Whole-genome sequencing identifies I-SceI-mediated transgene integration sites in Xenopus tropicalis snai2:eGFP line

Author:

Wang Jian12ORCID,Lu Congyu1ORCID,Wei Shuo1ORCID

Affiliation:

1. Department of Biological Sciences and Center for Bioinformatics and Computational Biology, University of Delaware, Newark, DE 19716, USA

2. Department of Computational Biology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA

Abstract

Abstract Transgenesis 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 Xenopus tropicalis genome in the hemizygous transgenic embryos. Two transgene integration loci adjacent to each other were identified in a noncoding 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.

Funder

National Institute of Health

Marshall University Genomics Core Facility

NIH

Publisher

Oxford University Press (OUP)

Subject

Genetics (clinical),Genetics,Molecular Biology

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