Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

Summary

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.

Abstract

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.

Protocol

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 technique (for necessary reagents and equipment see Table 2).

Injection pads:

1. Make a 2% agarose so....

Discussion

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.......