Whitehead Institute, 455 Main Street, Cambridge, MA 02142.
Whitehead Institute, 455 Main Street, Cambridge, MA 02142
G3 (Bethesda). 2019 May 7;9(5):1481-1486. doi: 10.1534/g3.119.300582.
The introduction of foreign DNA into cells and organisms has facilitated much of modern biological research, and it promises to become equally important in clinical practice. Locating sites of foreign DNA incorporation in mammalian genomes has proven burdensome, so the genomic location of most transgenes remains unknown. To address this challenge, we applied nanopore sequencing in search of the site of integration of (also known as ), a widely used fluorescent reporter in mouse germ line research. Using this nanopore-based approach, we identified the site of transgene integration near the telomere of Chromosome 9. This methodology simultaneously yielded an estimate of transgene copy number, provided direct evidence of transgene inversions, revealed contaminating genomic DNA within the transgene array, validated the integrity of neighboring genes, and enabled definitive genotyping. We suggest that such an approach provides a rapid, cost-effective method for identifying and analyzing transgene integration sites.
将外源 DNA 导入细胞和生物体极大地促进了现代生物学研究，而且它有望在临床实践中同样重要。在哺乳动物基因组中定位外源 DNA 整合的位置已被证明是繁琐的，因此大多数转基因的基因组位置仍然未知。为了解决这一挑战，我们应用纳米孔测序来寻找整合的位点（也称为 ），这是一种在小鼠生殖系研究中广泛使用的荧光报告基因。使用这种基于纳米孔的方法，我们确定了 转基因在染色体 9 端粒附近的整合位点。这种方法同时提供了转基因拷贝数的估计值，提供了转基因反转的直接证据，揭示了转基因阵列内的污染 基因组 DNA，验证了邻近基因的完整性，并能够进行明确的基因分型。我们建议，这种方法为鉴定和分析转基因整合位点提供了一种快速、具有成本效益的方法。
