Worm Breeder's Gazette 5(2): 44
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
I have developed the following procedure for introducing macromolecules into living worms with the kind assistance of Doug Melton. Immobilization. Adult worms are placed in a solution containing.1% Tricaine and.01% tetramisole for 30 min to 1 hr st room temperature. This treatment causes the worms to lie completely still for at least an hour after removal from the anaesthetic. Survival is 100%. Mounting procedure. To support the worms during injection, I line the worms up along the fire-polished edge of a coverslip attached to a slide by a thin film of vacuum grease. (Ed Hedgecock has devised a different mounting procedure. He applies fingernail polish to the edge of a coverslip on a slide, lets it set, and then breaks off the coverslip. This leaves a neat edge of hardened fingernail polish along which the worms can be arranged.) Injection of the worms is done under 3S Voltalef oil. To transfer from anaesthetic to oil, the worms are pipetted onto a 1mM tetramisole plate and excess fluid is drawn off with a piece of filter paper. It is important to draw off all extra fluid, because the worms can roll out from under the micropipette if they are covered by a thin film of liquid. I manipulate the worms, once transferred by toothpick to the slide, by using a fire polished micropipette tip. Micropipettes. Micropipettes are pulled using an electrical pipette puller (Shinohara). I use a borosilicate capillary tubing with an outside diameter of 1.2 mm and an inside diameter of 0.69 mm. The capillary tubing is pulled so that the shape of the pipette is approximately: [See Figure 1]. The freshly pulled pipette tips are then etched with 10% hydrofluoric acid (nasty stuff so be careful) and rinsed with absolute ethanol. To do this, the pipette is joined to a syringe by a teflon Biolab connector, and fluid is sucked into the pipette using the syringe. I pull the HF a short way into the tip 3 times fairly quickly and then rinse in ethanol. This gives a final tip diameter of 4-8 microns. The size of the tip can be estimated with little experience by the size of air bubbles that come out when rinsed in ethanol. Just before injection, the tip is cleaned with chromic acid and rinsed in injection buffer. If the tip clogs before you break it, clean it again with chromic acid. Injection apparatus. Liquid is drawn into and expelled from the pipette using a standard pressure injection system. Paraffin oil is used to fill a micrometer controlled syringe, connecting tubing, and the upper part of the micropipette. Teflon Biolab connectors are used to join these pieces together. The micropipette is held in a Leitz micromanipulator and injection is done while observing the worm through a Wild dissecting microscope at 50X. Injection results so far. 50-90% of the worms survive and produce progeny when injected with 50-100 pl of a non-toxic substance. Such substances include phosphate buffer (Graesmann and Graesmann, 1976), Tris buffer(Laskey and Gurdon, 1973), 32P-phosphate, 32P-alpha-dATP, and DNA at about 0.5 mg/ml. Injection of fluorescein and fluorescein- conjugated BSA kills the animal, but was used to demonstrate penetration in initial experiments. Injection of the fluoresceinated BSA showed that injection into the gonad is possible and that the injected molecules diffuse rapidly (<5 min) throughout the space injected (pseudocoelom, lumen of the somatic gonad, or the syncytial germ line tissue). Injection of the 32 P-phosphate and 32P-dATP was used to follow incorporation of injected molecules into progeny of the injected adult. Radioactively labelled animals were detected by autoradiography of whole worms dried down directly on their agar. In this way, it was shown that progeny were radioactively labelled by injection into either the pseudocoelom or the gonads of the parent. I am currently using microinjection to inject genomic clones of worm DNA that probably cover the unc-54 locus into unc-54 mutants in an attempt to achieve transformation. No positives yet.