Worm Breeder's Gazette 16(1): 33 (October 1, 1999)
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.
IBG, University of Colorado, Boulder, CO 80309
We have used the smg-1-inducible transgene system developed in Andy Fire!s lab [see WBG 14(5):26] to engineer transgenic worms that inducibly express the human beta amyloid peptide. We constructed a beta amyloid transgene (pAF30) using Fire lab vector L3809 (myo-3/long 3 prime UTR) and introduced it (along with the dominant rol-6 marker plasmid pRF4) into a smg-1(cc546 ts) strain. We ultimately recovered four independently integrated lines and characterized them at 16 and 23 degrees C. All of the lines showed complete paralysis/arrest at the non-permissive temperature (23 degrees), although they varied in their growth rate at 16 degrees. This phenotype is more extreme than that exhibited by any of the unc-54/beta peptide transgenic animals we have previously been able to recover. We have focused on one line (CL4176) that appeared healthiest at the permissive temperature. Removing smg-1 from this strain completely blocks the temperature sensitive phenotype, while re-introducing the smg-1 restores it, demonstrating that the temperature sensitivity of this strain is smg-1 dependent. We have used immunohistochemistry to characterize beta peptide expression in CL4176 animals grown at 16 degrees or upshifted at L2 to 23 degrees for 48 hrs. Expression is undetectable or weak in most animals grown at 16 degrees, typically with a mosaic pattern of expression. Upshifted animals show strong expression in all body wall muscle cells, comparable to that seen in. our original unc-54/beta peptide constructs (see Fay et al., J. Neurochem. 71:1616, 1998). Onset of paralysis occurs approximately 27 hours post-upshift; these animals may progress to adulthood, but do not produce eggs that hatch. We have not seen recovery of any animals if they are down-shifted back to 16 degrees. The take-home messages are: 1) The smg-1 system can be an effective method to express toxic transgenes, 2) It may be tricky to recover transgenic animals that show significant temperature induction but a complete absence of expression at the permissive temperature, and 3) The inducible strains we have been generated may be very useful in suppressor screens and timecourse analysis of beta amyloid-dependent gene expression.