Name: rnai mediated gene knockdown and transgenesis by microinjection in the necromenic nematode pristionchus pacificus
Uploaded: 2011-10-16T14:00:00.000+00:00
Duration: 6 min 57 s
Description: Watch this Scientific Journal Video about RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus at JoVE.com

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。

<ol>
	<li>Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., Mello, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+and+specific+genetic+interference+by+double-stranded+RNA+in+Caenorhabditis+elegans.">Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.</a> Nature. 391, 806-811 (1998).</li><li>Ahringer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+Genetics.">Reverse Genetics.</a> Wormbook. , (2006).</li><li>Mello, C. C., Kramer, J. M., Stinchcomb, D., Ambros, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+in+C.+elegans:+extrachromosomal+maintenance+and+integration+of+transforming+sequences.">Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences.</a> EMBO J. 10, 3959-3970 (1991).</li><li>Junio, A. J., Li, X., Massery, H. C., Nolan, T. J., Lamitina, S. T., Sundaram, M. V., Lok, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strongyloides+stercoralis:+cell-+and+tissue-specific+transgene+expression+and+co-transformation+with+vector+constructs+incorporating+a+common+multifunctional+3'+UTR.">Strongyloides stercoralis: cell- and tissue-specific transgene expression and co-transformation with vector constructs incorporating a common multifunctional 3' UTR.</a> Exp. Parasitology. 118, 253-265 (2008).</li><li>Schlager, B., Wang, X., Braach, G., Sommer, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+of+a+dominant+roller+mutant+and+establishment+of+DNA-mediated+transformation+in+the+nematode+Pristionchus+pacificus.">Molecular cloning of a dominant roller mutant and establishment of DNA-mediated transformation in the nematode Pristionchus pacificus.</a> Genesis. 47, 300-304 (2009).</li><li>Hong, R. L., Sommer, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pristionchus+pacificus:+a+well-rounded+nematode.">Pristionchus pacificus: a well-rounded nematode.</a> Bioessays. 28, 651-659 (2006).</li><li>Hong, R. L., Sommer, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoattraction+in+Pristionchus+nematodes+and+implications+for+insect+recognition.">Chemoattraction in Pristionchus nematodes and implications for insect recognition.</a> Curr. Biol. 16, 2359-2365 (2006).</li><li>Rudel, D., Riebesell, M., Sommer, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gonadogenesis+in+Pristionchus+pacificus+and+organ+evolution:+development,+adult+morphology+and+cell-cell+interactions+in+the+hermaphrodite+gonad.">Gonadogenesis in Pristionchus pacificus and organ evolution: development, adult morphology and cell-cell interactions in the hermaphrodite gonad.</a> Dev. Biol. 277, 200-221 (2005).</li><li>Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., Prasher, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+fluorescent+protein+as+a+marker+for+gene+expression.">Green fluorescent protein as a marker for gene expression.</a> Science. 11, 802-805 (1994).</li><li>Chaudhuri, J., Parihar, M., Pires-daSilva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Introduction+to+Worm+Lab:+from+Culturing+Worms+to+Mutagenesis.">An Introduction to Worm Lab: from Culturing Worms to Mutagenesis.</a> J. Vis. Exp. (47), e2293-e2293 (2011).</li><li>Evans, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+and+Microinjection.">Transformation and Microinjection.</a> Wormbook. , (2006).</li></ol>

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由于显著的差异，在器官形态的主要DNA序列，以及外源DNA。我们目前的研究中详细说明了如何引入稳定记者， 以前 施拉格等5所描述的基因，以及如何通过RNAi击倒相同的主导辊突变。我们的协议并不假定事先KNowledge的转基因或RNAi 在 C 线虫 。 在这项研究中（1〜2％）所示的F 1的转化率是可比以前的结果，使用P. PRL - 1标记pacificus（2-10％）5，但低于S 中发现的汇率粪类圆线虫 （3-22％）4。还有许多潜在的转基因技术在体育的改进pacificus。即使使用PRL - 1（tu92）主导辊标记同源的常用ROL - 6（su1006）在 C等位基因线虫的转基因P 中的有效性pacificus是明显比第一的 C少由梅洛和同事3，其中多个转基因F 1子代可以在专家手中获得每注入动物的线虫。有几个因素可以解释这种差异：（1）卵母细胞的数量较少，在终变期的P.女性生殖pacificus（AVE愤怒1％性腺）8，可能反映了较低的有丝分裂生殖细胞在体育过渡到减数分裂pacificus相比为 C。 线虫11。因此，可以利用较少的卵母细胞的DNA和RNA每次注射。每雌雄同体的整体降低育雏的大小，在P. pacificus PS312（〜200）相比，C。线虫 N2（〜300）可能也加剧了每注入动物的转基因F 1低。 （2）在注射外源DNA从F 1 F 2混合基因组DNA的要求建议一个更强大的基因沉默机制也可能参与在P. pacificus比在 C 线虫 。然而， 体育pacificus基因的表达似乎并不接受极端 S中发现的基因沉默粪类圆线虫转基因表达是有限的F 1动物4。目前，我们正在特征PPA - EGL - 4P的表达 ：GFP线：F 6只动物。一个简单的方法来提高转基因率是增加辊合作注入的标记（1纳克/μL的pRL3相比25-50纳克/μLpRF4线虫）的浓度。 能力操纵基因的功能，通过RNAi转基因有机体技术的两大支柱，提升 C。上述许多其他模式生物线虫 。我们希望我们目前的研究将大大提高体育pacificus遗传学研究的发展和提供方便说明，转基因和RNAi的行为模式。

<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

Universidad San Sebastian

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

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. This is due to its reproductive behavior: females damage the external surface of fruits and vegetables when they lay eggs and the hatched larvae feed on their pulp. Wild C. capitata populations are traditionally controlled through insecticide spraying and/or eco-friendly approaches, the most successful being the Sterile Insect Technique (SIT). The SIT relies on mass-rearing, radiation-based sterilization and field release of males that retain their capacity to mate but are not able to generate fertile progeny. The advent and the subsequent rapid development of biotechnological tools, together with the availability of the medfly genome sequence, has greatly boosted our understanding of the biology of this species. This favored the proliferation of new strategies for genome manipulation, which can be applied to population control.
In this context, embryo microinjection plays a dual role in expanding the toolbox for medfly control. The ability to interfere with the function of genes that regulate key biological processes, indeed, expands our understanding of the molecular machinery underlying medfly invasiveness. Furthermore, the ability to achieve germ-line transformation facilitates the production of multiple transgenic strains that can be tested for future field applications in novel SIT settings. Indeed, genetic manipulation can be used to confer desirable traits that can, for example, be used to monitor sterile male performance in the field, or that can result in early life-stage lethality. Here we describe a method to microinject nucleic acids into medfly embryos to achieve these two main goals.

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Diptera: Tephritidae) is a worldwide pest of agriculture. A deeper understanding of its biology is key to control medfly populations and thus reduce economic impact. Embryo microinjection is a fundamental tool allowing both germ-line transformation and reverse genetics studies in this species.

核酸的传递通过显微注射胚胎在世界范围内的农业害虫，<em&gt;地中海实蝇</em

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. ...

科研

遗传学

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

饲养并在隐藏甲壳虫双链RNA介导的基因敲除，<em&gt;白腹皮蠹</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

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 ...

RNA Interference in Aquatic Beetles as a Powerful Tool for Manipulating Gene Expression at Specific Developmental Time Points

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is applicable to a wide variety of organisms and is highly efficient in the species-rich order Coleoptera (beetles). Here, we summarize the necessary steps for developing this technique in a novel organism and illustrate its application to the different developmental stages of the aquatic diving beetle Thermonectus marmoratus. Target gene sequences can be obtained cost-effectively through the assembly of transcriptomes against a close relative with known genomics or de novo. Candidate gene cloning utilizes a specific cloning vector (the pCR4-TOPO plasmid), which allows the synthesis of double-stranded RNA (dsRNA) for any gene with the use of a single common primer. The synthesized dsRNA can be injected into either embryos for early developmental processes or larvae for later developmental processes. We then illustrate how RNAi can be injected into aquatic larvae using immobilization in agarose. To demonstrate the technique, we provide several examples of RNAi experiments, generating specific knockdowns with predicted phenotypes. Specifically, RNAi for the tanning gene laccase2 leads to cuticle lightening in both larvae and adults, and RNAi for the eye pigmentation gene white produces a lightening/lack of pigmentation in eye tubes. In addition, the knockdown of a key lens protein leads to larvae with optical deficiencies and a reduced ability to hunt prey. Combined, these results exemplify the power of RNAi as a tool for investigating both morphological patterning and behavioral traits in organisms with only transcriptomic databases.

RNA interference is a widely applicable, powerful technique for manipulating gene expression at specific developmental stages. Here, we describe the necessary steps for implementing this technique in the aquatic diving beetle Thermonectus marmoratus, from the acquisition of gene sequences to the knockdown of genes that affect structure or behavior.

水生甲壳虫中的RNA干扰是特定发育时间点操纵基因表达的有力工具

RNA interference (RNAi) remains a powerful technique that allows for the targeted reduction of gene expression through mRNA degradation. This technique is ...

环境科学

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although RNA interference (RNAi) has proved to be a powerful tool for studying WCR biology, it has its limitations. Specifically, RNAi itself is transient (i.e. does not result in long-term Mendelian inheritance of the associated phenotype), and it requires knowing the DNA sequence of the target gene. The latter can be limiting if the phenotype of interest is controlled by poorly conserved, or even novel genes, because identifying useful targets would be challenging, if not impossible. Therefore, the number of tools in WCR&#39;s genomic toolbox should be expanded by the development of methods that could be used to create stable mutant strains and enable sequence-independent surveys of the WCR genome. Herein, we detail the methods used to collect and microinject precellular WCR embryos with nucleic acids. While the protocols described herein are aimed at the creation of transgenic WCR, CRISPR/Cas9-genome editing could also be performed using the same protocols, with the only difference being the composition of the solution injected into the embryos.

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

Here we present protocols for collecting and microinjecting precellular western corn rootworm embryos for the purpose of performing functional-genomic assays such as germline transformation and CRISPR/Cas9-genome editing.

对西方玉米 Rootworm、 Diabrotica virgifera virgifera、生殖转化胚胎或 CRISPR/Cas9 基因组编辑的显微注射

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although ...

生物工程

Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing

The corn planthopper, Peregrinus maidis, is&#160;a pest of maize and a vector of several maize viruses. Previously published methods describe the triggering of RNA interference (RNAi) in P. maidis through microinjection of double-stranded RNAs (dsRNAs) into nymphs and adults. Despite the power of RNAi, phenotypes generated via this technique are transient and lack long-term Mendelian inheritance. Therefore, the P. maidis toolbox needs to be expanded to include functional genomic tools that would enable the production of stable mutant strains, opening the door for researchers to bring new control methods to bear on this economically important pest. However, unlike the dsRNAs used for RNAi, the components used in CRISPR/Cas9-based genome editing and germline transformation do not easily cross cell membranes. As a result, plasmid DNAs, RNAs, and/or proteins must be microinjected into embryos before the embryo cellularizes, making the timing of injection a critical factor for success. To that end, an agarose-based egg-lay method was developed to allow embryos to be harvested from P. maidis females at relatively short intervals. Herein are provided detailed protocols for collecting and microinjecting precellular P. maidis embryos with CRISPR components (Cas9 nuclease that has been complexed with guide RNAs), and results of Cas9-based gene knockout of a P. maidis eye-color gene, white, are presented. Although these protocols describe CRISPR/Cas9-genome editing in P. maidis, they can also be used for producing transgenic P. maidis via germline transformation by simply changing the composition of the injection solution.

Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing

Herein are protocols for collecting and microinjecting precellular corn planthopper embryos for the purpose of modifying their genome via CRISPR/Cas9-based genome editing or for the addition of marked transposable elements through germline transformation.

显微镜注射玉米飞虱、 游隼、用于CRISPR/Cas9基因组编辑的胚胎

The corn planthopper, Peregrinus maidis, is&#160;a pest of maize and a vector of several maize viruses. Previously published methods describe the ...

Culturing and Genetically Manipulating Entomopathogenic Nematodes

Entomopathogenic nematodes in the genera Heterorhabditis and Steinernema are obligate parasites of insects that live in the soil. The main characteristic of their life cycle is the mutualistic association with the bacteria Photorhabdus and Xenorhabdus, respectively. The nematode parasites are able to locate and enter suitable insect hosts, subvert the insect immune response, and multiply efficiently to produce the next generation that will actively hunt new insect prey to infect. Due to the properties of their life cycle, entomopathogenic nematodes are popular biological control agents, which are used in combination with insecticides to control destructive agricultural insect pests. Simultaneously, these parasitic nematodes represent a research tool to analyze nematode pathogenicity and host anti-nematode responses. This research is aided by the recent development of genetic techniques and transcriptomic approaches for understanding the role of nematode secreted molecules during infection. Here, a detailed protocol on maintaining entomopathogenic nematodes and using a gene knockdown procedure is provided. These methodologies further promote the functional characterization of entomopathogenic nematode infection factors.

Entomopathogenic nematodes live in symbiosis with bacteria and together they successfully infect insects by undermining their innate immune system. To promote research on the genetic basis of nematode infection, methods for maintaining and genetically manipulating entomopathogenic nematodes are described.

培养和遗传操纵昆虫致病线虫

Entomopathogenic nematodes in the genera Heterorhabditis and Steinernema are obligate parasites of insects that live in the soil. The main characteristic ...

免疫与感染

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, host-symbiont interactions, speciation, and venom synthesis. Despite the availability of several molecular tools, including CRISPR/Cas9, functional genetic studies are still limited in this organism. The major limitation of applying CRISPR/Cas9 technology in N. vitripennis stems from the challenges of embryonic microinjections. Injections of embryos are particularly difficult in this organism and in general in many parasitoid wasps, due to small embryo size and the requirement of a host pupa for embryonic development. To address these challenges, Cas9 ribonucleoprotein complex delivery into female&#160;ovaries by adult injection, rather than embryonic microinjection, was optimized, resulting in both somatic and heritable germline edits. The injection procedures were optimized in pupae and female wasps using either ReMOT Control (Receptor-Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules). These methods are shown to be effective alternatives to embryo injection, enabling site-specific and heritable germline mutations.

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

Here, we describe methods for efficient pupal and adult injections in Nasonia vitripennis as accessible alternatives to embryo microinjection, enabling functional analysis of genes of interest using either RNA-silencing via RNA interference (RNAi) or gene knockout via CRISPR/Cas9 genome editing.

纳索尼亚维特里彭尼斯的 Rnai 和克里斯普基因编辑的普帕尔和成人注射

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, ...

生物学

Application of RNA Interference in the Pinewood Nematode, Bursaphelenchus xylophilus

The pinewood nematode, Bursaphelenchus xylophilus, is one of the most destructive invasive species worldwide, causing the wilting and eventual death of pine trees. Despite the recognition of their economic and environmental significance, it has thus far been impossible to study the detailed gene functions of plant-parasitic nematodes (PPNs) using conventional forward genetics and transgenic methods. However, as a reverse genetics technology, RNA interference (RNAi) facilitates the study of the functional genes of nematodes, including B. xylophilus. 
This paper outlines a new protocol for RNAi of the ppm-1 gene in B. xylophilus, which has been reported to play crucial roles in the development and reproduction of other pathogenic nematodes. For RNAi, the T7 promoter was linked to the 5&#8242;-terminal of the target fragment by polymerase chain reaction (PCR), and double-stranded RNA (dsRNA) was synthesized by in vitro transcription. Subsequently, dsRNA delivery was accomplished by soaking the nematodes in a dsRNA solution mixed with synthetic neurostimulants. Synchronized juveniles of B. xylophilus (approximately 20,000 individuals) were washed and soaked in dsRNA (0.8 &#181;g/mL) in the soaking buffer for 24 h in the dark at 25 &#176;C.
The same quantity of nematodes was placed in a soaking buffer without dsRNA as a control. Meanwhile, another identical quantity of nematodes was placed in a soaking buffer with green fluorescent protein (gfp) gene dsRNA as a control. After soaking, the expression level of the target transcripts was determined using real-time quantitative PCR. The effects of RNAi were then confirmed using microscopic observation of the phenotypes and a comparison of the body size of the adults among the groups. The current protocol can help advance research to better understand the functions of the genes of B. xylophilus and other parasitic nematodes toward developing control strategies through genetic engineering.

Application of RNA Interference in the Pinewood Nematode, Bursaphelenchus xylophilus

Here, we introduce a detailed soaking method of RNA interference in Bursaphelenchus xylophilus to facilitate the study of gene functions.

RNA干扰在松林线虫、木犀牛线虫中的应用

The pinewood nematode, Bursaphelenchus xylophilus, is one of the most destructive invasive species worldwide, causing the wilting and eventual death of ...

RNAi in C. elegans

RNA interference (RNAi) is a widely used technique in which double stranded RNA is exogenously introduced into an organism, causing knockdown of a target gene. In the nematode, C. elegans, RNAi is particularly easy and effective because it can be delivered simply by feeding the worms bacteria that express double stranded RNA (dsRNA) that is complementary to a gene of interest. First, this video will introduce the concept of RNA interference and explain how it causes targeted gene knockdown. Then, we will demonstrate a protocol for using RNAi in C. elegans, which includes preparation of the bacteria and RNAi worm plates, culturing of the worms, and how to assess the effects of RNAi on the worms. RNAi is frequently used to perform reverse genetic screens in order to reveal which genes are important to carry out specific biological processes. Furthermore, automated reverse genetic screens allow for the efficient knockdown and analysis of a large collection of genes. Lastly, RNAi is often used to study the development of C. elegans. Since its discovery, scientists have used RNAi to make tremendous progress on the understanding of many biological phenomena.

RNA Interference RNAi in Caenorhabditis elegans

线虫RNA干扰的简介

RNA interference (RNAi) is a widely used technique in which double stranded RNA is exogenously introduced into an organism, causing knockdown of a target ...

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

摘要

探索更多视频

此视频中的章节

核酸的传递通过显微注射胚胎在世界范围内的农业害虫，

饲养并在隐藏甲壳虫双链RNA介导的基因敲除，

金小蜂金小蜂基因组操作的胚胎显微注射和移植技术

水生甲壳虫中的RNA干扰是特定发育时间点操纵基因表达的有力工具

对西方玉米 Rootworm、 Diabrotica virgifera virgifera、生殖转化胚胎或 CRISPR/Cas9 基因组编辑的显微注射

显微镜注射玉米飞虱、游隼、用于CRISPR/Cas9基因组编辑的胚胎

培养和遗传操纵昆虫致病线虫

纳索尼亚维特里彭尼斯的 Rnai 和克里斯普基因编辑的普帕尔和成人注射

RNA干扰在松林线虫、木犀牛线虫中的应用

线虫RNA干扰的简介

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

摘要

探索更多视频

此视频中的章节

核酸的传递通过显微注射胚胎在世界范围内的农业害虫，

饲养并在隐藏甲壳虫双链RNA介导的基因敲除，

金小蜂金小蜂基因组操作的胚胎显微注射和移植技术

水生甲壳虫中的RNA干扰是特定发育时间点操纵基因表达的有力工具

对西方玉米 Rootworm、 Diabrotica virgifera virgifera、生殖转化胚胎或 CRISPR/Cas9 基因组编辑的显微注射

显微镜注射玉米飞虱、 游隼、用于CRISPR/Cas9基因组编辑的胚胎

培养和遗传操纵昆虫致病线虫

纳索尼亚维特里彭尼斯的 Rnai 和克里斯普基因编辑的普帕尔和成人注射

RNA干扰在松林线虫、木犀牛线虫中的应用

线虫RNA干扰的简介

显微镜注射玉米飞虱、游隼、用于CRISPR/Cas9基因组编辑的胚胎