Methods to study toxic transgenes inC. elegans: an analysis of protease-dead separase in theC. elegansembryo

ABSTRACT We investigated whether the protease activity of separase, which is required for chromosome segregation, is also required for its other roles during anaphase in C. elegans given that non-proteolytic functions of separase have been identified in other organisms. We find that expression of protease-dead separase is dominant-negative in C. elegans embryos. The C. elegans embryo is an ideal system to study developmental processes in a genetically tractable system. However, a major limitation is the lack of an inducible gene expression system for the embryo. The most common method for embryonic expression involves generation of integrated transgenes under the control of the pie-1 promoter, using unc-119 as a selection marker. However expression of dominant-negative proteins kills the strain preventing analysis of mutants. We have developed two methods that allow for the propagation of lines carrying dominant-negative transgenes in order to study protease-dead separase in embryos. The first involves feeding gfp RNAi to eliminate transgene expression and allows propagation of transgenic lines indefinitely. Animals removed from gfp RNAi for several generations recover transgene expression and associated phenotypes. The second involves propagation of the transgene with the female specific pie-1 promoter via the male germline and analysis of phenotypes in embryos from F1 heterozygous hermaphrodites that express the protein. Using these methods, we show that protease-dead separase causes chromosome nondisjunction and cytokinesis failures. These methods are immediately applicable for studies of dominant-negative transgenes and should open new lines of investigation in the C. elegans embryo.