LARGE-SCALE CULTIVATION OF THE FREE-LIVING NEMATODE CAENORHABDITIS-ELEGANS

We describe conditions for reproducible large scale cultivation of the free living nematode, Caenorhabditis elegans, and downstream processing of large quantities of this tissue. We were able to grow and harvest 500 grams of purified worms from a 150-liter bioreactor. Cultivation on this scale was accomplished in three steps by using worms from 50 agar plates as inoculum for 4 liters of liquid media. Mature worms from the 4-liter culture were transferred to 20 liters of fresh liquid media. After three days of incubation, the 20-liter culture was used for inoculation of the 150-liter tank. At the time of harvest, a mixture of developmental stages of worms was present. Healthy, motile, egg-laden adults worms appeared to make up the major portion of the population. Membranes prepared from sucrose purified worms were biologically active in an ivermectin binding assay. The affinity of ivermectin (K(d)) and the density of receptors (B(max)) were comparable to values generated using membranes from worms grown on agar plates. The amount of membrane tissue obtained from the 500 gm worm harvest made it feasible to consider purification to homogeneity of a nematode protein such as the ivermectin receptor, which is not abundant in crude homogenates. In addition, RNA isolated from sucrose purified worms was translated in Xenopus oocytes. Several channel proteins expressed from this RNA have been characterized, including a chloride channel sensitive to ivermectin and glutamate.