Worm Breeder's Gazette 9(2): 102
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.
We have been using a brute force mutant screen to isolate long-lived (age) mutants. These experiments have provided several age mutants to be used in further studies. In a typical hunt DH26, fer-15(b26), is grown at elevated temperatures. This procedure enables us to screen several hundred F2 clones per hunt because elevated temperature prevents eggs from developing and contaminating the survival population. DH26 has survival curves which are quite similar to N2 and is therefore appropriate for use in isolating increased life-span mutants. We mutated DH26 hermaphrodites with EMS and isolated F1 progeny to small spotted NGM plates. F1 progeny were hypochlorited three days later and individual F2 animals were transferred to individual wells of 24-well (COSTAR) microtitre plates and allowed to develop for seven to ten days at 20 C in 0.6 ml Op50 10+E9 cells/ml. Then, a replicate of about 20 L1's were removed from each well and transferred, again in 0.6 ml of Op50 at 10+E9 cells/ml, to microtitre wells of replicate plates and allowed to grow at 25 C. Masters were kept at 16 C and allowed to form dauers. We monitored both control morbidity and culture volume twice a week, keeping the volume and food supply more or less constant (0.6 ml). Age mutants were picked if >10% of the worms in a well were alive after several wells containing control, unmutagenized DH26 hermaphrodites, contained only dead. After identifying possible mutants, worms were transferred from the master wells to NGM plates and a more detailed screen employed. Twenty-five animals were assayed for survival, (scored at least twice a week) and compared with survival of control strains. If survival curves of putative mutants were significantly different from control at the .05 level or less, then the strain passed the second screen. In the final screen, 50 worms were assayed by a standard survival assay. If survival differed significantly from control, we then accepted the strain as a mutant and froze it for further studies. We have isolated four significantly longer lived mutants from three hunts, (see data below). All four were isolated in the fer-15(b26) background. The mean life-span of DH26 is approximately 20 1.7 (S.E. ) days and the mutants are significantly different from the control with mean life spans of 26.3 0.98, 23.7 1.9, 24.9 2.2 and 25.4 1.7. Maximum life spans were 30 days for the control and 41, 33, 41 and 39 days, respectively, for the mutants. We are in the process of detailed screening on several other mutants from two subsequent hunts using TJ1003, zyg-9(b244), as well as DH26.