星号（*）表示新的或修改的步骤相比，Christensen 等人 1 1。材质设置<ol><li>在细胞培养过程需要大量的鸡蛋从妊娠的成年人中分离的。 C.成长线虫对8P琼脂平板NA22（可通过线虫遗传协会-长城电脑）接种细菌分离大量鸡蛋。在这些板用蛋白胨的量为8倍，通常是用于NGM板的量。较高的蛋白胨浓度更有效地维系着NA22细菌的生长，这出乎OP50-，生长在厚层。 </li></ol> 8P板的配方: 将3克氯化钠，20g的细菌用胰蛋白胨，1升无菌蒸馏水和高压釜30分钟25克琼脂。让介质凉爽，在55℃，然后加入无菌过滤的1毫升胆固醇（5毫克/毫升的EtOH），将1ml 1的MgCl 2的1毫升硫酸镁和25毫升KP缓冲液（500毫升储:5克K 2 HPO 4，30克KH 2 PO 4，pH值6.00）。倒液琼脂培养基中成10毫米培养皿中（25毫升/盘）。 <ol start="2"><li>第二天散布各富含蛋白胨琼脂板的整个表面上用1毫升NA22 E的在2XYT培养基（16克胰蛋白胨，10克酵母提取物，5克NaCl的1升的无菌水，pH 7.0）中于37℃培养的大肠杆菌过夜这些细菌构成了丰富的食物来源，会形成一层厚厚的支持大量妊娠成人的生长。离开种子板在室温下过夜，使细菌的生长。该过程可以通过在37℃下进行4-5小时来加速。 </li><li>饿死的动物转移到播种8P板。清洗过的动物用5-6毫升M9的缓冲区或水的匮乏NGM板加1-2毫升此悬液，以每8P板。 </li><li>允许生长的动物和乘法UNT金正日板是由妊娠成人人口在汇合。 </li><li>隔离要求下编制的蛋线虫用5-6的裂解溶液胚胎细胞，从妊娠的成年人。 </li></ol>裂解液配方 5毫升新鲜的漂白剂1.25毫升10 N的NaOH和18.5毫升的无菌H 2 O的该混合物必须准备每次使用前新鲜。 <ol start="6"><li>从洗净用鸡蛋缓冲妊娠成人孤立的蛋: </li></ol>鸡蛋缓冲配方 118 mM氯化钠，48毫米氯化钾，2毫米氯化钙2，2毫米氯化镁2，25毫米HEPES，pH值7.3，渗透压340毫渗量。 <ol start="7"><li>删除与几丁质酶处理周围的鸡蛋蛋壳。几丁质酶是在酸性pH值25最高活性的酶。在蛋缓冲液pH 6.5溶解几丁质酶（Sigma公司，目录号C6137）在2毫克/毫升的最终浓度。存储几丁质酶股票溶液在-20℃下在1ml等份在无菌的15毫升锥形管中。等分试样可被储存在-20℃至几个月。 </li><li> C.成长线虫培养细胞上覆盖着花生凝集素灭菌盖玻片（直径12 mm）。溶解花生植物血凝素在无菌水（0.5毫克/毫升）。花生凝集素溶液不应该过滤或高压灭菌。它也并不需要被用UV光处理。存储2毫升分装在-20°C可保存6个月。 </li><li>完整的细胞培养液（500毫升）中包含500个ML L-15培养基购自Gibco，将50ml胎牛血清（热灭活），7.7克蔗糖（45毫渗量），5毫升100微克100 U / mL青霉素和/毫升链霉素（2％）。在完全培养基中，然后用0.20微米的微孔过滤器过滤。 </li></ol>注意的是，蛋缓冲器和培养基分别具有340摩尔渗透压浓度和345毫渗透摩尔浓度。事实上，相反的哺乳动物细胞，C。线虫细胞中有相对高的渗透压需要准备的解决方案，在将要与所述细胞的细胞膜直接接触时，必须考虑到计数。这些试剂的配方进行了调整，以达到所需的渗透压，这是用一个渗压计1测量。这是没有必要使用一个渗透压力计如果这些配方是完全按照与护理用于制备这些试剂。然而，人们应该使用osmoter如果其他解决方案需要做好准备，他们的食谱这里不报，或在任何使用C的出版物线虫培养细胞。 2。鸡蛋隔离<ol><li>我们建议开始与至少四个8P板收集到足够的鸡蛋12井培养细胞（24孔板）。 </li><li>开始在手术前解冻的花生凝集素原液一管。置于高压灭菌的盖玻片在孔的底部的24孔板，加入200μl的花生凝集素的盖玻片。孵育1小时或uNTIL细胞准备进行电镀。完全取出花生凝集素和用1毫升无菌水灭菌一次洗井。彻底去除盖玻片花生凝集素是避免细胞聚集是必不可少的。 </li><li>洗妊娠关闭成人用无菌水高压灭菌的琼脂平板。收集悬浮成两个无菌锥形50ml管中。留在冰上的管中长达5分钟，以允许蠕虫沉淀在管（*）的底部。与转印塑料吸管除去水，并将其与新鲜无菌水高压灭菌取代。通过台式离心分离，在200×g离心（〜1200 rpm）离心10分钟（*）沉淀蠕虫。重复该最后步骤至少为3。 </li><li>传送蠕虫到无菌的15毫升锥形管中，沉淀它们在200×g离心（〜1200 rpm）离心10分钟（*）。不使用更高的离心速度，以避免收集细菌在管的底部，以及。有时候，离心后的虫还没有完全沉淀。 AF三每一清洗和去除之前上清液，将管5分钟冰上。在冷却水的蠕虫沉淀到管的底部。 </li><li>去除水中加5-6毫升裂解液（见材料设置）。摇动该悬浮液轻轻地5-10分钟，然后开始监控虫溶胞体视显微镜下，每2-3分钟。一滴悬浮液也可以放置在盖玻片，以方便检查。孵化时间取决于漂白剂的新鲜度;买小瓶装的漂白剂，每月开新瓶。 </li><li>当〜蠕虫的70-80％被溶解（从孵化开始10分钟），停止裂解反应中加入9mL的蛋缓冲液pH 7.3（见材料设置）。离心该悬浮液在200×g离心（〜1200 rpm）离心10分钟。从这点上有一个本生灯上，在板凳上，以防止再次污染的鸡蛋（*）的。 </li><li>小心取出用无菌塑料移液上清，洗涤沉淀3-4x，其中蛋缓冲区中，直到溶液澄清。确保每次洗涤过程中混合颗粒以及在蛋缓冲区。 </li><li>鸡蛋是从用30％蔗糖溶液的动物尸体分离。重悬浮颗粒在2毫升无菌鸡蛋缓冲和加入2ml 60％的蔗糖溶液（股票在无菌鸡蛋缓冲区）。拌匀，离心20分钟在200 XG（〜1200转）（*）。 </li><li>小心地从离心分离机取出试管。将卵悬浮在溶液的上方。使用P1000移液器和无菌针头，转移所有鸡蛋放入一个新的无菌的15 mL锥形管。 </li><li>加入10毫升无菌蛋缓冲到管和离心机在200×g离心（〜1200 rpm）离心10分钟。重复洗涤3×。请确保在鸡蛋缓冲区每次清洗时将鸡蛋完全悬浮。 </li></ol> 3。胚胎干细胞解离进行下一步骤的程序使用的是层流罩的无菌条件下进行。虽然动物袍菌上板，将洗涤液并用含漂白剂的裂解溶液处理，如果不是所有的细菌应消除大部分。因此，使用在层流罩在这一点上的程序可以防止新的卵悬浮液的污染。 <ol><li>重悬沉淀鸡蛋1毫升2毫克/毫升的几丁质酶（股票鸡蛋缓冲液pH 6.5（*）），并将它们转移到一个新的无菌的15 mL锥形管。摇动该管，在室温下10-30分钟。根据酶和房间的温度的新鲜度的精确的温育时间的变化，因此应为每个准备确定。建议在开始监视孵育10分钟后倒置细胞培养显微镜下的蛋。注:根据我们的经验，低pH值增加几丁质酶活性。为此，我们使用蛋缓冲液，pH 6.5以溶解几丁质酶（配方上面的报道，其中pH用NaOH调整至6.5）。 </li><li>时〜80％的蛋壳是由消化几丁质酶处理（ 图1 AB），通过离心分离沉淀的鸡蛋，在900×g离心（〜2500 rpm）离心3分钟（*）。使用P1000移液器和无菌针头，小心取出上清液，加3毫升的L-15培养基（*）。 </li><li>传送鸡蛋成直径6厘米的板，并用10毫升无菌注射器配备有18号针头轻轻地解离的细胞。将一滴悬浮液到一个新的塑料培养皿中，并通过在显微镜下观察监测解离度。在此过程中不要吸入空气进入注射器，以避免损坏电池。继续解离，直到〜80％的细胞解离。 </li><li>使用无菌5微米的微孔过滤器过滤悬浮液。细胞悬浮液必须经过过滤，以除去细胞团块，未消化的卵和孵出的幼虫。通过过滤器过滤额外4-5毫升新鲜的L-15介质恢复所有的细胞。不要使用在过滤步骤用力过猛，以免达maging过滤器和/或细胞。 </li></ol> 4。细胞培养<ol><li>通过离心分离沉淀的分离的细胞在900×g离心（〜2500 rpm）离心3分钟（*）。使用P1000移液器和无菌针头小心地取出所有上清。重悬细胞沉淀在完整的L-15培养基和板1毫升/。加入培养基中的量依赖于所用的8P板的数量，在平板上蠕虫的汇合处，以及将要在细胞上进行的实验的类型。细胞密度可以使用血细胞计数器确定。对于膜片钳记录电镀〜230000个细胞/ cm 2为最佳浓度。 </li><li>保持24孔板中含有湿纸巾，避免培养基蒸发特百惠塑料容器。存储容器，在湿润的培养箱中在20℃和环境空气。 </li><li>这些细胞通常是准备在24小时内试验时的形态分化和快递的绿色荧光蛋白标记物的离子是完整的。细胞可以保存在文化长达2周，但它们通常是最健康的长达7-9天电镀后。介质需要更换，每天一次，以保持健康的细胞。 </li></ol>

线虫是一种功能强大的模式生物破译参与发展，行为和衰老的遗传途径。其方便茎主要从难易程度可以遗传操作，并从它的循环寿命短。尽管它的便利性，C.线虫有其局限性。C.线虫细胞是微小的，在加压的角质层，限制了需要直接访问的细胞，例如电生理和药理学技术，或特定类型的细胞的分离，如通过微阵列分析的基因表达图谱方法的应用局限。该三线虫的细胞培?...

秀丽隐杆线虫 （ 线虫 ）是一款功能强大的模式生物研究细胞功能，分化和行为的分子基础。虽然它的基因组，代谢和生物合成途径类似于脊椎动物"，它的遗传和分子可追踪性都远远大于2。在它的优点是它的外形和简洁的解剖，其快速的生命周期（3天在25°C），短寿命（2周）和大量的后代（&gt; 200）。由于其雌雄同体的性质和生命周期短，分子和基因操作是直接在C。线虫 ，包括转基因动物3,4和诸如RNA干扰5基因敲低技术的应用的生成。C.线虫体和蛋壳是透明的。因此，细胞可以容易地可视化在成人和使用标准显微镜的胚胎。在过去的40年里，C。线虫 </em&gt;的社区已经为下创建的宝贵资源线虫的研究，包括收集了大量的突变体，基因敲除和转基因，解剖和发展6,7的详细说明，包括完整的重建神经系统8，和一个完全测序的基因组这是很好的说明，并将提供给整个社会（ <a href="http://www.wormbase.com" target="_blank">www.wormbase.com</a> ）。 尽管有许多优点，一些实验方法已在C挑战线虫 。这些包括需要访问到组织或细胞类型的细胞和分离的质膜的那些。事实上Ç。线虫组织都在其加压流体静骨架，这是不容易通过酶处理或洗涤剂消化密闭。在20世纪90年代末仪Goodman和珍妮特里士满率先为C的电生理记录的方法线虫神经元和肌肉细胞中原位 9,10。虽然这些方法给了我们重要的见解和神经元在体内的肌肉功能，它们是具有挑战性和低吞吐量。替代的方法来研究细胞功能的体内已经制定了，大多尤其是在使用基因编码的钙传感器，如GCamP和卡默莱昂11-13 体内钙成像。这些方法虽然不允许使用的药理学工具，因为它们是应用在完整的活的动物。 在培养C.第一次尝试线虫细胞在体外大规模被他的博士论文14的制备过程中由莱尔德布卢姆。不幸的是，难以与细胞到衬底的粘附性差遇到，不良细胞的分化和存活无法建立此早期协议作为一个强大的细胞培养方法。 1995年，埃德加和他的同事发表的程序由孤立的单个C和文化来研究细胞分化和形态发生。线虫胚胎15。通过用酶处理和手动解离的组合蛋壳的消化获得的胚胎干细胞，继续增殖，产生高达〜500个细胞15。随后，梁先生和同事培养一个小数字卵裂球的研究肠道形态。它们表明，1 体外分离ë卵裂球产生由通过心尖粘着连接16彼此进行交互创建了一个结构类似于肠腔偏振肠细胞。布氏及其同事还报道了培养℃的类似的方法。线虫胚胎细胞在体外 17。 在此基础上的早期作品，克里斯滕森和同事开发出一种强大的协议培养胚胎Ç。线虫细胞在体外 1。他们表明分离出来的C。线虫细胞可以分化成各种细胞类型和保持它们在体内具有的功能，包括细胞特异性标志物的表达。那些在体内具有挑战性的几种技术，可以在隔离C.应用线虫胚胎细胞。这些包括电1,18 19，成像和免疫化学技术20,21，以及通过荧光激活细胞的特定细胞类型分选（FACS）对细胞特异性cDNA文库22,23的结构的隔离。如RNA干扰（RNAi）的基因敲除技术可以对培养C.施加线虫细胞1，用叠氮基糖作为糖蛋白发现的一个工具了一种新的代谢标记方法最近已在体外培养的C.开发线虫细胞24。 总之，细胞培养方法扩展数组Ø可应用到的C. f技术线虫模型，以努力破译基因功能的生物体的情况下。我们在这里描述的协议C.培养线虫在体外 ，这在很大程度上是基于最早由克里斯蒂安森和他的同事1中所述的协议胚胎细胞。

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>Bacto Peptone</td>
			<td>VWR International Inc.</td>
			<td>90000-382</td>
			<td>&nbsp;</td>
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		<tr>
			<td>Difco Agar Granulated</td>
			<td>VWR International Inc.</td>
			<td>90000-784</td>
			<td>&nbsp;</td>
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		<tr>
			<td>Bacto Tryptone</td>
			<td>VWR International Inc.</td>
			<td>90000-284</td>
			<td>&nbsp;</td>
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			<td>Bacto Yeast Extract</td>
			<td>VWR International Inc.</td>
			<td>90000-724</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Leibovitz's L-15 Medium (1x) Liquid</td>
			<td>Invitrogen</td>
			<td>11415-064</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Invitrogen</td>
			<td>16140-063</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Sigma</td>
			<td>P4333-100ML</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Chitinase from Streptomyces Griseus</td>
			<td>Sigma</td>
			<td>C6137-25UN</td>
			<td>&nbsp;</td>
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			<td>NA22 Escherichia coli</td>
			<td>Caenorhabditis Genetics Center</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
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			<td>Peanut Lectin</td>
			<td>Sigma</td>
			<td>L0881-10MG</td>
			<td>&nbsp;</td>
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			<td>Sucrose</td>
			<td>Sigma</td>
			<td>57903-1KG</td>
			<td>&nbsp;</td>
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			<td>D-(+)Glucose</td>
			<td>Sigma</td>
			<td>67528-1KG</td>
			<td>&nbsp;</td>
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			<td>Ethylene glycol-bis (2-aminoethylether), N,N,N',N'- tetraacidic acid (EGTA)</td>
			<td>Sigma</td>
			<td>E0396-25G</td>
			<td>&nbsp;</td>
		</tr>
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			<td>Hepes</td>
			<td>Sigma</td>
			<td>H3375-500G</td>
			<td>&nbsp;</td>
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			<td>Cholesterol</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>57653-1KG</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333-500G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C1016-500G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266-100G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma</td>
			<td>M2643-500 g</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Sigma</td>
			<td>P2222-500G</td>
			<td>&nbsp;</td>
		</tr>
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			<td>KH2PO4</td>
			<td>Sigma</td>
			<td>P9791-500G</td>
			<td>&nbsp;</td>
		</tr>
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			<td>NaOH</td>
			<td>Sigma</td>
			<td>S8045-500G</td>
			<td>&nbsp;</td>
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		<tr>
			<td>KOH</td>
			<td>Sigma</td>
			<td>P1767-500G</td>
			<td>&nbsp;</td>
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			<td>Ethanol</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
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			<td>Autoclaved distilled H2O</td>
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			<td>EQUIPMENT</td>
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			<td>101-1000 &mu;l Blue Graduated Pipet Tips</td>
			<td>USA Scentific</td>
			<td>1111-2821</td>
			<td>&nbsp;</td>
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			<td>10 ml Sterilized Pipet Individually Wrapped</td>
			<td>USA Scentific</td>
			<td>1071-0810</td>
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			<td>Ergonomic Variable Volume (100-1000 &mu;l) Pipettor with tip ejector</td>
			<td>VWR International Inc.</td>
			<td>89079-974</td>
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		</tr>
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			<td>VWR International Inc.</td>
			<td>53498-103</td>
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			<td>USA Scentific</td>
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			<td>USA Scentific</td>
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			<td>Becton, Dickinson and Co.</td>
			<td>305482</td>
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			<td>Plastic Syringe Filters Corning 0,20 &mu;m pore size</td>
			<td>Corning</td>
			<td>431224</td>
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			<td>Acrodic 25 mm Syringe filter w/5 &mu;m versapor Membrane</td>
			<td>VWR International Inc.</td>
			<td>28144-095</td>
			<td>&nbsp;</td>
		</tr>
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			<td>60x15 mm Petri Dish Sterile</td>
			<td>VWR International Inc.</td>
			<td>82050-548</td>
			<td>&nbsp;</td>
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			<td>100x15 mm Petri Dish Sterile</td>
			<td>VWR International Inc.</td>
			<td>82050-912</td>
			<td>&nbsp;</td>
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			<td>12 mm Diameter Glass Coverslips</td>
			<td>VWR International Inc.</td>
			<td>48300-560</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Clear Cell Culture Plates 24 Well Flat Bottom w/lid</td>
			<td>Thomas scientific</td>
			<td>6902A09</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dumont #5- Fine Forceps</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td>&nbsp;</td>
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		<tr>
			<td>Centrifuge 5702</td>
			<td>Eppendorf</td>
			<td>022629883</td>
			<td>&nbsp;</td>
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		<tr>
			<td>Laminar Flow Hood</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
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			<td>Inverted Microscope with x10 objective</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ambient air humidified Incubator</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

a method for culturing embryonic c. elegans cells

线虫是一种强大的模型系统中，其中的遗传和分子生物学技术是很容易适用的。直到最近，虽然，需要直接访问到细胞和特定的细胞类型的隔离技术，无法在C应用线虫 。这种限制是由于这一事实，即组织内被加压的角质层这是不容易通过与酶和/或洗涤剂处理消化的局限。基于早期的先驱工作，莱尔德布鲁姆，克里斯滕森和同事1开发出一个强大的方法培养C.线虫胚胎干细胞在大规模。鸡蛋是从妊娠成人分离与漂白/ NaOH处理，随后用壳多糖酶处理以除去蛋壳。胚胎细胞，然后通过手动移液解离，并铺板于血清中丰富的介质基片覆盖的玻璃。在隔离牢房的24小时开始改变形态和表达细胞特异性马克分化RS。C.线虫细胞用这种方法培养的存活最多2周体外和已用于电生理，免疫和成像分析以及它们已被分类并用于微阵列分析。

线虫培养的细胞分化并表达细胞特异性标志物 Christensen和使用台盼蓝染色的同事证明，&gt; 99％的胚胎C的线虫细胞存活的分离过程。在9日和镀后22，分别为85％和65％，仍健在1。孤立的胚胎C.线虫的细胞必须坚持的衬底，以区分。细胞没有附着形成团块和它是否生存是不明确的。细胞的分化开始2-3小时电镀后并持续至24小时。 24小时内，细胞承?...

Watch this Scientific Journal Video about A Method for Culturing Embryonic C. elegans Cells at JoVE.com

A Method for Culturing Embryonic C. elegans Cells

我们在这里描述的修订协议，用于胚胎C.大型文化线虫的细胞。 C.胚胎线虫使用这种方法的细胞在体外培养时，出现分化和概括的基因的表达在细胞中的具体方式。需要直接访问到细胞中，或从其它组织中的特定细胞类型的分离技术可以对C.施加线虫培养细胞。

一种用于培养胚胎<em&gt; C。线虫</em&gt;细胞

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

a-method-for-culturing-embryonic-c-elegans-cells

Research

JoVE Journal

Biology

A Method for Culturing Embryonic C. elegans Cells

15.5K 次观看.  University of Miami. 我们在这里描述的修订协议，用于胚胎C.大型文化线虫的细胞。 C.胚胎线虫使用这种方法的细胞在体外培养时，出现分化和概括的基因的表达在细胞中的具体方式。需要直接访问到细胞中，或从其它组织中的特定细胞类型的分离技术可以对C.施加线虫培养细胞。 

视频: A Method for Culturing Embryonic C. elegans Cells

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 ...

科研

生物学

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration.
Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity 1. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration 2. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets 3, 4.
Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.

This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

一个线虫多巴胺神经元退化含量的应用验证潜在的帕金森氏症基因

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

为核型分析人类胚胎干细胞的染色体蔓延制备

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

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 ...

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and expression pattern of genes of interest or generation of tandem affinity purification (TAP) tagged versions of specific genes to facilitate their purification. Typically transgenes are generated by placing a promoter upstream of a GFP reporter gene or cDNA of interest, and this often produces a representative expression pattern. However, critical elements of gene regulation, such as control elements in the 3' untranslated region or alternative promoters, could be missed by this approach. Further only a single splice variant can be usually studied by this means. In contrast, the use of worm genomic DNA carried by fosmid DNA clones likely includes most if not all elements involved in gene regulation in vivo which permits the greater ability to capture the genuine expression pattern and timing. To facilitate the generation of transgenes using fosmid DNA, we describe an E. coli based recombineering procedure to insert GFP, a TAP-tag, or other sequences of interest into any location in the gene. The procedure uses the galK gene as the selection marker for both the positive and negative selection steps in recombineering which results in obtaining the desired modification with high efficiency. Further, plasmids containing the galK gene flanked by homology arms to commonly used GFP and TAP fusion genes are available which reduce the cost of oligos by 50% when generating a GFP or TAP fusion protein. These plasmids use the R6K replication origin which precludes the need for extensive PCR product purification. Finally, we also demonstrate a technique to integrate the unc-119 marker on to the fosmid backbone which allows the fosmid to be directly injected or bombarded into worms to generate transgenic animals. This video demonstrates the procedures involved in generating a transgene via recombineering using this method.

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.

生产 C。线虫</em通过重组工程与转基因 galK选择标记

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and ...

Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical studies are substantial, therefore it is essential that more predictive toxicology screens are developed and deployed early on in drug development (Greenhough et al 2010). Human hepatocytes represent the current gold standard model for evaluating drug toxicity, but are a limited resource that exhibit variable function. Therefore, the use of immortalised cell lines and animal tissue models are routinely employed due to their abundance. While both sources are informative, they are limited by poor function, species variability and/or instability in culture (Dalgetty et al 2009). Pluripotent stem cells (PSCs) are an attractive alternative source of human hepatocyte like cells (HLCs) (Medine et al 2010). PSCs are capable of self renewal and differentiation to all somatic cell types found in the adult and thereby represent a potentially inexhaustible source of differentiated cells. We have developed a procedure that is simple, highly efficient, amenable to automation and yields functional human HLCs (Hay et al 2008 ; Fletcher et al 2008 ; Hannoun et al 2010 ; Payne et al 2011 and Hay et al 2011). We believe our technology will lead to the scalable production of HLCs for drug discovery, disease modeling, the construction of extra-corporeal devices and possibly cell based transplantation therapies.

This article will focus on the generation of human hepatic endoderm from human embryonic stem cell populations.

肝细胞样细胞从人类胚胎干细胞群的鲁棒代

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi筛选识别胚后的表型 C。线虫</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

适用于培养人类胚胎和诱导多能干细胞的小鼠胚胎成纤维细胞的制备

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

一个系统<em&gt;体外</em培养的胚胎胰腺

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

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 ...

一种用于培养胚胎

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产生稳定的转基因C.使用微量注射线虫

一个线虫多巴胺神经元退化含量的应用验证潜在的帕金森氏症基因

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生成的基因 C。线虫通过基因枪转化

生产 C。线虫 galK选择标记

肝细胞样细胞从人类胚胎干细胞群的鲁棒代

RNAi筛选识别胚后的表型 C。线虫

适用于培养人类胚胎和诱导多能干细胞的小鼠胚胎成纤维细胞的制备

一个系统

基本的