Generation of Xenopus Transgenic Lines Using I-SceI

Transgenic Xenopus lines can be generated with high efficiency using the I-SceI meganuclease, an enzyme that recognizes an 18-base pair DNA sequence. To enable this, the desired transgene must be flanked by one or two I-SceI recognition sites either in the plasmid or a PCR product. Following a brief in vitro digestion with I-SceI, the entire reaction mixture is microinjected into fertilized eggs. Within the embryo, the enzyme facilitates genomic integration of the transgene through a mechanism that remains unknown. Embryonic development after injections generally proceeds normally and transgene integration occurs in up to 70% of embryos.