Worm Breeder's Gazette 10(3): 27
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.
As described in the last issue of WBG, we are obtaining transformants with a plasmid containing the gene fusion, act-4 5' /lacZ/act-4 3' The aims of these experiments are to use expression of lacZ as a screen for transformed animals, and also to study the regulation of act-4 gene expression. We have used our original transformed line to develop a reliable screen for transformants based on their ability to cleave the chromogenic substrate X-gal. Briefly, the F2 progeny of injected worms are washed off their plates and permeabilized in a solution of 5% SDS. They are then washed and incubated in a staining solution containing X-gal at 25 C for 3-5 hours. Worms containing the exogenous DNA turn strikingly blue while N2 nontransformed worms remain colorless. The hermaphrodites are killed by the procedure but progeny eggs within them remain viable and are recovered. Since developing the screen we have screened the F2 progeny of thirty injected worms. From those injections we have obtained three independent transformed lines. In one transformant the injected DNA has integrated into the genome, mapping to the X chromosome. The other two transformants appear to have extra-chromosomal exogenous DNA based on the pattern of inheritance of lacZ expression in subsequent generations. We have recently modified our original plasmid so that the chimeric gene lies between the inverted repeats of a Tc1 element. No transformed lines have been obtained, as yet, from this construction. The pattern of expression of -galactosidase transformants has been studied histochemically. With the possible exception of the most recent transformant, the pattern of staining seems constant among the transformed lines. When fixed adults are stained with X-gal, strong expression of lacZ appears to be tissue specific, with staining in body wall muscle and particularly vulval muscle and spermathaeca. There is expression in embryonic and larval animals as well, but it is not clear yet whether the adult tissue specificity applies to all developmental stages. We also don't know whether act-4/lacZ is actually expressed in all tissues but is more highly expressed or more easily detected in these tissues.