Transgenesis in Hydra to characterize gene function and visualize cell behavior

The green fluorescent protein (GFP) is a widely used reporter in gene expression and protein localization studies. GFP is a stable protein; this property allows its accumulation and easy detection in cells. However, this stability also limits its application in studies that require rapid reporter turnover. We created a destabilized GFP for use in such studies by fusing amino acids 422-461 of the degradation domain of mouse ornithine decarboxylase (MODC) to the C-terminal end of an enhanced variant of GFP (EGFP). The fusion protein, unlike EGFP, was unstable in the presence of cycloheximide and had a fluorescence half-life of 2 h. Western blot analysis indicated that the fluorescence decay of EGFP-MODC-(422-461) was correlated with degradation of the fusion protein. We mutated key amino acids in the PEST sequence of EGFP-MODC-(422-461) and identified several mutants with variable half-lives. The suitability of destabilized EGFP as a transcription reporter was tested by linking it to NFkappaB binding sequences and monitoring tumor necrosis factor alpha-mediated NFkappaB activation. We obtained time course induction and dose response kinetics similar to secreted alkaline phosphatase obtained in transfected cells. This result did not occur when unmodified EGFP was used as the reporter. Because of its autofluorescence, destabilized EGFP can be used to directly correlate gene induction with biochemical change, such as NFkappaB translocation to the nucleus.

The ability to visualize, track, and quantify molecules and events in living cells with high spatial and temporal resolution is essential for understanding biological systems. Only recently has it become feasible to carry out these tasks due to the advent of fluorescent protein technology. Here, we trace the development of highly visible and minimally perturbing fluorescent proteins that, together with updated fluorescent imaging techniques, are providing unprecedented insights into the movement of proteins and their interactions with cellular components in living cells.

Understanding the evolution of development in large part relies on the study of phylogenetically old organisms. Cnidarians, such as Hydra, have become attractive model organisms for these studies. However, despite long-term efforts, stably transgenic animals could not be generated, severely limiting the functional analysis of genes. Here we report the efficient generation of transgenic Hydra lines by embryo microinjection. One of these transgenic lines expressing EGFP revealed remarkably high motility of individual endodermal epithelial cells during morphogenesis. We expect that transgenic Hydra will become important tools to dissect the molecular mechanisms of development at the base of the Metazoan tree.