Worm Breeder's Gazette 12(2): 39 (January 1, 1992)

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.

Integrated Transgenic Lines Rescue Low Fertility in Age Strains

Pat Tedesco, Tom Johnson

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Institute for Behavioral Genetics, Box 447, University of Colorado, Boulder, CO 80309

Our main focus is on the positional cloning of age-1 .We based our initial cloning strategy on the cosegregation of Age and a reduced fertility (Fer) phenotype in backcrosses of age-1 to N2 ,making the assumption that the same mutational event gave rise to both traits. This assumption simplified the cloning because it is much easier to identify cosmids complementing the Fer trait than the Age trait. We showed that the fer-15 region was responsible for the reduced fertility by deficiency mapping and presented the corresponding physical map (WBG 11.4:14,15). Cosmid T13F9 detected end points of deficiencies immediately to the left and right of fer-15 ;thus T13F9 contains fer-15 ,while age-1 maps closer to unc-4 (WBG 11.4:74).

As a final proof that the Age phenotype is separable from Fer we made a series of transgenic animals using the rol-6 coinjection strategy; we also included an actin-4 plasmid ( pA4 ßGal,Jocelyn Shaw) that was reported to increase the frequency of line formation (Mello, WBG 11.3:12). The injections were successful when we coinjected T13F9 and we saw complementation for fer-15 ( b26 )at 25°C, although the level of complementation was low. At 20°C little effect on fertility was observed (Table 1). We isolated two independent integrated lines starting with one of the cosmid/ Rol-6 arrays and have found that one of these lines complements fertility quite well at 20°C and at 25°C. The other integrated line fails to complement at 20°C and has lower levels of complementation at 25°C. We were not able to get complementation using C26D10 which overlaps extensively with T13F9 and which was reported by Brister and L'Hernault (WBG 11.4:21) to contain fer-15 .

These results show that integrated arrays work better for complementation than do extrachromosomal arrays. The quantitative approach we've employed is useful because, unlike qualitative analyses, quantitative analysis can provide better insight into the the level of function of integrated arrays positioned in different chromosomal milieus. We are planning life span assays of these integrated transgenics and hope that they will confirm the fact that the Age and Fer traits are genetically separable.

[See Figure 1]

Figure 1