Worm Breeder's Gazette 17(1): 36 (October 1, 2001)

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.

A vector for pan-neuronal smg-1-dependent transgene expression.

Chris Link, Gin Fonte, Andrew Taft

IBG, University of Colorado, Boulder CO 80309

We have previously described construction of strains with smg-1-dependent muscle-specific expression of human beta amyloid peptide (Abeta) [see WBG 16(1) p33] using the expression system devised in Andy Fire's lab (see Fire lab '97 vector kit). We have now brewed up a new vector that has allowed us to similarly generate strains with smg-1 dependent pan-neuronal expression of Abeta and other neurodegenerative disease-associated proteins. We amplified and cloned a 3kb promoter fragment of the snb-1 gene into Fire vector pPD118.74, a promoterless GFP construct with an abnormally long (i.e., smg-1 dependent) 3' UTR. (Thanks to Chris Li for info on using this promoter fragment.) The GFP portion was then replaced with a newly designed multiple cloning site cassette containing a small intron (stolen from a previous Fire lab vector-based construct) upstream of the MCS, resulting in pCL35. A GFP coding cassette was re-inserted into this expression vector, and the resulting plasmid was used to transform smg-1(cc546ts) animals. Analysis of an integrated line (CL1234) containing this construct at 16 and 25 degrees C showed strong temperature-dependent induction of neuronal GFP. As previously reported in the original snb-1 paper (Nonet et al, 1998), we also see GFP expression in the spermatheca. As observed for our previous myo-3 driven smg-1 dependent constructs, the temperature induction is not absolute, and weak GFP expression (only visible under a compound epifluorescence scope) can be seen in transgenic animals raised at 16 degrees C.

We inserted an Abeta-coding minigene cassette into pCL35 and established integrated lines in a smg-1(546ts) background. These lines also show temperature background-dependent expression of Abeta. We are particularly happy about this, because we have had surprising difficulty getting lines with good pan-neuronal Abeta expression, and have had a long series of failures with unc-119 promoter-based constructs. We suspect (but have no direct evidence) that inclusion of the 5' UTR intron may have been important for getting good neuronal expression. Although including an intron is apparently not essential for myo-3 based constructs (our smg-1- dependent myo-3 driven lines do not contain an intron), this may be important for neuronal expression or expression with weaker promoters.

Literature cited

Nonet, M. L., Saifee, O., Zhao, H., Rand, J. B., & Wei, L. (1998). Synaptic transmission deficits in Caenorhabditis elegans synaptobrevin mutants. J Neurosci, 18(1), 70-80.