[42 Reporter Transgenes for Study of Oxidant Stress in Caenorhabditis elegans

By Christopher D. Link and Carolyn J. Johnson Introduction

For many studies of the effects of oxidant stress on cells it can be advantageous to visualize the transcriptional response of the cell in vivo in real time. In optically transparent model systems, gene expression can be directly visualized by the construction of reporter transgenes expressing green fluorescent protein (GFP), as originally demonstrated by Chalfie and colleagues.1 We describe both the general considerations involved in the construction of GFP reporter transgenes responsive to oxidative stress and the specific details of constructing a representative transgenic reporter in the model nematode worm Caenorhabditis elegans. Although the details of the representative reporter transgene apply specifically to C. elegans, the general approach should be applicable to many model systems.

Identification of Oxidant Stress-Responsive Genes

Construction of oxidative stress-responsive reporter transgenes first requires identification of oxidative stress-responsive genes. Candidate responsive genes can be identified by extrapolation from studies of other systems [e.g., a C. elegans GFP reporter transgene based on the small C. elegans heat shock protein 16 (HSP16) was found to be responsive to oxidative stress,2 an unsurprising result considering previous studies of mammalian small heat shock proteins3] or from direct gene

1 M. Chalfie, Y. Tu, G. Euskirchen, W. W. Ward, and D. C. Prasher, Science 263, 802 (1994).

2 C. D. Link, J. R. Cypser, C. J. Johnson, and T. E. Johnson, Cell Stress Chaperones 4,235 (1999).

3 X. Preville, H. Shultz, U. Knauf, M. Gaestel, and A. P. Arrigo, J. Cell Biochem. 69,436 (1998).

for the expression range of interest. Thus, the topmost and bottommost entries in this sorted list provide a convenient starting point for evaluation and further experimentation when examined by the critical eye of the researcher.

Acknowledgments

Supported by NIH GM 27345, the Surgery Wound Healing Research Program, and U.S. Surgical, Tyco Healthcare Group. The Laboratory of Molecular Medicine is the research wing of the Center for Minimally Invasive Surgery.

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