CONSTRUCTION AND EVALUATION OF A TRANSGENIC hsp16-GFP-lacZ CAENORHABDITIS ELEGANS STRAIN FOR ENVIRONMENTAL MONITORING

Abstract

A novel integrated transgenic Caenorhabditis elegans strain (PC161) incorporates a double reporter construct with green fluorescent protein (GFP) and lacZ genes fused in-frame into the second exon of the hsp16-1 gene. This construct also includes the Simian Virus 40 (SV40) nuclear localization signal such that the fusion protein accumulates in the nuclei of expressing cells. The PC161 strain was used to monitor the effects of several known stressors, including heat, cadmium, and microwave radiation. The time course of induction was similar for both reporters but was strongly influenced by pretreatment conditions. The PC161 worms kept at 15 degrees C beforehand showed a steady increase in reporter expression (up to at least 16 h) when heated to 30 degrees C. However, if washed on ice prior to heat stress at 30 degrees C, PC161 worms showed a much steeper rise in reporter expression, reaching a maximum after 2.5 h and then plateauing. Heat shock induced strong expression of both reporter genes in all tissues apart from the germ line and early embryos. A highly significant linear dose-response relationship was observed for both transgenes with increasing cadmium concentrations (5-100 microg/ml). Prolonged exposure to microwave radiation (750 MHz and 0.5 W for 16 h) also induced expression of both transgenes at 25 and (to some extent) 27 degrees C, but only beta-galactosidase activity was detectable at 23 degrees C, and neither reporter was detectably expressed at 21 degrees C. Throughout all exposures, the lacZ reporter product was more readily detectable than coexpressed GFP. However, the GFP reporter affords opportunities to monitor the stress response in living worms.

Low‐intensity microwave irradiation does not substantially alter gene expression in late larval and adult Caenorhabditis elegans

“…Similar experiments on strain PC161 (an hsp-16.1::GFP:lacZ double reporter strain) [David et al, 2003] revealed a similar but smaller up-regulation after 6 h at 30ºC (data not shown).…”

Continuous wave and simulated GSM exposure at 1.8 W/kg and 1.8 GHz do not induce hsp16‐1 heat‐shock gene expression in Caenorhabditis elegans

“…This underlines the wide applicability of transgenic strains carrying hsp16::reporter genes [e.g. PC161, David et al, 2003;CL2070, Link et al, 1999PC72, Stringham et al, 1994] as general biomarkers of stress, since the nematode HSP16 proteins provide a first line of defence against multiple stresses. These roles may not necessarily be identical for orthologous small hsp's in vertebrates.…”

A small temperature rise may contribute towards the apparent induction by microwaves of heat-shock gene expression in the nematodeCaenorhabditis Elegans

“…Recent data indicates that fluorescence signals from GFP reporters (e.g. in the hsp16-1::GFP::lacZ double-reporter strain PC161; David et al, 2003) can be measured from as few as 100 worms per well in a 96-well microplate format suitable for high-throughput screening. These features allow extensive replication under near-identical test conditions, which should allow even small responses to be picked up unambiguously.…”

“…Other differences between shielded and exposed conditions may contribute to this contrast. (David et al, 2003). All chemicals were Ultrapure grade from Sigma, and plastic disposables were from Nunc Ltd. unless otherwise stated.…”