Microirradiation for Precise, Double-strand Break Induction in vivo in Caenorhabditis elegans .

DNA double-strand breaks (DSBs) are toxic lesions that every cell must accurately repair in order to survive. The repair of DSBs is an integral part of a cell life cycle and can lead to lethality if repaired incorrectly. Laser microirradiation is an established technique which has been used in yeast, mammalian cell culture, and Drosophila cell culture to study the regulation of DSB repair. Up to our studies, this method has not been adapted for use in a whole, live, multicellular organism to study this repair in vivo. We have recently shown that this system can be used for study of the recruitment of vital repair proteins to microirradiation-induced breaks in the transparent nematode Caenorhabditis elegans. With the integration of microirradiation and imaging technology, we can precisely induce DSBs in target nuclei and study the recruitment of fluorescently tagged repair proteins from the time of damage induction. Whole, live worms are plated and immobilized for targeting of nuclei, and immediately following induction the targeted region can be imaged for up to an hour and a half post-microirradiation. This method is the first that allows for study of DNA repair protein kinetics in vivo in an intact organism, which can be adapted in numerous ways to allow for study of repair kinetics in various aspects of the repair process.