Using C31 Integrase to Mediate Insertion of DNA in Xenopus Embryos

The two most common methods used to generate transgenic Xenopus embryos, restriction enzyme-mediated insertion, and I-SceI meganuclease take advantage of relatively common but spatially unpredictable double-stranded breaks in sperm, egg, or early embryo genomes. These methods also tend to insert multimeric copies of the transgene. An alternative is to use bacteriophage- or transposon-derived integrase or recombinase to mediate more site-specific insertion of the transgene. The use of phiC31 integrase requires a defined sequence for insertion and is compatible with insertion of a single copy of the transgene. We describe the protocol we use to facilitate phiC31 integrase transgene insertion including the use of insulator sequences to reduce position effect disruption of transgene activity.