Name: culturing caenorhabditis elegans in axenic liquid media and creation of transgenic worms by microparticle bombardment
Uploaded: 2014-08-02T15:00:00.000+00:00
Duration: 8 min 26 s
Description: Watch this Scientific Journal Video about Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment at JoVE.com

这项工作是由HealthGrants DK85035和DK074797（H）全国学院的支持。

1，蠕虫菌株<ol><li> C.获得线虫野生型布里斯托尔 N2菌株的秀丽隐杆线虫遗传学中心（CGC）（http://www.cbs.umn.edu/cgc），并保持他们在NGM板接种大肠杆菌大肠杆菌菌株OP50 7。注意:转基因虫株IQ6011（Phrg-1 :: GFP），如前所述11利用所产生。 IQ6011可从相应的作者提出要求。 </li></ol> 2:改性C的线虫居住和繁殖培养基（mCeHR） 准备mCeHR液体介质所描述的12岁以下。这种无菌液体介质允许蠕虫无需任何额外的食物来源增长。进行使用严格的无菌条件下，例如在层流罩无菌液体培养基和无污染的蠕虫的所有操作。 <ol><li>获得超巴氏杀菌脱脂牛奶从一家杂货店。使用冷藏亲管而不是室温产物。此前在mCeHR媒体，连胜用琼脂平板上脑心浸液（BHI）牛奶和孵化3天在30°C和37°C到检查不育。奶转移至50毫升无菌试管中并储存于-80℃下</li><li>准备以下每个组件，并结合顺序并给予准备1升mCeHR量（ 表1A）:10毫升2毫米胆碱二酸柠檬酸，将10毫升的维生素和生长因子组合（见配方表1B），将10ml 2.4毫肌醇 ，将10毫升2毫氯高铁血红素，氯化加入250毫升去离子水。通过0.22μm过滤器单元施加吸力和过滤器。 </li><li>加入20毫升的核酸混合物（配方见表1C），加入100毫升矿物组合（配方在表1D），20毫升170毫克/毫升的乳清蛋白水解物，加入20ml的必需氨基酸，将10毫升的非必需氨基酸的酸，将20ml的450 mM的KH 2 PO 4，加入50ml 1.45 M D-葡萄糖，加入10ml 1M的HEPES，钠盐，加入250毫升去离子水。 </li><li>适用于吸入到过滤消毒的组件。取下过滤器，并加入1毫升5毫克/毫升的胆固醇。确保介质的pH值约为6-6.5。注:此溶液可以贮存于-80℃下长达一年。 </li><li>添加20％（V / V），超巴氏杀菌的有机脱脂牛奶的mCeHR介质后方可使用。开等分牛奶时，使用严格的无菌技术（例如在层流罩）。存储介质在4°C。 </li></ol> 3，准备C.线虫文化在无菌mCeHR液体培养基<ol><li>生长在1060毫米NGM板蠕虫直到有许多妊娠蠕虫和OP50 大肠杆菌的最小量大肠杆菌在板上。 </li><li>通过用5毫升的M9缓冲液（配方见表1F）漂洗各板取出的蠕虫，并结合在50ml锥形管中。 </li><li>允许蠕虫为settle通过离开管静置5〜10分钟，然后小心地取出含有大肠杆菌上清液大肠杆菌 。 </li><li>重复步骤3.2和3.3倍持续的过程之前，除去尽可能多的剩余细菌的可能。 </li><li>悬浮在0.1N的NaCl的蠕虫在一个体积是3的倍数，例如1.5毫升3毫升或6毫升其中个别蠕虫可在管看出。 </li><li>通过加入5 1N NaOH和5％的次氯酸钠在1点02分06秒比对虫悬浮液（漂白剂）溶液漂白蠕虫。例如，加入1ml 5的1N NaOH:2毫升5％次氯酸钠:6毫升蜗杆悬浮液。添加到蠕虫停牌前混合NaOH和漂白剂。 </li><li>涡旋该溶液大力直到妊娠蠕虫溶解并只蛋的悬浮液（约5至10分钟）内可见。监视用相衬显微镜用10X物镜的过程。 </li><li>之后，蠕虫已经完全溶解后，立即将沉淀鸡蛋在800×g离心45秒，在4°C。除去上清液，并用10ml无菌水通过离心沉淀，在800×g离心45秒，在4℃下冲洗沉淀两次</li><li>漂白后，卵丸粒转移到含有10ml补充有100微克/毫升四环素mCeHR媒体的25cm 2组织培养瓶。采用无菌技术进行此过程。如果需要，添加更多的抗生素，包括250微克/毫升萘啶酸，以防止细菌生长在无菌液体培养。 </li><li>孵育所述液体培养物在20℃下在一个摇动的培养箱中在约70转每分钟。 </li><li>检查蠕虫日常要注意发展阶段和增长速度。 注:蠕虫最初将生长缓慢，需要7〜10天，成为妊娠。然而，由于蠕虫使其恢复到液体培养基中的世代时间将减少到大约4天的野生型（N2）的蠕虫。 </li><li>之后，蠕虫有德韦大步，以在液体介质中的妊娠阶段，蠕虫培养转移到锥形管中，并使用吊桶式转子在4℃下沉淀的蠕虫在800×g离心5分钟。 </li><li>去除上清，并轻轻重悬沉淀蠕虫中的0.1M NaCl的相同体积。例如，使用10 ml 0.1 M氯化钠蠕虫在10毫升mCeHR的培养。允许蠕虫定居5分钟冰上。 </li><li>进行与步骤3.5-3.8上述的漂白过程，并允许蠕虫在无菌液体培养基中生长的第二代。如果有必要，再加入抗生素以抑制细菌的生长。 </li><li>再次使蠕虫成为妊娠然后如步骤3.12和3.13中所述沉淀和重新悬浮的蠕虫在0.1M NaCl的，​​如在步骤3.5-3.8中所述，然后再漂白，以产生一个同步的人口。生长在液体介质中的L1幼虫蠕虫不从现在开始抗生素。 </li></ol> 4，同步从液体蠕虫文化<ol><li>颗粒和悬浮在步骤3.12和3.13上述在0.1 M氯化钠的蠕虫。按照步骤3.5-3.8上述漂白剂。 </li><li>重悬卵沉淀在10毫升M9的缓冲区，并允许幼虫孵化O / N在20°C在一个旋转平台摇床。注:卵孵化成幼虫的L1，但不会进一步发展，在L1阶段同步蠕虫。 </li><li>保持和继代培养的蠕虫，沉淀的L1幼虫在800×g离心5分钟，然后重悬幼虫在10毫升mCeHR的，并转移到的25cm 2烧瓶中。允许蠕虫在20℃旋转平台上的成长。维持3000蠕虫/毫升/厘米2的最大密度，以确保足够的营养的蠕虫。 注:在此阶段，抗生素不能当程序进行了使用在层流罩无菌技术，以保持无菌条件要求。蠕虫应每天检查监测的增长。 </li></ol> 5，免费用于无菌培养基培养子弹声和解冻蠕虫<ol><li>冻结无菌异步蠕虫病毒培养或在液氮中同步L1幼虫。沉淀的蠕虫在800×g离心5分钟，然后以0.5毫升每小瓶悬浮S中缓冲液（6.45毫KHPO 4，43.55毫KHPO 4，146.25 mM氯化钠）。添加第缓冲器等体积的含30％体积/体积甘油到每个小瓶中，前长期储存在液态氮的蠕虫转移到-80℃。冻结每毫升在每个小瓶中约1×10 6个蠕虫。 </li><li>通过培养瓶中，在37℃下进行约2分钟，直到几乎所有的冰的融化解冻蠕虫。在层流罩，蠕虫转移到含有mCeHR无菌瓶中，把它们摆放在20°C </li></ol> 6，确定高铁血红素浓度对生长及繁殖的mCeHR的影响<ol><li>使用无菌mCeHR媒体来测定C的营养生长依赖线虫 。为了确定影响Ø对蠕虫生长和发育f​​血红素浓度，使用同步无菌氮气蠕虫或感兴趣的其他菌株。如第4段所述的L1幼虫同步。 </li><li>在接下来的一天，小球和计数同步L1幼虫并稀释至1蠕虫每微升无菌介质中。使10mM的氯高铁血红素氯在0.3M的NH 4 OH，用浓盐酸调节pH至8。 </li><li>加入800微升mCeHR媒体的24孔板。加100蠕虫向每个孔中加入100μl的介质。在浓度范围为0μM，1.5μM至1,000μM，一式三份添加氯化高铁​​血红素到每个孔中。确保所有孔中含有0.3M的NH 4 OH的浓度相同。吹吸，以确保100 L1幼虫被加入到每个孔之前轻轻重悬的蠕虫。 </li><li>每天检查蠕虫，并注意在每口井的增长在每个浓度。计数的蠕虫数目后孵育9天，并绘制对应于每个血红素蠕虫/毫升数浓度（ 图1）。 </li></ol>氯化血红素浓度7。对血红素传感器蠕虫影响<ol><li>含孵 ​​育4同步L1血红素传感器蠕虫（Phrg-1 :: GFP）在mCeHR微米，8微米，10微米和20微米高铁血红素。 </li><li>图片用荧光共聚焦显微镜，48小时培养后（ 图2）的蠕虫。 </li></ol> 8，利用mCeHR产生转基因C。线虫使用微粒轰击注意:用于生成和执行微粒轰击使用UNC-119（ED3）中mCeHR蠕虫生长在图3中13中概述的步骤。 <ol><li>蠕虫准备<ol><li>将大约1×10 6异步UNC-119（ED3）蠕虫在90ml mCeHR媒体在175 平方厘米瓶前一周轰击。允许蠕虫生长在20°C O呐晃动平台〜70转（ 图3-1）。 注:一个星期后，该蠕虫密度应显著大于许多成年妊娠蠕虫。约3×10 7个蠕虫将需要用于微粒轰击。 </li><li>寒意JM109 大肠杆菌大肠杆菌接种于冰上10厘米NGM板。 </li><li>转让无菌蠕虫两个50毫升锥形管和离心蠕虫在800×g离心2分钟。吸去上清，重悬在每个管中的蠕虫在50毫升M9缓冲液（ 图3-2）。 </li><li>允许成虫通过重力沉淀为10至15分钟，在冰上。 注:2毫升颗粒现在应该包含&gt; 90％的成虫。这2毫升颗粒应该有大约5×10 6蠕虫和足以让一个微粒轰击。 </li><li>大衣冷冻JM109的整个表面接种NGM板与2毫升颗粒蠕虫。允许被完全吸收的液体，然后离开板对投诉进行（ 图3-3）前30分钟在冰上。 </li></ol></li><li>金颗粒的制备<ol><li>称量金颗粒30毫克成硅化离心管中。加入1 ml 70％的乙醇和旋涡振荡管5分钟。 </li><li>允许管坐15分钟，然后离心以6,000×g离心10秒以沉淀的金颗粒。使用移液管尖端通过仔细触摸笔尖管相反的一侧的金颗粒去除上清。 </li><li>用1毫升无菌水，涡旋混合1分钟洗涤金颗粒并短暂离心​​管中以6000×g离心10秒。重复该洗涤步骤两次，然后重悬金颗粒在0.5ml的无菌50％的甘油。 注意:在金颗粒的最终浓度为60毫克/毫升，它可以在4℃下存放1至2个月</li></ol></li><li>准备DNA的金颗粒混合<ol><li>结合10微克所要的质粒DNA与10及＃956;克硅化1.5 ml离心管的UNC-119救援质粒。加入50微升的去离子水和100μl的良好悬浮的金颗粒溶液，然后涡旋混合1分钟。 </li><li>加入150μl的2.5M的CaCl 2并再次旋涡振荡溶液1分钟。向此溶液中，加入60微升的0.1M亚精胺并涡旋混合3〜5分钟。 </li><li>脉冲离心溶液以6,000×g离心10秒，去除上清液，加入300μl的70％乙醇中。旋涡以及1分钟，脉搏离心机在6000×g离心，去除170微升100％乙醇的上清，重悬。大力旋涡振荡5〜10分钟，然后离心脉冲，去除上清溶液。 </li></ol></li><li>轰击（ 图3-3） 注:此协议进行了材料/设备表中指定的粒子输送系统。遵循颗粒输送设备中可用的指令。 <ol> &lt;LI&gt;将薄纸片成15厘米的板。使用镊子，浸7大载体分别为100％的乙醇，奠定他们在组织干。 </li><li>彻底清洁基因枪室，大载体支架和目标板用70％乙醇。清洁和高压灭菌每个轰击过程之前停止屏幕。 </li><li>加载macrocarrriers到使用镊子，然后按入底座使用该座位工具的持有人。 </li><li>均匀地散布20微升DNA包被的金颗粒在每个大载体的中心，然后使溶液完全干燥。 </li><li>清洁压力分压器与乙醇的两部分，并组装用乙醇清洗破裂盘。螺丝组装压力分频器和爆破片入轰击室。 </li><li>组装的蒸压停止屏与大载体支架和放置设备上所需的金属架子和锁定到位。 </li><li>将组装好的MACRocarrier设备进入下面的压力分配器所分配的时隙基因枪腔和配合的压力分频器。 </li><li>大盘开NGM板的蠕虫到目标板架，并关闭该室的门。 </li><li>轰击蠕虫，调整压力至该打破断裂盘需要的。打开真空至28英寸汞柱的压力，然后按下开火按钮，直到磁盘破裂。 </li><li>释放真空并从装置中取出NGM板。从板块洗净蠕虫和再分配中接种JM109 大肠杆菌 2010厘米NGM板大肠杆菌 （ 图3-4）。 </li><li>允许蠕虫生长10〜14天，在25°C和饿死的板块。蠕虫从UNC-119缺陷救出将具有正常运动，并且可以在此时被识别。因为这些代表独立的转基因株系（FIGUR挑选单独的板块至少10"野生型"蠕虫病毒的进一步分析Ë3-5）。 </li></ol></li></ol>

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Current protocols in toxicology editorial board, Mahin D. Maines. , (2007).</li><li>Schweinsberg, P. J., Grant, B. 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=elegans+gene+transformation+by+microparticle+bombardment+WormBook+the+online+review+of+C.">elegans gene transformation by microparticle bombardment WormBook the online review of C.</a> elegans biology. , 1-10 (2013).</li><li>Rao, A. U., Carta, L. K., Lesuisse, E., Hamza, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+heme+synthesis+in+a+free-living+eukaryote.">Lack of heme synthesis in a free-living eukaryote.</a> Proceedings of the National Academy of Sciences of the United States of America. 102, 4270-4275 (2005).</li><li>Szewczyk, N. J., Kozak, E., Conley, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemically+defined+medium+and+Caenorhabditis+elegans.">Chemically defined medium and Caenorhabditis elegans.</a> BMC biotechnology. 3, 19 (2003).</li><li>Szewczyk, N. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+development+and+lifespan+extension+as+features+of+metabolic+lifestyle+alteration+in+C+elegans+under+dietary+restriction.">Delayed development and lifespan extension as features of metabolic lifestyle alteration in C elegans under dietary restriction.</a> The Journal of experimental biology. 209, 4129-4139 (2006).</li><li>White, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HRG1+is+essential+for+heme+transport+from+the+phagolysosome+of+macrophages+during+erythrophagocytosis.">HRG1 is essential for heme transport from the phagolysosome of macrophages during erythrophagocytosis.</a> Cell metabolism. 17, 261-270 (2013).</li><li>Chen, C., Samuel, T. K., Sinclair, J., Dailey, H. A., Hamza, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+intercellular+heme-trafficking+protein+delivers+maternal+heme+to+the+embryo+during+development+in+C+elegans.">An intercellular heme-trafficking protein delivers maternal heme to the embryo during development in C elegans.</a> Cell. 145, 720-731 (2011).</li><li>Praitis, V., Casey, E., Collar, D., Austin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creation+of+low-copy+integrated+transgenic+lines+in+Caenorhabditis+elegans.">Creation of low-copy integrated transgenic lines in Caenorhabditis elegans.</a> Genetics. 157, 1217-1226 (2001).</li><li>Berezikov, E., Bargmann, C. I., Plasterk, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homologous+gene+targeting+in+Caenorhabditis+elegans+by+biolistic+transformation.">Homologous gene targeting in Caenorhabditis elegans by biolistic transformation.</a> Nucleic acids research. 32, e40 (2004).</li><li>Semple, J. I., Biondini, L., Lehner, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+transgenic+nematodes+by+bombardment+and+antibiotic+selection.">Generating transgenic nematodes by bombardment and antibiotic selection.</a> Nature Methods. 9, 118-119 (2012).</li></ol>

作者宣称没有任何竞争的经济利益或利益冲突。

在这个协议中，我们提出一个修改的无菌液体介质mCeHR，允许快速下线虫一代生产大量的蠕虫病毒。该媒体展示了几个优点蠕虫生长没有污染E。大肠杆菌或细菌的副产品和可被利用的营养和毒理学研究。利用E的大肠杆菌或其他细菌在这些研究中有几个缺点。例如，细菌的生长可以在各种条件下的改变和细菌代谢可能正在检测的分子，混杂结果的解释。因此，在确定的培...

土壤线虫， 线虫 ，是在众多的研究中所使用的遗传毒理学一个强大的模式生物。作为其第1 mm的尺寸，四天，便于栽培的快速生成时间，以及大的后代数量结果，这些线虫已被用于许多遗传和药理屏1,2。研究人员利用这个蠕虫利用来识别分子和保守的途径在脊椎动物系统。这些途径包括细胞死亡信号，老化和促进新陈代谢的途径，以及神经系统3-6。另外，C的透明度线虫允许使用荧光蛋白记者，它可以直接可视化，分 ​​析基因表达模式和蛋白质定位的转基因品系的产生。 在许多研究中这种线虫培养使用线虫生长培养基（NGM）板一个固体琼脂为基础的表面或升iquid培养物接种有大肠杆菌作为食物来源7,8。这些细菌的食物来源可以混淆生化和毒理学研究，细菌副产品影响结果的解释干扰。为了避免这些复合效应，C.线虫可以培养在无菌液体介质内，不含细菌作为食物来源的。使用这种媒体，我们成功地培养数以百万计的高度同步的蠕虫许多标准C。线虫协议，包括在C差异调节基因芯片分析线虫暴露于不同浓度的血红素和生产利用基因轰击转基因蠕虫。这种介质化学定义和由埃里克·克莱格博士9制定了原来的配方修改。使用这种mCeHR媒体，我们已经成功地确定参与血红素的动态平衡的基因，称为血红素反应基因（HRG次）10，其将还没有可行的，其中利用接种有大肠杆菌 NGM琼脂平板上正常生长条件大肠杆菌 。 在这个协议中，我们描述的程序，引入和维护C。生长在大肠杆菌线虫大肠杆菌的无菌mCeHR并利用此方法来获得大量的蠕虫病毒用于生产转基因C。使用微粒轰击线虫线。这表明使用无菌介质中，用于确定C的营养需求的效用，我们也存在研究线虫利用血红素作为一个例子。这些研究表明，利用mCeHR介质允许对大量C的快速增长线虫由蠕虫的研究人员利用许多下游应用。

<table><tbody><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;#8226;6H2O</td><td>Sigma</td><td>M-2393</td><td></td></tr><tr><td>Sodium citrate</td><td>Sigma</td><td>S-4641</td><td></td></tr><tr><td>Potassium citrate monohydrate</td><td>Sigma</td><td>P-1722</td><td></td></tr><tr><td>CuCl&lt;sub&gt;2&lt;/sub&gt;&amp;#8226;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher</td><td>C455-500</td><td></td></tr><tr><td>MnCl&lt;sub&gt;2&lt;/sub&gt;&amp;#8226;4H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher</td><td>M87-100</td><td></td></tr><tr><td>ZnCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>Z-0152</td><td></td></tr><tr><td>Fe(NH4)&lt;sub&gt;2&lt;/sub&gt;(SO4)&lt;sub&gt;2&lt;/sub&gt;&amp;#8226;6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>F-1018</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;#8226;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher</td><td>C70-500</td><td></td></tr><tr><td>Adenosine 5 -monophosphate, sodium salt</td><td>Sigma</td><td>A-1752</td><td></td></tr><tr><td>Cytidine 5 -phosphate</td><td>Sigma</td><td>C-1006</td><td></td></tr><tr><td>Guanosine 2&#039; - and 3&#039; -monophosphate</td><td>Sigma</td><td>G-8002</td><td></td></tr><tr><td>Uridine 5 -phosphate, disodium salt</td><td>Sigma</td><td>U-6375</td><td></td></tr><tr><td>Thymine</td><td>Sigma</td><td>T0376</td><td></td></tr><tr><td>N-Acetylglucosamine</td><td>Sigma</td><td>A3286</td><td></td></tr><tr><td>DL-Alanine</td><td>Fisher</td><td>S25648&amp;nbsp;</td><td></td></tr><tr><td>p-Aminobenzoic Acid</td><td>Sigma</td><td>A-9878</td><td></td></tr><tr><td>Biotin</td><td>Sigma</td><td>B-4639</td><td></td></tr><tr><td>Cyanocobalamine (B-12)</td><td>Sigma</td><td>V-2876</td><td></td></tr><tr><td>Folinate (Ca)</td><td>Sigma</td><td>F-7878</td><td></td></tr><tr><td>Niacin</td><td>Sigma</td><td>N-0761</td><td></td></tr><tr><td>Niacinamide</td><td>Sigma</td><td>N-3376</td><td></td></tr><tr><td>Pantetheine</td><td>Sigma</td><td>P-2125</td><td></td></tr><tr><td>Pantothenate (Ca)</td><td>Sigma</td><td>P-6292</td><td></td></tr><tr><td>Pteroylglutamic Acid (Folic Acid)</td><td>ACRCS</td><td>21663-0100</td><td></td></tr><tr><td>Pyridoxal 5&#039;-phosphate</td><td>Sigma</td><td>P-3657</td><td></td></tr><tr><td>Pyridoxamine 2HCl</td><td>Sigma</td><td>P-9158</td><td></td></tr><tr><td>Pyridoxine HCl</td><td>Sigma</td><td>P-6280</td><td></td></tr><tr><td>Riboflavin 5-PO&lt;sub&gt;4&lt;/sub&gt;(Na)</td><td>Sigma</td><td>R-7774</td><td></td></tr><tr><td>Thiamine HCl</td><td>Sigma</td><td>T-1270</td><td></td></tr><tr><td>DL-6,8-Thioctic Acid</td><td>Sigma</td><td>T-1395</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>P-5379</td><td></td></tr><tr><td>Choline di-acid citrate</td><td>Sigma</td><td>C-2004</td><td></td></tr><tr><td>&lt;em&gt;myo&lt;/em&gt;-Inositol</td><td>Sigma</td><td>I-5125</td><td></td></tr><tr><td>D-Glucose</td><td>Sigma</td><td>G-7520</td><td></td></tr><tr><td>Lactalbumin enzymatic hydrolysate</td><td>Sigma</td><td>L-9010</td><td></td></tr><tr><td>Brain Heart Infusion</td><td>BD</td><td>211065</td><td></td></tr><tr><td>Hemin chloride</td><td>Frontier Scientific</td><td>H651-9</td><td></td></tr><tr><td>HEPES, sodium salt</td><td>Sigma</td><td>H-3784</td><td></td></tr><tr><td>Cholesterol</td><td>J.T. Baker</td><td>F676-05</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids</td><td>Invitrogen</td><td>11140-076</td><td></td></tr><tr><td>MEM Amino Acids Solution</td><td>Invitrogen</td><td>11130-051</td><td></td></tr><tr><td>Nalidixic acid sodium salt</td><td>Sigma</td><td>N4382</td><td></td></tr><tr><td>Tetracycline Hydrochloride</td><td>MP Biomedicals</td><td>2194542</td><td></td></tr><tr><td>Biolistic Delivery System</td><td>BioRad</td><td>165-2257</td><td></td></tr><tr><td>Gold particles (Au Powder)&amp;nbsp;</td><td>&amp;nbsp;Ferro Electronic Material Systems</td><td>6420 2504, JZP01010KM</td><td></td></tr><tr><td>or</td><td></td><td></td><td></td></tr><tr><td>Gold Particles 1.0 &amp;mu;m</td><td>BioRad</td><td>&amp;nbsp;165-2263</td><td></td></tr></tbody></table>

culturing caenorhabditis elegans in axenic liquid media and creation of transgenic worms by microparticle bombardment

在这个协议中，我们提出了所需的材料，并作出修改C程序的。Ëlegans习惯化和媒介的复制（mCeHR）。此外，该步骤用于暴露和驯化C。生长在大肠杆菌线虫大肠杆菌对无菌液体介质描述。最后，利用无菌C.下游实验线虫说明这个程序的优点。分析并确定C的能力线虫营养需求是由不断增长的N2野生型蠕虫在无菌液体介质具有不同的血红素浓度的说明。这个过程可以与其它营养物质被复制到确定为蠕虫生长和发育的最佳浓度，或者确定药物治疗的毒理效应。不同的血红素浓度对野生型蠕虫生长的影响，通过定性显微镜观察和定量t一下确定他的蠕虫病毒，在每个血红素浓度上升的。此外，不同的养分浓度的影响可以通过利用表达的变化感兴趣的养分回应荧光传感器蠕虫检测。此外，大量的蠕虫被容易地制造，适用于使用微粒轰击转基因线虫的产生。

C.培养在营养成分的测定无菌液体介质辅助工具所要求的蠕虫，而不由大肠杆菌产生的次生代谢产物的干扰线虫大肠杆菌 。野生型N2蠕虫使其恢复到mCeHR媒体三代以内并呈增长媲美生长在NGM细菌平板蠕虫。事实上，这些蠕虫成为4天内为带有3.5天，生长在OP50细菌的蠕虫相比，妊娠。 使用mCeHR的一个优点被认为在研究，审查这些蠕虫14的确切营养需要?...

Watch this Scientific Journal Video about Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment at JoVE.com

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

线虫通常生长在固体琼脂平板或液体培养基接种大肠杆菌大肠杆菌 。为了防止细菌副产品混杂毒理学和营养研究，我们利用的无菌液体介质，CeHR，成长和同步大量的蠕虫病毒对各种下游应用。

培养<em&gt;秀丽隐杆线虫</em &gt;在无菌液体介质与创造转基因蠕虫通过微粒轰击

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

culturing-caenorhabditis-elegans-axenic-liquid-media-creation

Research

JoVE Journal

Biology

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

16.9K 次观看.  University of Maryland.  线虫通常生长在固体琼脂平板或液体培养基接种大肠杆菌大肠杆菌 。为了防止细菌副产品混杂毒理学和营养研究，我们利用的无菌液体介质，CeHR，成长和同步大量的蠕虫病毒对各种下游应用。 

视频: Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

线虫线虫线虫-一个多用途的体内模型研究宿主-微生物相互作用

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

科研

遗传学

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

产生稳定的转基因C.使用微量注射线虫

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

生物学

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

rna 干扰和热休克诱导转录因子表达在重编程生殖细胞到神经元中的应用C. 线虫

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

发育生物学

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing A&beta;42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

一<em&gt;线虫</em&gt;研究Amylopathy模型系统

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

神经科学

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

生成的基因 C。线虫通过基因枪转化

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

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

Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions

The nematode Caenorhabditis elegans is a model system for host-microbe and host-microbiome interactions. Many studies to date use batch digests rather than individual worm samples to quantify bacterial load in this organism. Here it is argued that the large inter-individual variability seen in bacterial colonization of the C. elegans intestine is informative, and that batch digest methods discard information that is important for accurate comparison across conditions. As describing the variation inherent to these samples requires large numbers of individuals, a convenient 96-well plate protocol for disruption and colony plating of individual worms is established.

Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions

This protocol describes a 96-well disruption of individual bacterially colonized Caenorhabditis elegans following cold paralysis and surface bleaching&#160;to remove external bacteria. The resulting suspension is plated on agar plates to allow accurate, medium-throughput quantification of bacterial load in large numbers of individual worms.

使用单蠕虫数据量化 秀丽隐杆线虫的异质性 - 细菌相互作用

The nematode Caenorhabditis elegans is a model system for host-microbe and host-microbiome interactions. Many studies to date use batch digests rather ...

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid nematode growth media (NGM), allowing the efficient collection of population-level data for survival and other physiological phenotypes, and periodic sampling of subpopulations for fluorescent biomarker quantification. Limitations to this approach are the inability to (1) follow individual worms over time to develop age trajectories for phenotypes of interest and (2) monitor fluorescent biomarkers directly in the context of the culture environment. Alternative culture approaches use liquid culture or microfluidics to monitor individual animals over time, in some cases including fluorescence quantification, with the tradeoff that the culture environment is contextually distinct from solid NGM. The WorMotel is a previously described microfabricated multi-well device for culturing isolated worms on solid NGM. Each worm is maintained in a well containing solid NGM surrounded by a moat filled with copper sulfate, a contact repellent for C. elegans, allowing longitudinal monitoring of individual animals. We find copper sulfate insufficient to prevent worms from fleeing when subjected to aversive interventions common in aging research, including dietary restriction, pathogenic bacteria, and chemical agents that induce cellular stress. The multi-well devices are also molded from polydimethylsiloxane, which produces high background artifacts in fluorescence imaging. This protocol describes a new approach for culturing isolated roundworms on solid NGM using commercially available polystyrene microtrays, originally designed for human leukocyte antigen (HLA) typing, allowing the measurement of survival, physiological phenotypes, and fluorescence across the lifespan. A palmitic acid barrier prevents worms from fleeing, even in the presence of aversive conditions. Each plate can culture up to 96 animals and easily adapts to a variety of conditions, including dietary restriction, RNAi, and chemical additives, and is compatible with automated systems for collecting lifespan and activity data.

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Here, we present a protocol to culture isolated individual nematodes on solid media for lifelong physiological parameter tracking and fluorescence quantification. This culture system includes a palmitic acid barrier around single-worm wells to prevent animals from fleeing, allowing the use of aversive interventions, including pathogenic bacteria and chemical stressors.

在固体培养基上长期培养单个 秀丽隐杆线虫 ，用于纵向荧光监测和厌恶干预

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid ...

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices

The nematode Caenorhabditis elegans is among the most common model systems used in aging research owing to its simple and inexpensive culture techniques, rapid reproduction cycle (~3 days), short lifespan (~3 weeks), and numerous available tools for genetic manipulation and molecular analysis. The most common approach for conducting aging studies in C. elegans, including survival analysis, involves culturing populations of tens to hundreds of animals together on solid nematode growth media (NGM) in Petri plates. While this approach gathers data on a population of animals, most protocols do not track individual animals over time. Presented here is an optimized protocol for the long-term culturing of individual animals on microfabricated polydimethylsiloxane (PDMS) devices called WorMotels. Each device allows up to 240 animals to be cultured in small wells containing NGM, with each well isolated by a copper sulfate-containing moat that prevents the animals from fleeing. Building on the original WorMotel description, this paper provides a detailed protocol for molding, preparing, and populating each device, with descriptions of common technical complications and advice for troubleshooting. Within this protocol are techniques for the consistent loading of small-volume NGM, the consistent drying of both the NGM and bacterial food, options for delivering pharmacological interventions, instructions for and practical limitations to reusing PDMS devices, and tips for minimizing desiccation, even in low-humidity environments. This technique allows the longitudinal monitoring of various physiological parameters, including stimulated activity, unstimulated activity, body size, movement geometry, healthspan, and survival, in an environment similar to the standard technique for group culture on solid media in Petri plates. This method is compatible with high-throughput data collection when used in conjunction with automated microscopy and analysis software. Finally, the limitations of this technique are discussed, as well as a comparison of this approach to a recently developed method that uses microtrays to culture isolated nematodes on solid media.

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices

Presented here is an optimized protocol for culturing isolated individual nematodes on solid media in microfabricated multi-well devices. This approach allows individual animals to be monitored throughout their lives for a variety of phenotypes related to aging and health, including activity, body size and shape, movement geometry, and survival.

多孔设备固体培养基上分离秀 丽隐杆线虫 的长期培养和监测

The nematode Caenorhabditis elegans is among the most common model systems used in aging research owing to its simple and inexpensive culture techniques, ...

C. elegans Maintenance

Ceanorhabditis elegans has been, and is still, used to great success as a model organism for studying a variety of developmental, genetic, molecular and even physical phenomena. In order to use C. elegans to its full potential, proper care and attention to the basic maintenance of this powerful organism is essential.
In this video you will learn the basic housing and feeding requirements of C. elegans, how to correctly handle and manipulate worms using a worm pick and how to freeze and recover important worm stocks. Towards the end of the video we will visit a few applications of modifying the housing, feeding and manipulation of these important animals.

Maintenance of Caenorhabditis elegans

秀丽隐杆线虫的实验室基本培养

Ceanorhabditis elegans has been, and is still, used to great success as a model organism for studying a variety of developmental, genetic, molecular and ...

培养

摘要

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此视频中的章节

线虫线虫线虫-一个多用途的体内模型研究宿主-微生物相互作用

产生稳定的转基因C.使用微量注射线虫

rna 干扰和热休克诱导转录因子表达在重编程生殖细胞到神经元中的应用C. 线虫

一

生成的基因 C。线虫通过基因枪转化

培养和遗传操纵昆虫致病线虫

使用单蠕虫数据量化秀丽隐杆线虫的异质性 - 细菌相互作用

在固体培养基上长期培养单个秀丽隐杆线虫，用于纵向荧光监测和厌恶干预

多孔设备固体培养基上分离秀丽隐杆线虫的长期培养和监测

秀丽隐杆线虫的实验室基本培养

培养

摘要

探索更多视频

此视频中的章节

线虫线虫线虫-一个多用途的体内模型研究宿主-微生物相互作用

产生稳定的转基因C.使用微量注射线虫

rna 干扰和热休克诱导转录因子表达在重编程生殖细胞到神经元中的应用C. 线虫

一

生成的基因 C。线虫通过基因枪转化

培养和遗传操纵昆虫致病线虫

使用单蠕虫数据量化 秀丽隐杆线虫的异质性 - 细菌相互作用

在固体培养基上长期培养单个 秀丽隐杆线虫 ，用于纵向荧光监测和厌恶干预

多孔设备固体培养基上分离秀 丽隐杆线虫 的长期培养和监测

秀丽隐杆线虫的实验室基本培养

使用单蠕虫数据量化秀丽隐杆线虫的异质性 - 细菌相互作用

在固体培养基上长期培养单个秀丽隐杆线虫，用于纵向荧光监测和厌恶干预

多孔设备固体培养基上分离秀丽隐杆线虫的长期培养和监测