Wang, JianLu, CongyuWei, Shuo2022-03-302022-03-302022-02-16Jian Wang, Congyu Lu, Shuo Wei, Whole-genome sequencing identifies I-SceI-mediated transgene integration sites in Xenopus tropicalis snai2: eGFP line, G3 Genes | Genomes | Genetics, 2022;, jkac037, https://doi.org/10.1093/g3journal/jkac0372160-1836https://udspace.udel.edu/handle/19716/30738This article has been accepted for publication in G3: Genes | Genomes | Genetics Published by Oxford University Press. The version of record is available at: https://doi.org/10.1093/g3journal/jkac037Transgenesis with the meganuclease I-SceI is a safe and efficient method, but the underlying mechanisms remain unclear due to the lack of information on transgene localization. Using I-SceI, we previously developed a transgenic Xenopus tropicalis line expressing enhanced green fluorescent protein driven by the neural crest-specific snai2 promoter/enhancer, which is a powerful tool for studying neural crest development and craniofacial morphogenesis. Here we carried out whole-genome shotgun sequencing for the snai2: eGFP embryos to identify the transgene integration sites. With a 19x sequencing coverage, we estimated that 6 copies of the transgene were inserted into the X. tropicalis genome in the hemizygous transgenic embryos. Two transgene integration loci adjacent to each other were identified in a non-coding region on Chromosome 1, possibly as a result of duplication after a single transgene insertion. Interestingly, genomic DNA at the boundaries of the transgene integration loci contains short sequences homologous to the I-SceI recognition site, suggesting that the integration was not random but probably mediated by sequence homology. To our knowledge, our work represents the first genome-wide sequencing study on a transgenic organism generated with I-SceI, which is useful for evaluating the potential genetic effects of I-SceI-mediated transgenesis and further understanding the mechanisms underlying this transgenic method.en-USwhole-genome sequencingtransgenesisI-SceIXenopus tropicalissnai2: eGFP lineArticle