Worm Breeder's Gazette 9(1): 85
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.
Amber alleles of the gene tra-3 provide a sensitive assay for amber suppression; without suppression, tra-3 animals are intersexual and sterile above 20 C. Expression of suppressor tRNA either in the mother or zygote results in animals that are phenotypically 'rescued' and hence fertile at 20 C. The initial goal was to assay for transient expression of DNA following microinjection. Kumble, et. al., (Nature 299, 456) had shown that injection of a tRNA fraction from the sup-7 strain into the distal gonad arm of tra-3 animals rescued some of the resulting progeny. I obtained similar results upon injection of the sup-7 gene contained in a supercoiled plasmid. The observed rescue resulted almost entirely from transient expression since none of the rescued progeny expressed sup-7 in their own gonads. In optimizing transient expression, a strong dependence on the site of injection was observed. The most efficient transient expression was obtained following injection near the rapidly dividing nuclei around the distal tip. Some transient expression was also obtained following injection near the bend in the gonad arm and into oocytes. Surprisingly, injection into the center bulge of the distal arm, which until now has been standard for most studies, yielded little or no expression. Transient expression should provide a tool useful in analyzing expression of genes active during oogenesis and early embryogenesis. For most applications, however, one needs transgenic lines expressing a DNA segment of interest. Hence a large number of tra-3 animals were injected with sup-7 containing DNA constructs and transformed progeny selected by continued growth at 20 C. To date, ten independent transgenic lines have been established; each has continued to breed since establishment (ten to forty generations). The nature of injections seem to be critical for stable transformation; the most successful sets of injections have been those in which each worm was injected multiple times (six to ten) aiming for nuclei around the turn in the gonad. Although the first transformants were from injections of sup-7 cloned inside the ends of the transposable element Tc1, it has subsequently proved unnecessary to have any Tc1 sequences in cis or trans. Indeed the highest frequencies so far have been observed after injection of a 4kb sup-7 plasmid with no transposon elements; three transgenic lines were derived from 169 injected parents. At present, doing multiple injections near the gonadal turn, I can inject and recover about 80 animals/day. Including time to follow the injected animals and maintain reagents and stocks, about two and one- half days of full-time work are needed per transformed line. The existing transformants are being characterized both genetically and by Southern blots. Homozygous strains have been derived for five of the ten transformants. In four cases the suppressor locus has been mapped to a chromosome by standard genetics; one suppressor maps on chromosome I, one on chromosome III and two on chromosome II. Of the transformants so far analyzed using Southern blots, each appears to contain several copies of the injected plasmid; these copies appear to have been linearized at different places and joined together. Although some rearrangements are observed, it is encouraging that large (4-7 Kb) fragments from the initial plasmids have apparently been preserved without internal rearrangement. In making some of the transformed strains, the sup-7 containing plasmid had been co-injected with a second, unselected plasmid. In the two such strains so far analyzed, sequences from the second plasmid are also present in the transformed line. This may be useful in co-introducing various genes of interest.