Worm Breeder's Gazette 17(1): 31 (October 1, 2001)
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.
|1||Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville, MD|
|2||Dept. of Molecular and Cellular Biology, University of Arizona, Tucson, AZ|
One of the limitations of C. elegans transgene studies has been the difficulty in obtaining stable (i.e., integrated), low copy number lines. Praitis et al. (Genetics 2001, 157:1217-26) have adapted biolistic transformation (bombardment with DNA-coated gold microparticles) to worms for this purpose by selecting for rescue of unc-119(ed3) lethality on starved plates. The identification of selectable markers in addition to unc-119(ed3)would facilitate the construction of lines with multiple integrated transgenes. In a pilot experiment, we attempted to rescue the temperature-sensitive, sperm-specific sterility (Spe) of the spe-26(hc138ts) mutation via biolistic transformation. We used the spe-26 plasmid pJV145, which, by microinjection, complements the Spe phenotype. Synchronized populations of spe-26(hc138ts) were grown at 15 degrees until young adulthood, subjected to bombardment, allowed to recover for two hours, transferred to new plates, then shifted to 25 degrees. Otherwise, conditions for bombardment were identical to the published protocol. The large number of worms quickly depleted the available OP50, so we added an OP50 "soup" (1ml of a centrifuged culture containing ~50% bacteria by volume) every day or two as needed to prevent dauer arrest. F1 progeny of the bombarded worms should be sterile unless rescued. We obtained four independent fertile lines from 20 bombardments. PCR screening of single fertile worms with plasmid-specific primers confirmed its presence in all four lines. In three of the lines, the high frequency of sterility in subsequent generations suggested extrachromosomal maintenance; those lines were not characterized further. The fourth line exhibited stable maintenance of fertility, and Southern blot analysis confirmed the integration of a single copy of the transgene. Current efforts include testing of a different spe-26 allele (it112ts) and different bombardment parameters to increase the efficiency of integrated rescue.