这项工作是从美国匹兹堡大学和美国国立卫生研究院授予AG028977 ALF的种子资金支持

概述传统的转基因蠕虫所产生的 C.显微注射到转基因DNA 线虫生殖1,2,3,4。虽然成功，这种方法需要专门的设备和经验与蠕虫解剖和显微注射技术的发展。最近枪法已发展作为另一种方法，它使用DNA包覆的金颗粒引入外源DNA的生殖细胞 5,6 。这种方法需要较少的时间在实践方面的投资，成为成功的。 C.枪法线虫生成转基因蠕虫已经在个人蠕虫水平的低效率，所以取得成功，需要大量的动物。这些都可以在几个方面成长，但我们用鸡蛋板，以减少工作和涉及的板块数目。我们的协议，开始准备鸡蛋板和蠕虫，然后集中轰击在Bio - Rad PDS-1000/He七溴系统使用协议。我们调整我们的协议，在工作Berezikov 等 7的一部分。 随着轰击，UNC - 119基因通常被用作转基因动物的标志。携带这种突变的蠕虫病毒株，严重不协调，勉强移动，矮胖的外观，而且也无法形成的dauer幼虫8。的dauer是一个备用的幼虫阶段，它是用来延迟在不利条件下，发展生殖成人，和成的dauer项是由多个基因的9调节。运载UNC - 119基因的几个菌株，可从三线虫遗传学中心（CGC，明尼苏达州明尼阿波利斯）。原DP38株（UNC - 119（ED3））也进行无关的dauer形成构（DAF - C）突变可能影响下游实验。几个实验室有异交这种突变，HT1593应变是从政府总部大楼。我们发现，转基因动物是比较容易识别与DP38应变，并趋向于使用这样的压力下，为创造转基因动物。必要时，我们使用HT1593交配的转基因蠕虫删除DAF - C突变。 DP38蠕虫增长是在协议中最慢的步骤之一，所以我们在保持增长的板块的股票，而准备的转基因。 UNC - 119感兴趣的基因标记的表达式允许易于识别基础上的正常蠕动和车身尺寸 5救援转基因动物表达。 UNC - 119基因，可从几个来源， 使用UNC - 119基因组DNA的载体，UNC - 119启动子和cDNA和基因组DNA 为较小的C briggsiae基因用于8,10。最近，我们描述了一个简单的协议中添加一个UNC - 119盒式任何含有氨苄青霉素抗性基因的质粒通过同源重组11和方法来修改蠕虫fosmids进行CRE - LOX重组的UNC - 119基因产生转基因动物12。质粒可以从Addgene公司（马萨诸塞州剑桥，）。 虽然在执行单一的轰炸所涉及的努力是重要的，但管理的的，额外的工作，涉及多个单日轰炸是最小的。因此，我们经常集群生产转基因蠕虫，以减少产生每所涉及的工作。 1。蛋盘的准备突变的UNC - 119蠕虫难以增长，因为他们行动不便，他们往往饿死一个板块的部分，而板块的其他部分仍然包含食品标准NGM板。鸡蛋板厚的食物层允许UNC - 119蠕虫病毒更容易抓取，并接管所有的板7蛋板解决这个问题。鸡蛋板也有大量的蠕虫病毒的生长，使更少的板需要13的支持受益。一般5个鸡蛋板足以增长一炮击中的蠕虫病毒。下面的配方使得大约50板，但可减半少，如果需要的话。 鸡蛋板可以成为容易受到污染，所以我们在板使用的抗生素和抗真菌药物，只用次氯酸钠播种板的处理产生的鸡蛋。 第1天： <ol><li>准备在一个500毫升的瓶子磅（400毫升）。高压灭菌器为20分钟。 </li><li>倒〜50 10厘米NGA的板使用1公升NGA的2％琼脂。我们每公升和链霉素添加制霉菌素10毫升至200微克/毫升13。 </li><li> 200μg/ mL链霉素，增长40毫升OP50 - 1 LB培养过夜。这株链霉素抗性和从总部大楼。 </li><li>高压灭菌器为500毫升一瓶第二天。 </li></ol>第2天： <ol><li> 10个鸡蛋分开蛋黄进入无菌瓶。我们用一个蛋黄分离器（可在家庭商店，包括目标）。摇匀。 </li><li>将量400毫升用LB。动摇</li><li>在60 ° C孵育1小时。 </li><li>冷却至室温。 </li><li> OP50 - 1添加40毫升。动摇</li><li>分发5-8毫升每盘混合。 </li><li>允许板在室温下静置过夜。 </li></ol>第3天： <ol><li>从板轻轻倒出剩余的液体。允许干在室温下用盖子上过夜。 </li></ol>第4天： <ol><li>置于4℃直至所需的板成箱或塑料袋和存储。这些板块出现被确定为1-2个月使用。 </li></ol> 2。播种蛋板<ol><li>加入集中OP50最近饿死板展开6厘米NGA的板DP38蠕虫。对于一个单一的轰炸中，我们用小板〜6种子5个鸡蛋板。对于多个轰炸，我们用小板，而不是种子丰富的蛋白胨2板播种前鸡蛋板。 </li><li>隔离通过使用次氯酸钠13,14 DP38蠕虫卵。重悬在无菌的S -基肥或M9缓冲区13,14鸡蛋。 </li><li>新增的蛋（每盘&gt; 10,000）鸡蛋板的适当数量。增长20 ° C时为7-10天使用轰击的蠕虫卵板。板块将准备使用一次的蠕虫已经开始明确的食品板块。这些板块确有困难的，饿死和更长的持续时间增长将提高蠕虫产量。 </li></ol>轰击我们准备的黄金储备时间提前，并保持在4 ° C下使用存储。还需要unspotted，并发现了10厘米NGA的板。 unspotted板需要提前做好浇，让其彻底干燥，以促进吸收与蠕虫添加液体。 在一天的轰击，我们洗掉，然后开始准备的DNA涂层的金颗粒的蠕虫。然后，我们添加的蠕虫unspotted NGA的板，完成洗的金颗粒和他们转移到macrocarriers。 金粒子的制备： <ol><li>称取60mg金颗粒在1.7mL管和70％的乙醇浸泡15分钟。自旋简要沉淀的金颗粒。 </li><li>在无菌水清洗3次，每次旋转简要收集黄金。 </li><li>重悬在50％的无菌甘油1ML的金牌。这是股市的黄金粒子的解决方案，可以存放在4个月为° C。 </li></ol>蠕虫： <ol><li>关闭的S -基肥或M9缓冲的鸡蛋板浮法DP38蠕虫，并转移至50ml锥形管。 </li><li>在板凳上的管子，直到蠕虫在底部。然后吸出缓冲区，并添加新鲜的S -基肥或M9。洗2〜3次，直到缓冲区是比较明确的碎片。保持在这一步的蠕虫病毒的DNA涂层，直到完成。 </li><li>吸离开约2-3毫升每轰击缓冲区的蠕虫。转移至10厘米unspotted NGA的冰板。重要的是最大限度地减少传输量衙前板将需要完全晾干后方可轰击。一定要板的整个表面覆盖的蠕虫。 </li><li>允许干冰板。冰蠕虫防止结块，同时干燥。 </li></ol>这一步很重要：板应干燥，衙前板均匀分散的蠕虫。上冰蠕虫，阻止他们对板块移动，聚集和成桩。 DNA的制备（之一轰击）： <ol><li>在一个1.7mL管混合： <ul><li> 50μL的黄金颗粒。之前加入彻底重悬。 </li><li> 50μL的DNA（10 - 15ug）。我们看到圆形或线性DNA之间的差别不大。通常我们使用DNA纯化Qiagen公司midiprep盒（QIAGEN公司，瓦伦西亚，CA）。 </li><li> 50μL2.5M氯化钙2。这个解决方案是在室温下几个星期的稳定。 </li><li> 20μL0.1M精胺。 亚精胺是在水溶液中不稳定。我们70μl精胺分装并储存于-20 ° C我们再加入1ml到水权使用前获得一个0.1M的解决方案。该解决方案必须准备新鲜的每个轰击。 我们通常在1.7mL管旋涡的金粒子，在低速，然后添加氯化钙，DNA和亚精胺/鱼精蛋白中滴加而震荡。对于磷酸钙转染的人，这种技术很熟悉。 我们也使用鱼精蛋白，而不是精胺，具有非常好的结果15。我们准备一个新的解决方案（1mg/ml的）在水和我们Ë50μL，而不是精胺20μL。如果我们用鱼精蛋白的话，我们通常在室温下孵育10分钟，而不是放置在冰上的混合物。鱼精蛋白并不需要冰冻保存，是一种粉末，而不是液体。 </li></ul></li><li>孵育30min后在冰的混合物，并定期混合保持悬浮液中的金颗粒。然后简单地旋转和吸出上清液。 </li><li> 300μL70％乙醇洗。自旋简要。 </li><li>洗涤用1ml 100％乙醇。自旋简要。 </li><li>悬浮在170μl100％的乙醇。 </li></ol>准备macrocarriers： <ol><li>冲洗7 macrocarriers（Bio - Rad公司实验室，大力士，CA），2 - 丙醇。放在纸巾上，并让他们在室温下干燥。 </li><li> DNA /金混合物添加在每个macrocarrier中心20μL。之前确定涡权移液，悬浮液中保持的黄金。 </li><li>让乙醇蒸发。 </li></ol> 3。轰击请务必执行一个空白轰击实验前冲洗氦气通过系统。 <ol><li>真空泵的开启和关闭真空泵陷阱。我们使用无油真空泵。 </li><li>打开氦气罐。检查压力≥2200psi。 </li><li>湿破裂盘，在2 - 丙醇（1350 psi）时，放置在保留第七溴适配器。 </li><li>拧入PDS - 1000系统的适配器，并与所提供的扭矩扳手拧紧。 </li><li>将7成的持有人，座位他们提供的工具macrocarriers。持有人，然后把一个七溴停止屏幕的底部。从上面的第二个架子上，翻转超过持有和地点。黄金发现一侧macrocarriers必须朝下，在这一点上。 </li><li> macrocarrier持有人的七溴适配器网点顶部的孔对齐。 </li><li>将衙前板涂轰击室的最低货架上的蠕虫（盖关闭）。 </li><li> PDS - 1000真空流量旋钮完全打开。 VAC按按钮，直到室达到在HG 26。然后切换到保持原来位置，以保持真空。 </li><li>按住直到磁盘破裂的消防按钮。会发生这种情况，当压力达到1350psi。有时，这需要5-20秒发生。松开按钮。 </li><li>发行真空室（泄位置），并删除板。 </li><li>打开真空和氦关闭。 </li><li>消防几次，直到该行不包含氦（氦压力表应标志0 PSI）。 </li><li>关闭PDS - 1000系统。 </li></ol>蠕虫轰击后的恢复： <ol><li>保留在20 ° C下20分钟的轰炸板。 </li><li>从盘用10 mL的S -基肥或M9缓冲浮动的蠕虫病毒。 </li><li> 0.5ML蠕虫放在20 - 10厘米发现NGA的板块。 </li><li>保留在20 ° C或室温为2周左右（22-24℃）前检查获救蠕虫。 </li><li>在解剖显微镜UNC - 119（+）表型的蠕虫的画面。筛查的最佳时间是后板都饿死以来获救的蠕虫非救出蠕虫有生存优势。 </li><li>各10厘米的板中救出蠕虫生成一个单独的行。 </li></ol> 4。代表性的成果该协议获得转基因动物方面的成功，依赖于特定的基因。发起人：GFP记者转基因，我们获得了20行。一个较为典型的结果是3-10线。高达30％的转基因株系的综合线，但是这是随机的，我们将执行多个单日的轰炸，如果一个集成线路是特别期望。

<ol>
	<li>Evans, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Wormbook. , 1-15 (2006).</li><li>Berkowitz, L. A., Knight, A. L., Caldwell, G. A., Caldwell, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+stable+transgenic+C.+elegans+using+microinjection.">Generation of stable transgenic C. elegans using microinjection.</a> J Vis Exp. , (2008).</li><li>Mello, C., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+transformation.">DNA transformation.</a> Methods Cell Biol. 48, 451-482 (1995).</li><li>Mello, C. C., Kramer, J. M., Stinchcomb, D., Ambros, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+in+C.elegans:+extrachromosomal+maintenance+and+integration+of+transforming+sequences.">Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences.</a> EMBO J. 10, 3959-3970 (1991).</li><li>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>Wilm, T., Demel, P., Koop, H. U., Schnabel, H., Schnabel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ballistic+transformation+of+Caenorhabditis+elegans.">Ballistic transformation of Caenorhabditis elegans.</a> Gene. 229, 31-35 (1999).</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 Res. 32, e40-e40 (2004).</li><li>Maduro, M., Pilgrim, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+cloning+of+unc-119,+a+gene+expressed+in+the+Caenorhabditis+elegans+nervous+system.">Identification and cloning of unc-119, a gene expressed in the Caenorhabditis elegans nervous system.</a> Genetics. 141, 977-988 (1995).</li><li>Riddle, D. L., Swanson, M. M., Albert, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interacting+genes+in+nematode+dauer+larva+formation.">Interacting genes in nematode dauer larva formation.</a> Nature. 290, 668-671 (1981).</li><li>Maduro, M., Pilgrim, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservation+of+function+and+expression+of+unc-119+from+two+Caenorhabditis+species+despite+divergence+of+non-coding+DNA.">Conservation of function and expression of unc-119 from two Caenorhabditis species despite divergence of non-coding DNA.</a> Gene. 183, 77-85 (1996).</li><li>Ferguson, A. A., Fisher, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrofitting+ampicillin+resistant+vectors+by+recombination+for+use+in+generating+C.+elegans+transgenic+animals+by+bombardment.">Retrofitting ampicillin resistant vectors by recombination for use in generating C. elegans transgenic animals by bombardment.</a> Plasmid. 62, 140-145 (2009).</li><li>Zhang, Y., Nash, L., Fisher, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified,+robust,+and+streamlined+procedure+for+the+production+of+C.+elegans+transgenes+via+recombineering.">A simplified, robust, and streamlined procedure for the production of C. elegans transgenes via recombineering.</a> BMC Dev Biol. 8, 119-119 (2008).</li><li>Lewis, J. A., Fleming, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+culture+methods.">Basic culture methods.</a> Methods Cell Biol. 48, 3-30 (1995).</li><li>Sulston, J. E., Horvitz, H. R., Wood, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The nematode, Caenorhabditis elegans. , 587-606 (1988).</li><li>Sivamani, E., DeLong, R. K., Qu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protamine-mediated+DNA+coating+remarkably+improves+bombardment+transformation+efficiency+in+plant+cells.">Protamine-mediated DNA coating remarkably improves bombardment transformation efficiency in plant cells.</a> Plant Cell Rep. 28, 213-221 (2009).</li><li>Johnston, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biolistic+transformation:+microbes+to+mice.">Biolistic transformation: microbes to mice.</a> Nature. 346, 776-777 (1990).</li></ol>

基因枪法是一个简单的的方法，引入外源DNA的许多生物，包括 C。 线虫 1,5,6,7,16。依靠黄金颗粒，形成了一个复杂的DNA 与氯化钙存在。阳离子多胺，如亚精胺，保护核酸在体内降解的DNA。既然精胺是一种不稳定的分子，它是重要的存储在小分装于-20 ° C，并在执行轰击之前做出正确的解决方案。正如我们在轰炸协议描述，鱼精蛋白可以用来代替亚精胺提供外源DNA 1...

The volume for a solution in, <a href="http://www.jove.com/video/2090">Generation of Transgenic C. elegans by Biolistic Transformation</a>, was incorrect.

Erratum: Generation of Transgenic C. elegans by Biolistic Transformation

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Gold particles</td>
<td>&nbsp;</td>
<td>Inbio gold</td>
<td>BD021</td>
<td>1micron</td>
</tr>
<tr>
<td>Midiprep kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>12143</td>
<td>&nbsp;</td>
<tr>
<td>Spermidine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S4139</td>
<td>&nbsp;</td>
<tr>
<td>Protamine</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td> P4505</td>
<td>&nbsp;</td>
<tr>
<td>Macrocarriers</td>
<td>&nbsp;</td>
<td>Biorad</td>
<td>165-2335</td>
<td>&nbsp;</td>
<tr>
<td>PDS-1000/He Hepta System</td>
<td>&nbsp;</td>
<td>Biorad</td>
<td>165-2257</td>
<td>&nbsp;</td>
<tr>
<td>Rupture disk</td>
<td>&nbsp;</td>
<td>Biorad</td>
<td>165-2330</td>
<td>&nbsp;</td>
<tr>
<td>Nystatin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>N1638</td>
<td>&nbsp;</td>
<tr>
<td>Streptomycin </td>
<td>&nbsp;</td>
<td>MP</td>
<td>100556</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

generation of transgenic c. elegans by biolistic transformation

使用自由生活线虫C.的实验室的数量线虫是迅速增长。这种生物模型的普及是由于快速的一代人的时间和寿命短，维护容易和便宜，完全测序的基因组，和RNAi的资源和突变的动物数组。此外，分析了C.线虫基因组中发现了蠕虫和高等脊椎动物之间极大的相似性，这表明蠕虫病毒的研究可能是一个重要的辅助手段，在整个小鼠培养细胞进行的研究。一个强大的蠕虫病毒的研究和重要组成部分，是利用转基因动物的研究，基因定位和功能的能力。可以创建可以通过蠕虫生殖显微注射，或通过利用基因枪法转基因动物。炮击是一个较新的技术并不太熟悉的实验室。在这里，我们描述了一个简单的协议生成的Bio - Rad公司PDS - 1000系统使用的金粒子枪法转基因蠕虫。到雌雄同体生殖与DNA显微注射法相比，该协议已经不需要特殊技能，从运营商方面识别蠕虫解剖或执行显微注射​​的优势。通常从一个单一的轰炸进一步多个转基因株系。此外，在对比，以显微注射法，基因枪法染色体外的阵列和集成的转基因与转基因动物。能够获得综合的转基因株系，可避免使用整合外源DNA诱变协议。总之，基因枪法可以代转基因动物的一个极具吸引力的一种方法，尤其是对不感兴趣的投资需要的时间和精力，成为显微注射技术熟练的调查。

Watch this Scientific Journal Video about Generation of Transgenic C. elegans by Biolistic Transformation at JoVE.com

Generation of Transgenic C. elegans by Biolistic Transformation

转基因蠕虫常用<em&gt; C。线虫</em&gt;研究。描述的是一个简单而有效，协议引入蠕虫使用DNA包覆的金粒子枪法转基因。参与的努力和轰炸的结果相媲美注射代转基因动物。

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 ...

generation-of-transgenic-c-elegans-by-biolistic-transformation

Research

JoVE Journal

Biology

Generation of Transgenic C. elegans by Biolistic Transformation

21.8K 次观看.  University of Pittsburgh. 转基因蠕虫常用 C。线虫研究。描述的是一个简单而有效，协议引入蠕虫使用DNA包覆的金粒子枪法转基因。参与的努力和轰炸的结果相媲美注射代转基因动物。

视频: Generation of Transgenic C. elegans by Biolistic Transformation

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

Paradigms for Pharmacological Characterization of C. elegans Synaptic Transmission Mutants

The nematode, Caenorhabditis elegans, has become an expedient model for studying neurotransmission. C. elegans is unique among animal models, as the anatomy and connectivity of its nervous system has been determined from electron micrographs and refined by pharmacological assays. In this video, we describe how two complementary neural stimulants, an acetylcholinesterase inhibitor, called aldicarb, and a gamma-aminobutyric acid (GABA) receptor antagonist, called pentylenetetrazole (PTZ), may be employed to specifically characterize signaling at C. elegans neuromuscular junctions (NMJs) and facilitate our understanding of antagonistic neural circuits.
Of 302 C. elegans neurons, nineteen GABAergic D-type motor neurons innervate body wall muscles (BWMs), while four GABAergic neurons, called RMEs, innervate head muscles. Conversely, thirty-nine motor neurons express the excitatory neurotransmitter, acetylcholine (ACh), and antagonize GABA transmission at BWMs to coordinate locomotion. The antagonistic nature of GABAergic and cholinergic motor neurons at body wall NMJs was initially determined by laser ablation and later buttressed by aldicarb exposure. Acute aldicarb exposure results in a time-course or dose-responsive paralysis in wild-type worms. Yet, loss of excitatory ACh transmission confers resistance to aldicarb, as less ACh accumulates at worm NMJs, leading to less stimulation of BWMs. Resistance to aldicarb may be observed with ACh-specific or general synaptic function mutants. Consistent with antagonistic GABA and ACh transmission, loss of GABA transmission, or a failure to negatively regulate ACh release, confers hypersensitivity to aldicarb. Although aldicarb exposure has led to the isolation of numerous worm homologs of neurotransmission genes, aldicarb exposure alone cannot efficiently determine prevailing roles for genes and pathways in specific C. elegans motor neurons. For this purpose, we have introduced a complementary experimental approach, which uses PTZ.
Neurotransmission mutants display clear phenotypes, distinct from aldicarb-induced paralysis, in response to PTZ. Wild-type worms, as well as mutants with specific inabilities to release or receive ACh, do not show apparent sensitivity to PTZ. However, GABA mutants, as well as general synaptic function mutants, display anterior convulsions in a time-course or dose-responsive manner. Mutants that cannot negatively regulate general neurotransmitter release and, thus, secrete excessive amounts of ACh onto BWMs, become paralyzed on PTZ. The PTZ-induced phenotypes of discrete mutant classes indicate that a complementary approach with aldicarb and PTZ exposure paradigms in C. elegans may accelerate our understanding of neurotransmission. Moreover, videos demonstrating how we perform pharmacological assays should establish consistent methods for C. elegans research.

This video demonstrates how to employ two neural stimulants, aldicarb and pentylenetetrazole (PTZ), in complementary ways to study synaptic function in the nematode, C. elegans. This complementary approach may also be used to shed light on evolutionarily conserved mechanisms for modulating neuronal synchrony and has implications for epilepsy and seizures.

为线虫突触传递的突变体的药理特性的范式

The nematode, Caenorhabditis elegans, has become an expedient model for studying neurotransmission. C. elegans is unique among animal models, as the ...

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be produced to contain any desired DNA or RNA sequence and can be prepared to include a wide variety of base and sugar modifications. Moreover, oligos can be designed to mimic specific nucleic acid alterations and thus, can serve as important tools to investigate effects of DNA damage and mechanisms of repair. We found that Thermo Scientific Dharmacon RNA-containing oligos with a length between 50 and 80 nucleotides can be particularly suitable to study, in vivo, functions and consequences of chromosomal RNA/DNA hybrids and of ribonucleotides embedded into DNA. RNA/DNA hybrids can readily form during DNA replication, repair and transcription, however, very little is known about the stability of RNA/DNA hybrids in cells and to which extent these hybrids can affect the genetic integrity of cells. RNA-containing oligos, therefore, represent a perfect vector to introduce ribonucleotides into chromosomal DNA and generate RNA/DNA hybrids of chosen length and base composition. Here we present the protocol for the incorporation of ribonucleotides into the genome of the eukaryotic model system yeast /Saccharomyces cerevisiae/. Yet, our lab has utilized Thermo Scientific Dharmacon RNA-containing oligos to generate RNA/DNA hybrids at the chromosomal level in different cell systems, from bacteria to human cells.

This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.

改造使用含RNA寡核苷酸基因组DNA的RNA / DNA杂交一代

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

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 ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

线虫趋含量的

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

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 ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using "Freeze-Cracking"

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

抗体在染色<em&gt; C。线虫</em&gt;使用"冻结开裂"

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr&#160;(simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

对缺血/再灌注在缺氧，饥饿型号<em&gt; C。线虫</em

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...

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

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

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

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

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 ...

Generation of Transgenic Hydra by Embryo Microinjection

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology1. Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.

Generation of Transgenic Hydra by Embryo Microinjection

Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi.