounds are as critical as genetic humanized, fluorescent reporter, and conditional knockout mouse models. Therefore, this article presents the protocol for the zygote microinjection of CRISPR factors and double-stranded DNA donors in C57BL/6J mice for generating gene cassette knock-in and floxed mice. This article exclusively focuses on nuclear injection rather than cytoplasmic injection. In addition to zygote microinjection, we outline the timeline for the production process and peripheral techniques such as induction of superovulation and embryo transfer.

The present protocol describes zygote microinjection of CRISPR-Cas9 and donor DNA to efficiently produce gene cassette knock-in and floxed mice.

受精卵显微注射用于在小鼠中产生基因盒敲入和絮状等位基因

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily ...

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.

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

Gene-targeting mutagenesis is now possible in a wide range of organisms using genome editing techniques. Here, we demonstrate a protocol for targeted gene mutagenesis using transcription activator like effector nucleases (TALENs) in Astyanax mexicanus, a species of fish that includes surface fish and cavefish.

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 ...

Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture. This is perhaps best exemplified by the numerous convergent features of gymnotiforms and mormyrids, two species-rich teleost clades that produce and detect weak electric fields and are called weakly electric fish. In the 50 years since the discovery that weakly electric fish use electricity to sense their surroundings and communicate, a growing community of scientists has gained tremendous insights into evolution of development, systems and circuits neuroscience, cellular physiology, ecology, evolutionary biology, and behavior. More recently, there has been a proliferation of genomic resources for electric fish. Use of these resources has already facilitated important insights with regards to the connection between genotype and phenotype in these species. A major obstacle to integrating genomics data with phenotypic data of weakly electric fish is a present lack of functional genomics tools. We report here a full protocol for performing CRISPR/Cas9 mutagenesis that utilizes endogenous DNA repair mechanisms in weakly electric fish. We demonstrate that this protocol is equally effective in both the mormyrid species Brienomyrus brachyistius and the gymnotiform Brachyhypopomus gauderio by using CRISPR/Cas9 to target indels and point mutations in the first exon of the sodium channel gene scn4aa. Using this protocol, embryos from both species were obtained and genotyped to confirm that the predicted mutations in the first exon of the sodium channel scn4aa were present. The knock-out success phenotype was confirmed with recordings showing reduced electric organ discharge amplitudes when compared to uninjected size-matched controls.

Here, a protocol is presented to produce and rear CRISPR/Cas9 genome knockout electric fish. Outlined in detail are the required molecular biology, breeding, and husbandry requirements for both a gymnotiform and a mormyrid, and injection techniques to produce Cas9-induced indel F0 larvae.

沉默的火花:CRISPR/Cas9基因组编辑在弱电鱼

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these ...

Embryo Injection Technique for Gene Editing in the Black-Legged Tick, Ixodes scapularis

Ticks can transmit various viral, bacterial, and protozoan pathogens and are therefore considered vectors of medical and veterinary importance. Despite the growing burden of tick-borne diseases, research on ticks has lagged behind insect disease vectors due to challenges in applying genetic transformation tools for functional studies to the unique biology of ticks. Genetic interventions have been gaining attention to reduce mosquito-borne diseases. However, the development of such interventions requires stable germline transformation by injecting embryos. Such an embryo injection technique is lacking for chelicerates, including ticks. Several factors, such as an external thick wax layer on tick embryos, hard chorion, and high intra-oval pressure, are some&#160;obstacles that previously prevented embryo injection protocol development in ticks. The present work has overcome these obstacles, and an embryo injection technique for the black-legged tick, Ixodes scapularis, is described here.&#160;This technique can be used to deliver components, such as CRISPR/Cas9, for stable germline transformations.

Embryo Injection Technique for Gene Editing in the Black-Legged Tick, Ixodes scapularis

The present protocol describes a method for injecting tick embryos. Embryo injection is the preferred technique for genetic manipulation to generate transgenic lines.

用于黑腿蜱、肩突硬蜱基因编辑的胚胎注射技术

Ticks can transmit various viral, bacterial, and protozoan pathogens and are therefore considered vectors of medical and veterinary importance. Despite ...