We would like to acknowledge the collaborative nature of the Hunter lab, and thank them for the helpful discussion and assistance that made development of this technique possible and fun. We would like to thank Nigel Delaney and the Caenorhabditis Genetics Center for worm and bacterial strains. This work was supported by National Institutes of Health GM089795 grant to CPH.

1. Preparation of Materials
The material necessary for vampiric reverse microinjection is similar to that required for standard microinjection techniques used to make transgenic C. elegans strains 1. Although some reagents (e.g. assay plates) are made the day of experimental transfer, many of the materials must be coordinately prepared over an 8 day period (see Table 1 for a time table). As such, it is important to plan ahead carefully when using this techniq...

<ol>
	<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. (18), e833-e833 (2008).</li><li>Hirose, T., Nakano, Y., Nagamatsu, Y., Misumi, T., Ohta, H., Ohshima, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+GMP-dependent+protein+kinase+EGL-4+controls+body+size+and+lifespan+in+C+elegans.">Cyclic GMP-dependent protein kinase EGL-4 controls body size and lifespan in C elegans.</a> Development. 130, 1089-1099 (2003).</li><li>Kimble, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+cell+lineage+following+laser+ablation+of+cells+in+the+somatic+gonad+of+Caenorhabditis+elegans.">Alterations in cell lineage following laser ablation of cells in the somatic gonad of Caenorhabditis elegans.</a> Dev. Biol. 87, 286-300 (1981).</li><li>Hegreness, M., Shoresh, N., Hartl, D., Kishony, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+equivalence+principle+for+the+incorporation+of+favorable+mutations+in+asexual+populations.">An equivalence principle for the incorporation of favorable mutations in asexual populations.</a> Science. 311, 1615-1617 (2006).</li><li>Labrousse, A., Chauvet, S., Couillault, C., Kurz, C. L., Ewbank, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+is+a+model+host+for+Salmonella+typhimurium.">Caenorhabditis elegans is a model host for Salmonella typhimurium.</a> Curr. Biol. 10, 1543-1545 (2000).</li><li>Kamath, R. S., Martinez-Campos, M., Zipperlen, P., Fraser, A. G., Ahringer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+of+specific+RNA-mediated+interference+through+ingested+double-stranded+RNA+in+Caenorhabditis+elegans.">Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans.</a> Genome. Biol. 2, (2001).</li><li>Min, K., Kang, J., Lee, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+feeding+RNAi+method+for+simultaneous+knock-down+of+more+than+one+gene+in+Caenorhabditis+elegans.">A modified feeding RNAi method for simultaneous knock-down of more than one gene in Caenorhabditis elegans.</a> Biotechniques. 48, 229-232 (2010).</li><li>Zhuang, J. J., Hunter, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specificity+of+Caenorhabditis+elegans+Enhanced+RNAi+Mutants.">Tissue-specificity of Caenorhabditis elegans Enhanced RNAi Mutants.</a> Genetics. 188 (1), 235-237 (2011).</li><li>Way, J. C., Chalfie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mec-3,+a+homeobox-containing+gene+that+specifies+differentiation+of+the+touch+receptor+neurons+in+C.+elegans.">mec-3, a homeobox-containing gene that specifies differentiation of the touch receptor neurons in C. elegans.</a> Cell. 54, 5-16 (1988).</li><li>Kamath, R. S., Fraser, A. G., Dong, Y., Poulin, G., Durbin, R., Gotta, M., Kanapin, A., Bot, N. L. e., Moreno, S., Sohrmann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+functional+analysis+of+the+Caenorhabditis+elegans+genome+using+RNAi.">Systematic functional analysis of the Caenorhabditis elegans genome using RNAi.</a> Nature. 421, 231-237 (2003).</li><li>Winston, W. M., Molodowitch, C., Hunter, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+RNAi+in+C.+elegans+requires+the+putative+transmembrane+protein+SID-1.">Systemic RNAi in C. elegans requires the putative transmembrane protein SID-1.</a> Science. 295, 2456-2459 (2002).</li></ol>

No conflicts of interest declared.

We have presented here a novel method that enables the isolation and characterization of extracellular fluid from the model organism C. elegans. The technique starts with the genetic or physical manipulation of donor worms to increase their total volume of extracellular fluid. Extracellular fluid is then isolated using a modified microinjection technique. The worms are mounted for microinjection using dry agarose pads to hold the worms still during the procedure. The physical attachment to the pad uses the d...

<table border="1">
 <tbody>
 <tr>
 <td>Day</td>
 <td>Worm Prep</td>
 <td>Material Prep</td>
 </tr>
 <tr>
 <td>-7 or prior</td>
 <td>Maintain clean, well fed clr-1(e1745) and N2 worms</td>
 <td>Make injection pads, NGM plates, NGM +Carb/IPTG plates, OP50 LB stock</td>
 </tr>
 <tr>
 <td>-6</td>
 <td></td>
 <td>Streak out RNAi food on LB + Carb</td>
 </tr>
 <tr>
 <td>-5</td>
 <td></td>
 <td>Inoculate 3 mL overnight with RNAi food</td>
 </tr>
 <tr>
 <td>-4</td>
 <td>Move embryos to RNAi plate</td>
 <td>Seed RNAi plate</td>
 </tr>
 <tr>
 <td>-3</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>-2</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>-1</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>0</td>
 <td>Shift worms to 25 for 4 hours</td>
 <td>Make assay plates</td>
 </tr>
 <tr>
 <td></td>
 <td>Move worms to clean NGM plates</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td>Vampiric transfer</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td>Recover recipient</td>
 <td></td>
 </tr>
 <tr>
 <td>+1</td>
 <td>Remove transfer recipient</td>
 <td></td>
 </tr>
 <tr>
 <td>+2</td>
 <td>Score Progeny</td>
 <td></td>
 </tr>
 </tbody>
 </table>
Table 1. Material preparation timeline.
<table border="1">
 <tbody>
 <tr>
 <td>Equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Picoliter Pressure Injector</td>
 <td>Warner Instruments</td>
 
 <td>65-0001 &nbsp;(PLI-100)</td>
 </tr>
 <tr>
 <td>Flaming/Brown micropipette Puller</td>
 <td>Sutter Instruments</td>
 <td>P97</td>
 </tr>
 <tr>
 <td>Axiovert 200</td>
 <td>Zeiss</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td colspan="3">Reagents </td>
 </tr>
 <tr>
 <td>Mineral Oil</td>
 <td>EM Science</td>
 <td>MX1560-1</td>
 </tr>
 <tr>
 <td>22x50 no 1&frac12;&nbsp; Cover Glass</td>
 <td>Corning</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>SeaKem LE Agarose</td>
 <td>Lonza</td>
 <td>50004&nbsp;</td>
 </tr>
 <tr>
 <td>Borosilicate Glass Capillaries</td>
 <td>World Precisions Instruments</td>
 <td>1B100F-4</td>
 </tr>
 <tr>
 <td>Sodium Hypochlorite Solution (5% available Chlorine)</td>
 <td>J.T. Baker</td>
 <td>9416-01</td>
 </tr>
 <tr>
 <td colspan="3">C. elegans and Bacterial Strains </td>
 </tr>
 <tr>
 <td>clr-1(e1745)II&nbsp;9</td>
 <td>The Caenorhabditis Genetics Center (CGC) </td>
 <td>CB3241 </td>
 </tr>
 <tr>
 <td>glp-1 RNAi vector 10 </td>
 <td>Source Bioscience (Ahringer Feeding Library)</td>
 <td>F02A9.6</td>
 </tr>
 <tr>
 <td>pal-1 RNAi vector11 </td>
 <td>&nbsp;</td>
 <td>pHC187</td>
 </tr>
 <tr>
 <td>OP50-GFP&nbsp;5 </td>
 <td>The Caenorhabditis Genetics Center (CGC) </td>
 <td>OP50-GFP </td>
 </tr>
 <tr>
 <td>YFP E. coli 4 </td>
 <td>&nbsp;</td>
 <td>MC4100-YFP</td>
 </tr>
</tbody>
 </table>
 Table 2. Specific reagents and equipment.

vampiric isolation of extracellular fluid from caenorhabditis elegans

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.

Watch this Scientific Journal Video about Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans at JoVE.com

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The model organism C. elegans uses pseudocoelomic fluid as a passive circulatory system. Direct assay of this fluid has not been previously possible. Here we present a novel technique to directly assay the extracellular space, and use systemic silencing signals during an RNAi response as a proof of principle example.

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

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10.1K Görüntüleme.  Harvard University. Model organizma C. elegans Pasif bir dolaşım sistemi olarak pseudocoelomic sıvısı kullanır. Bu sıvı Doğrudan testi daha önce mümkün olmamıştır. Burada doğrudan tahlil ekstrasellüler boşluğa bir roman tekniği sunmak ve ilke örnek bir kanıtı olarak RNAi yanıtı sırasında sistemik susturulması sinyalleri kullanır.