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.

Inducible expression of human beta amyloid using the smg-1(ts) system.

Chris Link, Amy Fluet, Carolyn Johnson

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

  The take-home messages are:
1)  The smg-1 system can be an effective method to express toxic
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.