大范围核酸酶I-SceI转基因是一种安全有效的方法,但由于缺乏有关转基因定位的信息,潜在的机制仍不清楚.使用I-SceI,我们先前开发了一种转基因非洲爪鱼品系,表达由神经c特异性snai2启动子/增强子驱动的增强的绿色荧光蛋白,这是研究神经c发育和颅面形态发生的有力工具。这里,我们对snai2:eGFP胚胎进行了全基因组鸟枪测序,以鉴定转基因整合位点.有19倍的测序覆盖率,我们估计,在半合子转基因胚胎中,有6个转基因拷贝被插入到热带非洲爪鱼基因组中.在1号染色体的非编码区中鉴定出两个彼此相邻的转基因整合基因座,这可能是单个转基因插入后重复的结果。有趣的是,转基因整合基因座边界处的基因组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,而是可能由序列同源性介导的。据我们所知,我们的工作代表了用I-SceI产生的转基因生物的第一个全基因组测序研究,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。
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.