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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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 border="0" cellpadding="0" cellspacing="0" width="754">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:359px;">MgCl2.6H2O</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">M-2393</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium citrate</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">S-4641</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium citrate.H2O</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">P-1722</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">CuCl2.2H2O</td>
			<td style="width:108px;">Fisher</td>
			<td style="width:121px;">C455-500</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MnCl2.4H2O</td>
			<td style="width:108px;">Fisher</td>
			<td style="width:121px;">M87-100</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">ZnCl2</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">Z-0152</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Fe(NH4)2(SO4)2.6H2O</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">F-1018</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">CaCl2.2H2O</td>
			<td style="width:108px;">Fisher</td>
			<td style="width:121px;">C70-500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adenosine 5 -monophosphate, sodium salt</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">A-1752</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytidine 5 -phosphate</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">C-1006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Guanosine 2 - and3&#160; -monophosphate</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">G-8002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Uridine 5 -phosphate, disodium salt</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">U-6375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thymine&#160;</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">T0376</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">N-Acetylglucosamine</td>
			<td style="width:108px;">Sigma</td>
			<td>A3286</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">DL-Alanine</td>
			<td style="width:108px;">Fisher</td>
			<td>S25648&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">p-Aminobenzoic Acid</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">A-9878</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Biotin</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">B-4639</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Cyanocobalamine (B-12)</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">V-2876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Folinate (Ca)&#160;</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">F-7878</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Niacin</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">N-0761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Niacinamide</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">N-3376</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Pantetheine</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">P-2125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Pantothenate (Ca)</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">P-6292</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Pteroylglutamic Acid (Folic Acid)</td>
			<td style="width:108px;">ACRCS</td>
			<td style="width:121px;">21663-0100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Pyridoxal 5&#39;-phosphate</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">P-3657</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Pyridoxamine.2HCl</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">P-9158</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Pyridoxine.HCl</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">P-6280</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:359px;">Riboflavin 5-PO4(Na)</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">R-7774</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Thiamine.HCl</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">T-1270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">DL-6,8-Thioctic Acid</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">T-1395</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:359px;">KH2PO4</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">P-5379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Choline di-acid citrate</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">C-2004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">myo-Inositol</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">I-5125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">D-Glucose</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">G-7520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lactalbumin enzymatic hydrolysate</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">L-9010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Brain Heart Infusion</td>
			<td style="width:108px;">BD</td>
			<td style="width:121px;">211065</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:359px;">Hemin chloride</td>
			<td style="width:108px;">Frontier Scientific</td>
			<td style="width:121px;">H651-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">HEPES, Na salt</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">H-3784</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Cholesterol</td>
			<td style="width:108px;">J.T. Baker</td>
			<td style="width:121px;">F676-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Non-Essential Amino Acids</td>
			<td style="width:108px;">Invitrogen</td>
			<td>11140-076</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Amino Acids Solution</td>
			<td style="width:108px;">Invitrogen</td>
			<td>11130-051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nalidixic acid sodium salt</td>
			<td style="width:108px;">Sigma</td>
			<td>N4382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tetracycline Hydrochloride</td>
			<td style="width:108px;">MP Biomedicals</td>
			<td>2194542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biolistic Delivery System</td>
			<td style="width:108px;">BioRad</td>
			<td>165-2257</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">Gold particles (Au Powder)&#160;</td>
			<td style="width:108px;">&#160;Ferro Electronic Material Systems</td>
			<td style="width:121px;">6420 2504, JZP01010KM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">or</td>
			<td style="width:108px;"></td>
			<td style="width:121px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gold Particles 1.0 &#956;m</td>
			<td style="width:108px;">BioRad</td>
			<td>&#160;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

Measuring Caenorhabditis elegans Life Span on Solid Media

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has emerged as a principal model used to study the biology of aging. Because virtually every biological subsystem undergoes functional decline with increasing age, life span is the primary endpoint of interest when considering total rate of aging. In nematodes, life span is typically defined as the number of days an animal remains responsive to external stimuli. Nematodes can be propagated either in liquid media or on solid media in plates, and techniques have been developed for measuring life span under both conditions. Here we present a generalized protocol for measuring life span of nematodes maintained on solid nematode growth media and fed a diet of UV-killed bacteria. These procedures can easily be adapted to assay life span under various common conditions, including a diet consisting of live bacteria, dietary restriction, and RNA interference.

Measuring Caenorhabditis elegans Life Span on Solid Media

In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

测量线虫固体介质的寿命

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode ...

科研

生物学

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 ...

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

分离和<em在体外激活线虫精子

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

时间推移显微镜早期胚胎发育线虫

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

基本的<em&gt;线虫</em&gt;方法：同步和观察

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

前列腺素的提取与分析<em&gt;线虫</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

人鼻粘膜上皮细胞在气液界面培养

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode&#8217;s ATP levels.1-3 Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.4,5 In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

A protocol is described for in vivo detection of effects of mitochondrial inhibitors in the model organism Caenorhabditis elegans and for identification of potential enhancing compounds. This protocol can be used to screen drug libraries for compounds modulating mitochondrial function.

可筛选的<em&gt;在体内</em&gt;测定线粒体调节剂使用转基因生物发光<em&gt;秀丽隐杆线虫</em

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

栽培<em&gt;秀丽隐杆线虫</em&gt;在三个维度实验室

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

线虫的耗氧率测定

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...