Worm Breeder's Gazette 15(1): 33 (October 1, 1997)
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.
UT Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75235-9148 e-mail: richard@eatworms.swmed.edu
To express a transgene only in cells of interest at the time of
interest, we add a smg-sensitive (RNA destabilizing) extension to a
specific construct and transform that into a temperature-sensitive smg
mutant. At 15 degrees C, smg-1(cc546) is active and degrades smg-
sensitive transcripts (aberrant transcripts containing premature stop
codons or long 3' UTRs), limiting translation of these transcripts. At
25 C, smg-1(cc546) is inactive, allowing accumulation and translation
of these transcripts. A cell-specific promoter limits expression of the
construct to the tissue of interest. (see also Getz et al., WBG 14#5,
p26, and Zwaal et al., IWM97, p128.)
We have tested this method by expressing GFP under two
different promoters. The first, from myo-2, is a strong pharyngeal
muscle-specific promoter. Upon injection at a concentration of 4 ng/ul,
worms show low but detectable fluorescence when grown at 15 C, and an
estimated 8 to 10-fold increase in fluorescence at 25 C. With higher
concentrations the difference is less obvious (at least by eye) because
of bright staining even at 15 C. With lower concentrations not all the
transgenic lines contained the GFP construct (probably the construct
just wasn't incorporated by chance, because of the low copy number).
The smg-tagged GFP was also expressed under the mek-1 promoter, which
drives expression predominantly in the gut, the pharyngeal muscles and
some as yet unidentified neurons. At 25 ng/ul, there was faint
fluorescence at both 15 C and 25 C. At 75 ng/ul, there was faint
fluorescence at 15 C (although 3 or 4 neurons were consistently
visible), but very bright fluorescence in the proper tissues at 25 C.
Together these results show that this method can be used for stage- and
tissue-specific expression of transgenes engineered to be
smg-sensitive, but that one needs to fiddle around a bit to find
the optimal concentra tion for injection.
Visualization of GFP requires high levels of expression,
whereas to study biologically active proteins (e.g. those involved in
signal transduction) one might need much lower expression. Indeed,
injection of a smg-tagged, dominant active form of mek-1 under the
myo-2 promoter gave fewer transgenic F1s then expected, and transgenics
were very starved and grew very slow (in concordance with a role for
mek-1 in pharyngeal pumping, see Koga et al., WBG 14#2, p72). No lines
containing the transgene could be obtained (presumably due to selection
against presence of the transgene). One line was obtained with
smg-tagged, dominant active mek-1 under its own promoter, injected
at 4 ng/ul (20-fold lower than the GFP construct). This transgene was
subsequently integrated into the genome. Worms are healthy at 15 C,
but die as embryos or early larvae at 25 C. At 20 C, most of the
worms are fairly healthy, but at room temperature (a nice 23C down
here in Texas), the worms are very sick and there is a high degree of
larval lethality. This shows that transgenes with severe effects on the
animal's general well-being can be used, and that intermediate
temperatures can be helpful in obtaining intermediate phenotypes.