The coupling of transgenes to heat shock promoters is a common method for regulating C. elegans gene expression. Gene induction can be performed in a temporally defined manner, through timing of heat shock, and in a tissue-specific manner, via targeted rescue in hsf-1 mutants. However, cell-specific targeting methods are limited by the availability of cell-specific promoters. Here we present a method for evoking gene expression in single cells by local activation of heat shock. Our technique builds on the IR-LEGO method by Kamei et al (Nat. Meth. 2009) in which a continuous-wave, focused infrared laser beam is used to locally induce a heat shock response. However, continuous irradiation generates significant heating away from the laser focus due to the diffusion of heat, which increases the total heated volume. We have developed an improved method using a pulsed infrared laser that minimizes off-target heating by allowing time for thermal energy to dissipate between laser pulses. The resultant total heated volume is ~32 times smaller than that of IR-LEGO. Using a Phsp-16.2::GFP transgene as a reporter of heat shock we have optimized parameters to achieve a single cell gene induction rate of over 75% in seam cells (n=30), almost doubling the previously reported efficiency. To test the spatial resolution of our method, we successfully used it to induce GFP expression in the nerve ring neuron ADL in L2 larvae, and we observed no off-target induction despite the presence of neighboring neurons within 5 microns. To explore applications in lineage tracing, we used our method to individually heat shock each cell of the four-cell embryo. Resulting GFP expression patterns at bean stage were consistent with the expected positions of the descendants of the heat-shocked cell. In addition to direct transgene expression through heat shock, we also used an Phsp-16.2::FLP construct in combination with an FRT-flanked expression cassette to induce permanent GFP expression in single cells. Our laser-induced heat shock method is modest in cost and similar in technical complexity to widely used laser ablation systems. We therefore expect that our technique will be a broadly useful tool for C. elegans research.

Affiliation:
- University of Pennsylvania, Philadelphia, PA.