Department of Biological Sciences and Center for Bioinformatics and Computational Biology, University of Delaware, Newark, DE 19716, USA.
Department of Computational Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
G3 (Bethesda). 2022 May 6;12(5). doi: 10.1093/g3journal/jkac037.
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.
利用 meganuclease I-SceI 进行转基因是一种安全、高效的方法，但由于缺乏转基因定位的信息，其潜在机制仍不清楚。我们之前利用 I-SceI 开发了一种表达 snai2 启动子/增强子驱动的增强型绿色荧光蛋白的转基因非洲爪蟾（Xenopus tropicalis）品系，这是研究神经嵴发育和颅面形态发生的有力工具。在这里，我们对 snai2:eGFP 胚胎进行了全基因组鸟枪法测序，以鉴定转基因的整合位点。在 19 倍测序覆盖度下，我们估计在半合子转基因胚胎中，有 6 个拷贝的转基因插入到了非洲爪蟾基因组中。在染色体 1 的非编码区域鉴定到了两个相邻的转基因整合位点，可能是由于单个转基因插入后发生了复制。有趣的是，转基因整合位点边界处的基因组 DNA 含有与 I-SceI 识别位点同源的短序列，这表明整合不是随机的，而可能是由序列同源性介导的。据我们所知，我们的工作代表了首次利用 I-SceI 对转基因生物进行的全基因组测序研究，这有助于评估 I-SceI 介导的转基因的潜在遗传效应，并进一步了解这种转基因方法的潜在机制。
