作者非常感谢协助与显微注射，以及匿名审稿的有见地的意见RJ索默和X王。这项工作是支持由NIH资助SC2GM089602。

1。转基因：DNA的制备<ol><li>占主导地位的合作注入标记：pRL3 [PPA - PRL - 1（ tu92）] pRL3质粒是一个占主导地位的合作注入的视觉识别的成功转型事件的标记。这种质粒编码为主导的PPA - PRL - 1基因密切相关的SQT - 1 C.胶原蛋白基因的突变等位基因 （ tu92） 线虫和野生型正弦运动转换成沿顺时针方向扭蠕虫的体轴1,5的议案。转化的动物是非常相似流行的显性选择标记ROL - 6（su1006） 在 C在过去20年来的线虫。 </li><li>转基因目标记者：PPA - EGL - 4P：GFP 有兴趣的基因可以与转录或翻译的GFP编码序列 9 。在这项研究中，PPA - EGL - 4promoter：：GFP（EGL - 4的cGMP依赖的蛋白激酶）WAs一起使用，以确定是否2 kb的片段翻译起始的上游， 可以赋予P. GFP表达pacificus PS312。这GFP载体含有GFP的编码区与修改 内含子和多用途PPA - RPL - 23的3&#39;UTR结束符5请小月王和Ralf J.索默（马克斯-普朗克研究所，图宾根，德国，欧盟）提供。 </li><li>基因组DNA：PS312 野生型体育此外pacificus PS312基因组DNA是需要获得稳定的转基因继承，特别是从转基因 F 1动物的F 2 后裔 5 。这是尚未确定，如果基因组DNA的形式复杂的额外染色体阵列与两个转基因（共注射标记PRL - 3和PPA - EGL - 4P：GFP记者） 类似稳定楼2转基因株系观察C。线虫 3。然而，要求基因组DNA和转基因份额identica升凝聚力出挑强烈建议形成复杂的额外染色体阵列宿主细胞可以识别传输正确的DNA（gDNA的准备使用西格玛G1N10试剂盒）。 </li><li> DNA酶切 用限制性内切酶线性的PPA - EGL - 4P：GFP载体DNA和生产兼容与合作注入标记pRL3和PS312的gDNA粘出挑。混合攻丝，不震荡。一小时在37 ° C。 </li></ol><table border="1"><tbody><tr><td> pRL3 </td><td> 4微克</td></tr><tr><td> 10xbuffer </td><td> 10μL </td></tr><tr><td> PSTI（10 U /μL） </td><td> 4μL </td></tr><tr><td> DH 2 O </td><td> 〜 </td></tr><tr><td> 总计： </td><td> 加入100μL </td></tr><tr><td> PPA - EGL - 4P：：GFP </td><td> 4微克</td></tr><tr><td> 10X缓冲</tD&gt; <td> 10μL </td></tr><tr><td>萨利（10 U /μL） </td><td> 4μL </td></tr><tr><td> DH 2 O </td><td> 〜 </td></tr><tr><td> 总计： </td><td> 100μL </td></tr><tr><td> 基因组DNA PS312 </td><td> 10微克</td></tr><tr><td> 10X缓冲</td><td> 10μL </td></tr><tr><td>的Pst I和Sal I（10 U /μL） </td><td> 8μL </td></tr><tr><td> DH 2 O </td><td> 〜 </td></tr><tr><td> 总计： </td><td> 100μL </td></tr></tbody></table> 表1。限制性酶切混合物。 <ol start="5"><li> DNA沉淀和准备<ol><li> 1:10醋酸钠（3米NaCOOCH 3 pH值5.2）和2.5倍体积的100％的乙醇来消化产品;增加20毫微克/糖原μL看到颗粒容易。 </li><li>离心14000 RPM为15分钟，在4 ° C。 </li><li>小费慢慢的离心管倒置，然后攻上纸巾，确保颗粒是安全的，在右下角管和乙醇倒出上清液。 </li><li>加入1毫升75％的乙醇。 </li><li>立即旋转14000转5分钟，在4 ° C。 </li><li>慢慢引爆的离心管倒置，然后攻上纸巾，确保颗粒在右下角管安全和乙醇倒出上清液。 </li><li>干颗粒在真空离心14,000 5分钟的转速在25-30 ° C，直到干燥和完全不含乙醇。 </li><li>添加30μLTE或无菌DH 2℃前一个旋涡混合简要留在4℃过夜。 </li><li>表2描述了如何创建从每个注射纯化的DNA注射液。 </li><li>贮存于-20 ° C在解冻后的混合物jection，降速在5分钟14,000 RPM管摆脱碎片微粒可能阻塞注射针头。 </li></ol></li></ol><table border="1"><tbody><tr><td> 遗传物质 </td><td> 集中 </td></tr><tr><td> PPA - EGL - 4P：：GFP（贝） </td><td> &gt; 5 ng /μL的（0.1-10ng/μl） </td></tr><tr><td> pRL3（PSTI） </td><td> &gt; 1纳克/μL </td></tr><tr><td>基因组DNA PS312（PSTI +贝） </td><td> &gt; 60毫微克/μL </td></tr><tr><td> DH 2 O </td><td> 〜 </td></tr><tr><td> 总计： </td><td> 30μL </td></tr></tbody></table> 表2。PPA - PRL - 1注射的混合物 2。 RNA干扰：在体外双链RNA的合成<ol><li>使用PCR扩增感兴趣的基因。 RHL091 agtggatccGAAGGTCCATACGGGAGC - 5&#39; - 3&#39;BamHI位（网站）和RHL092 5&#39; - tatctgcagGTGAGGAGTACCAGGAGAG - 3“（PSTI网站）用于扩增一个464 bp的基因片段，从pRL3含有PPA - PRL - 1（tu92）等位基因向量（位置3-466 ）。 </li><li>用BamHI和PstI消化的PCR产物。 </li><li>结扎片段BamHI位/ PSTI消化pL4440 RNAi的喂养载体2。 </li><li>主管细胞转换成插入载体和增长氨苄青霉素的LB媒体板（50微克/毫升）。 </li><li>选择经PCR成功插入载体，使用的T7引物。 </li><li>使用两侧的T7 PCR产物含有PRL - 1基因，使双链RNA双链RNA）使用块的IT RNAi的合成试剂盒（Invitrogen公司）。 </li><li> dsRNA的浓度是最好的，如果&gt; 200纳克/μL，最高可达1微克/μL注射。 </li><li>贮存于-20 ° C解冻后的混合物注射，5分钟降速管摆脱碎片微粒可能阻塞注射液在14000转ñ针。 </li></ol> 3。显微注射：注射转基因和/或双链RNA协议<ol><li>注射针头<ol><li>设置Narishige针拔取器（型号：PC - 10）的温度： <ol><li>底部的旋钮转到“2号取暖器。” </li><li>打开“第二加热器ADJ。”直到面板旋钮读取55.0-60.0 ° C的长短不一的锥形针头。 </li></ol></li><li>底部旋钮转到“步骤1”（在一个步骤中提取的针）。 </li><li>负载针进入会议厅，并确保它是安全的。 </li><li>按“开始”按钮（红色按钮），并允许所有的权重（这应该使两个相反的方向相同的针）拉针。 </li><li>从担保播放，卫生署，以防止灰尘积累针在塑料培养板腔和存储针。 </li></ol></li><li>安装垫<ol><li>在1.5 ml离心管，使2％的崇高的琼脂solution的宝琼脂加入0.02克至1毫升H 2 O琼脂可以保存于4 ° C的一年。 </li><li>调匀融化在热块琼脂设置为&gt; 88 ° C。 </li><li>用1毫升的微管，置于载玻片中间的液体高贵的琼脂2％的下降（费舍尔，12 - 544 - F）。 </li><li>拼合由另一载玻片放置在顶部的下拉。 </li><li>直到有一层五载玻片重复“落步”。 </li><li>删除每个玻片上滑动的玻璃幻灯片交相辉映。 </li><li>干扁平化玻片上的琼脂板在真空烘箱在70 ° 4小时至过夜C（或在室温下过夜）。 </li><li>多次注射垫和存储以供将来使用。 </li></ol></li><li>微量注射雌雄同体性腺</li></ol><img alt="图1" src="/files/ftp_upload/3270/3270fig1.jpg" /> 图1。对体育的一个示意图pacificus ANATomy。明确的有核细胞是女性生殖细胞（卵母细胞）和灰色阴影部分是肠道。所示，但只有一个远端前性腺通知远端的两个性腺跨近中期体对方背部。 <img alt="图2" src="/files/ftp_upload/3270/3270fig2.jpg" /> 图2。显微注射的图片（一）注射针头的重点是与拉毛细管件行权。轻轻抚摸该边针的针尖，针尖应打破，同时仍​​保留了尖锐点。 （二）进入前针插入性腺只是第一次注射的肠道曲率以上。 （三）进入底部的针插入性腺略低于肠道曲率，第二次注射。 <ol start="3"><li><ol><li>确保对比度和DIC的剪切设置查看雌雄同体性腺的理想选择。 </li><li> 1.0-2.0μL的注射液混合装入拉升毛细管针。 </li><li>放置在操纵的显微注射针;氮气罐的压力表应设置为0.5-1秒（10-20 psi）的理想条件。 </li><li>针尖突破稍微触摸到一个薄拉到的毛细管管放在幻灯片（图2A）边缘。 <ol><li>针断路器幻灯片：最薄的部分，一把拉住毛细管置于载玻片上一滴油</li></ol></li><li>针一旦被打破，它可以在空闲的位置，而你挑你的蠕虫垫（P. pacificus注射他们干出前5分钟的窗口和死亡）。 </li><li>使用全板的J4年轻成虫，倒入足够的石蜡油（重），以刚好盖住OP50细菌草坪中的所有蠕虫。 </li><li>选择一个蠕虫病毒和它放置在注射垫。 </li><li>显微镜蠕虫将载玻片;位置蠕虫的性腺针的方向和需要远离外阴LE（图2B，2C）。 </li><li>显微镜视图的位置，下针。 </li><li>顶部性腺在同一焦平面（图2B）和针的位置。 </li><li>轻轻捅蠕虫和泵注射液; ooctyes对远端尖端分离表明一个成功的注射（优化转基因或双链RNA传递，你应该看到注射后的扩张在继续，直到它向远端的性腺波TIP）。 </li><li>重新定位较低的性腺和重复注射针注入，但是由于性腺是卵母细胞的扩散将是不可见的肠道，从而更难以实现（图2C）。 </li><li>正常的蠕虫表明注射后成功注入，不应该有任何损坏的生殖腺（凸出外阴或体腔内部弹出）。 </li><li>放置在怠速位置退针。 </li><li>准备救援蠕虫。 </li><li>添加M9的缓冲区下降到注入蠕虫是放在垫（0.5μL）。 </li><li>使用，你挑一些OP50细菌，触摸与OP50的蠕虫，应该坚持。我们不使用吸管口。 </li><li>放置到钢板接种50μL点OP50的蠕虫病毒，2-4蠕虫等板块可以获救。 </li></ol></li><li>注射后的存储和转基因的筛选<ol><li>商店注入蠕虫（0）20 ° C〜4天产卵和成长起来的F 1。 </li><li>第四天为选定的表型转基因， 表达PPA - PRL - 1显性标记的滚动动物的搜索屏幕上的画面蠕虫。 </li><li>选择单一独立的，与所观察到的表型到新的种子板块和存储F 1“在25秒动物° C，这个温度鼓励形成稳定的线从 F 1 &#39;号</li><li>从独立的F 1线的单F 2 。个别F 2 </sub&gt;行也有类似的基因传输速率。我们经常获得&gt; 15楼2线每改造F 1动物。 </li><li>在25 ° C存储后代</li><li>轧辊的传输速率通常保持不变后的F 4代，因此它是在这个时候需要确定的目标基因的表达水平（PPA - EGL - 4P：GFP：）为每个行。 GFP的表达可能不占主导地位的滚子表型的显性程度的关联。 </li></ol></li><li>注射后的存储和RNAi的表型的筛选<ol><li>商店注入蠕虫（0）20 °〜4天，奠定和成长 F 1 C 。 </li><li>屏幕的F 1第四天蠕虫选定的表型（RNAi技术敲除表型，可能在我们的情况与目标基因的活性损失， 损失PPA - PRL - 1的表型渗透剂的表型搜索屏幕）。 </li><li>根据我们的经验，非辊击倒表型丢失&gt; 97％的F 2。 </li></ol></li></ol> 4。代表性的成果： 1。结果，转基因<table border="1"><tbody><tr><td> 会议 </td><td> 注射P 0 </td><td> F 1压路机 </td><td> ％转基因F 2 </td><td> 后楼2％的平均传输 </td></tr><tr><td> 1 </td><td> 60 </td><td> 2 </td><td> （1）0％ （2）13％ </td><td>不适用 13％± 10 </td></tr><tr><td> 2 </td><td> 40 </td><td> 1 * </td><td> （3）0％ </td><td>不适用</td></tr><tr><td> 3 </td><td> 40 </td><td> 0 </td><td> 0％ </td><td>不适用</td></tr><tr><td> 4 </td><td> 40 </td><td> 1 </td><td> （4）0％ </td><td>不适用</tD&gt; </tr><tr><td> 5 </td><td> 20 </td><td> 0 </td><td> 0％ </td><td>不适用</td></tr><tr><td> 6 </td><td> 40 </td><td> 1 </td><td> （5）26％ </td><td> 22％± 10 </td></tr></tbody></table> *男性辊没有交叉; NA：不适用 ：GFP（萨尔I酶切），pRL3（滚子）（PST我消化）和基因组DNA PS312（PstI和SalI酶切）：PPA - EGL - 4P注入PS312结果见表3。 <img alt="图3" src="/files/ftp_upload/3270/3270fig3.jpg" /> 图3（a）野生型（乙，丙）稳定的F 4 pRL3; PPA - EGL - 4P：：GFP转基因线显示头部神经元GFP表达。 2。 RNA干扰的结果<img alt="图4" src="/files/ftp_upload/3270/3270fig4.jpg" /> 图4。核糖核酸注射PRL - 1突变体显示完整击倒。“滚动式”运动（一）PRL - 1的滚筒增益功能突变 tu92 。 （二）“撞倒”PRL - 1突变体表现出正常的野生型的姿势和运动。 （三）注入PRL - 1（底部）也有更长的身体比PRL - 1突变体（顶部）。 （四）注射PRL - 1（底部）身体也比野生型PS312（顶部）。更长的身体表型中也观察到的ROL - 5（SQT - 1）的RNAi敲除的 C. 线虫氮气（数据未显示）。 <table border="1"><tbody><tr><td></td><td> 滚 </td><td> 非卷 </td></tr><tr><td>一个PRL - 1双链RNA（200纳克/微升） </td><td> 13 </td><td> 21 </td></tr><tr><td>乙PRL - 1双链RNA（1000纳克/μL） </td><td> 9 </td><td> 16 </td></tr><tr><td>控制</td><td> 20 </td><td> 2 </td></tr></tbody></table> 表4。 </s仲&gt;摘要PPA - PRL - 1双链RNA注射。 （一）[双链RNA] = 200毫微克/μL;（二）[双链RNA] = 1000纳克/微升。 P = Fisher精确检验，双尾，为200和1000 ng /μL的注射，分别为0.0019和0.041。

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没有利益冲突的声明。

P. pacificus人群中找到全球各种金龟子甲虫物种的密切联系，是一个模型之间的自由生活线虫和寄生线虫中间。 体育的力量作为一个新兴的模式生物pacificus在其遗传和物理图谱，促进不偏不倚正向遗传筛选分离突变体的位置映射整合打下（即不只是以前线虫为特征的候选基因）6,10。然而，C线虫基因技术是不会轻易转让给体育pacificus由于显著?...

<table border="1"><tbody><tr><th>产品名称： </th><th>产品编号： </th><th>供应商： </th></tr><tr><td> DMI3000注射显微镜</td><td> DMI3000 </td><td>徕卡</td></tr><tr><td>微量操纵器（直接驱动） </td><td> Narashige BC - 3球联合</td><td> Tritech研究</td></tr><tr><td>针拖轮</td><td> Narashige的PC - 10 </td><td> Tritech研究</td></tr><tr><td>微量™全数字多压力系统</td><td> Narashige MINJ - D </td><td> Tritech研究</td></tr><tr><td> GeneElute™哺乳动物的基因组DNA小量试剂盒</td><td> G1N70 </td><td>西格玛</td></tr><tr><td> pJet克隆喷射套件</td><td> K1231 </td><td> Fermentas公司</td></tr><tr><td> GeneJET质粒小量制备试剂盒</td><td> K0502 </td><td> Fermentas公司</td></tr><tr><td> DNA清洁浓缩™ </td><td> D4005 </td><td> ZYMO研究</td></tr><tr><td> BLOCK - IT™的RNAi TOPO ®转录试剂盒</td><td> K3500 - 01＆K3650 - 01 </td><td> Invitrogen公司</td></tr><tr><td> DIFCO™琼脂高贵</td><td> DF0142 - 15 - 2 </td><td>费舍尔Scientifi </td></tr><tr><td>显微镜盖玻片（1.5 - 0.160.19毫米厚;尺寸：50 x 45毫米） </td><td> 12 - 554 - F </td><td> Fisher Scientific则</td></tr><tr><td>玻璃毛细管（长丝） </td><td> 615000 </td><td>上午系统</td></tr><tr><td>石蜡油（重） </td><td> O122 - 1 </td><td> Fisher Scientific则</td></tr><tr><td> KH 2 PO 4 </td><td> P386 - 500 </td><td> Fisher Scientific则</td></tr><tr><td> NA 2 HPO 4 </td><td> AC20651 - 5000 </td><td> Fisher Scientific则</td></tr><tr><td>氯化钠</td><td> BP3581 </td><td> Fisher Scientific则</td></tr><tr><td> 硫酸镁 </td><td> M80 - 500 </td><td> Fisher Scientific则</td></tr></tbody></table>

rnai mediated gene knockdown and transgenesis by microinjection in the necromenic nematode pristionchus pacificus

虽然它是，越来越多的新兴模式生物实惠，进一步深入的基因功能和表达分析RNA干扰和稳定的转基因获得完全基因组测序仍然在许多物种由于特定的解剖和分子细胞生物学的有机体有限。例如，外冠集团线虫线虫 3，稳定传输的非线虫品种的转基因株系，其中包括没有在这似是而非的门（线虫）报道，除粪类 圆 线虫4 Pristionchus pacificus 5。为了方便的P.作用不断扩大pacificus的发展，演变和行为6-7的研究，我们在这里描述当前用于显微注射转基因动物和基因敲除通过RNAi的方法。性腺一样<em&gt; C。线虫和其他大多数线虫，P 的性腺pacificus合胞和能够纳入直接显微注射交付时的卵母细胞的DNA和RNA。不像C。线虫然而，稳定的转基因继承和躯体表达在体育pacificus需要的基因组与目标的转基因和共注射标记 5的两端互补的核酸内切酶酶切的DNA自我此外。此外载体的基因组DNA是类似粪类圆线虫 4和C 的生殖细胞转基因表达的要求线虫 。但是，目前尚不清楚如果自己的基因组DNA为动物的具体要求是因为体育pacificus SOMA是非常有效的，在沉默不复杂的多拷贝基因或染色体外体育阵列pacificus需要在有丝分裂过程中适当的动粒组装的基因组序列。两个武装（腹侧迁移didelphic）雌雄同体性腺进一步复杂化的能力， 在 8个人蠕虫都注入生殖腺。我们也证明了利用显微注射，以击倒一个显性突变（滚子，tu92）由双链RNA（dsRNA）的注入性腺获得非滚动F 1子代。不像C。线虫 ，但最喜欢的其他线虫， 体育pacificus PS312是不能接受系统性的RNAi通过喂养和浸泡，因此双链RNA必须注射到合胞性腺管理。在当前的研究中，我们希望描述的显微注射过程中， 需要变换PPA - EGL - 4启动子：：GFP融合的记者和击倒主导辊PRL - 1（tu92）突变的视觉信息的协议。

Watch this Scientific Journal Video about RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus at JoVE.com

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

在模式生物，转基因操纵基因的功能，而RNAi能够敲除特定的mRNA转录1-2。该协议的目的是为了说明需要稳定传输的DNA和瞬态双链RNA引入necromenic线虫技术<em&gt; Pristionchus pacificus</em在进化，发育和行为生物学的研究。

通过显微注射介导的RNAi Necromenic线虫基因敲除和转基因 Pristionchus pacificus</em

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression ...

rnai-mediated-gene-knockdown-transgenesis-microinjection-necromenic

Research

JoVE Journal

Biology

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

16.4K 次观看.  California State University. 在模式生物，转基因操纵基因的功能，而RNAi能够敲除特定的mRNA转录1-2。该协议的目的是为了说明需要稳定传输的DNA和瞬态双链RNA引入necromenic线虫技术 Pristionchus pacificus

视频: RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

Localized RNAi and Ectopic Gene Expression in the Medicinal Leech

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to perturb gene expression patterns in selected segments of leech embryos, leaving the untreated segments as internal controls.
The pneumatic capillary gun can be used to reach internal layers of cells at early stages of development without opening the specimen. As a method for localized introduction of substances into living tissues, the biolistic delivery with the gun has several advantages: it is fast, contact-free and non-destructive. In addition, a single capillary gun can be used for independent delivery of different substances. The delivery region can have lateral dimensions of ~50-150 &#181;m and extends over ~15 &#181;m around the mean penetration depth, which is adjustable between 0 and 50 &#181;m. This delivery has the advantage of being able to target a limited number of cells in a selected location intermediate between single cell knock down by microinjection and systemic knockdown through extracellular injections or by means of genetic approaches.
For knocking down or knocking in the expression of the axon guidance molecule Netrin, which is naturally expressed by some central neurons and in the ventral body wall, but not the dorsal domain, we deliver molecules of dsRNA or plasmid-DNA into the body wall and central ganglia. This procedure includes the following steps: (i) preparation of the experimental setup for a specific assay (adjusting the accelerating pressure), (ii) coating the particles with molecules of dsRNA or DNA, (iii) loading the coated particles into the gun, up to two reagents in one assay, (iv) preparing the animals for the particle delivery, (v) delivery of coated particles into the target tissue (body wall or ganglia), and (vi) processing the embryos (immunostaining, immunohistochemistry and neuronal labeling) to visualize the results, usually 2 to 3 days after the delivery.
When the particles were coated with netrin dsRNA, they caused clearly visible knock-down of netrin expression that only occurred in cells containing particles (usually, 1-2 particles per cell). Particles coated with a plasmid encoding EGFP induced fluorescence in neuronal cells when they stopped in their nuclei.

In this video, we show a procedure for an accurate biolistic delivery of reagents into live tissue with a novel miniature gene gun. We are knocking down the expression of the axon guidance molecule Netrin in leech embryos by delivering molecules of dsRNA into the ventral body wall and ganglia of single segments.

本地化的RNAi和药用水蛭异位基因表达

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to ...

科研

生物学

Microinjection of Zebrafish Embryos to Analyze Gene Function

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

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

斑马鱼的胚胎显微注射来分析基因的功能

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

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

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

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

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

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

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory tool, the RNAi pathway appears to be promising for therapeutic utilization. For development of RNA interference (RNAi)-based therapies, delivery of RNAi-mediating agents to target cells is one of the major obstacles. A novel strategy to overcome this hurdle is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria, e.g. Escherichia coli, to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. A first-generation tkRNAi-mediating vector, TRIP, contains the bacteriophage T7 promoter for expression regulation of a therapeutic shRNA of interest. Furthermore, TRIP has the Inv locus from Yersinia pseudotuberculosis that encodes invasin, which permits natural noninvasive bacteria to enter &beta;1-integrin-positive mammalian cells and the HlyA gene from Listeria monocytogenes, which produces listeriolysin O. This enzyme allows the therapeutic shRNA to escape from entry vesicles within the cytoplasm of the target cell. TRIP constructs are introduced into a competent non-pathogenic Escherichia coli strain, which encodes T7 RNA polymerase necessary for the T7 promoter-driven synthesis of shRNAs. A well-characterized cancer-associated target molecule for different RNAi strategies is ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). This ABC-transporter acts as a drug extrusion pump and mediates the "classical" ABCB1-mediated multidrug resistance (MDR) phenotype of human cancer cells which is characterized by a specific cross resistance pattern. Different ABCB1-expressing MDR cancer cells were treated with anti-ABCB1 shRNA expression vector bearing E. coli. This procedure resulted in activation of the RNAi pathways within the cancer cells and a considerable down regulation of the ABCB1 encoding mRNA as well as the corresponding drug extrusion pump. Accordingly, drug accumulation was enhanced in the pristine drug-resistant cancer cells and the MDR phenotype was reversed. By means of this model the data provide the proof-of-concept that tkRNAi is suitable for modulation of cancer-associated factors, e.g. ABCB1, in human cancer cells.

For development of RNA interference (RNAi)-based therapies, a novel strategy was developed, transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells. Here, tkRNAi was successfully applied for reversal of classical ABCB1-mediated multidrug resistance (MDR) of cancer cells.

细菌提供的RNAi效应：Transkingdom RNA干扰

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

激活细胞凋亡的细胞色素的细胞质显微注射 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

慢病毒介导的击倒在体外</em人造血干细胞>红细胞

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

农杆菌介导的病毒诱导的基因沉默含量在棉花

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. 
This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis1. 
The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

In this protocol we combine RNAi-mediated gene silencing with an in vivo diuresis assay to study the effects knockdown of genes of interest has on mosquito fluid excretion.

RNAi介导的基因敲除和<em&gt;在体内</em在成年女性利尿含量<em&gt;埃及伊蚊</em&gt;蚊子

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

全基因组基因缺失<em&gt;链球菌血统</em通过高通量PCR

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

使用屏幕在车身尺寸规例中的线虫基因介导的RNA干扰取食策略<em&gt; C.线虫</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...