molecular characterization of a transgenic animal generated using the meganuclease I-SceI

We report, to our knowledge, the first detailed molecular characterization of a transgenic animal generated using the meganuclease I-SceI, which is commonly used for transgenesis. The snai2:eGFP line, developed previously by our group, is a powerful tool to tudy normal/pathological neural crest development and craniofacial morphogenesis. Using whole-genome sequencing, we identified two transgene integration loci adjacent to each other in the X. tropicalis genome, likely as a result of tandem duplication following an initial transgene nsertion. Our analyses further show that the genomic DNA at the integration sites contains short sequences that are homologous to the I-SceI recognition site, including the 3 prime-overhang generated by digestion, suggesting that this transgenesis event is not random but mediated by microhomology. These results provide not only the much needed genetic information on the snai2:eGFP line, but also important insight into the underpinning molecular mechanisms for ISceI-mediated transgenesis.