Infrared Laser-Mediated Gene Induction at the Single-Cell Level in the Regenerating Tail of Tadpoles.

We describe a precise and reproducible gene-induction method in the amphibian, Tetrapod amphibians are excellent models for studying the mechanisms of three-dimensional organ regeneration because they have an exceptionally high regenerative ability. However, spatial and temporal manipulation of gene expression has been difficult in amphibians, hindering studies on the molecular mechanisms of organ regeneration. Recently, however, development of a transgenic system with a heat-shock-inducible gene has enabled the manipulation of specific genes. Here, we applied an infrared laser-evoked gene operator (IR-LEGO) system to the regenerating tail of tadpoles. In this method, a local heat shock by laser irradiation induces gene expression at the single-cell level. After amputation, tadpoles regenerate a functional tail, including spinal cord. The regenerating tail is flat and transparent enabling the targeting of individual cells by laser irradiation. In this protocol, a single neural progenitor cell in the spinal cord of the regenerating tail is labeled with heat-shock-inducible green fluorescent protein (GFP). Gene induction at the single-cell level provides a method for rigorous cell-lineage tracing and for analyzing gene function in both cell-autonomous and noncell-autonomous contexts. The method can be modified to study the regeneration of limbs or organs in other amphibians, including , newts, and salamanders.