Laboratory of Animal Models for Human Diseases, National Institutes of Biomedical Innovation, Health and Nutrition,7-6-8 Saito-Asagi, Ibaraki, Ibaraki, Osaka 568-0085, Japan.
Exp Anim. 2020 Aug 5;69(3):279-286. doi: 10.1538/expanim.19-0118. Epub 2020 Feb 11.
Transgene insertion patterns are critical for the analysis of transgenic animals because the influence of transgenes may change depending on the insertion pattern (such as copy numbers and orientations of concatenations) and the insertion position in the genome. We previously reported a genomic walking strategy to locate transgenes in the genomes of transgenic mice (Exp. Anim. 53: 103-111, 2004) and to analyze transgene insertion patterns (Exp. Anim. 55: 65-69, 2006). With such strategies, however, we could not determine the copy number of transgenes or global genome modification induced by transgene insertion due to read-length limitation. In this study, we used a long-read sequencer (MinION, Oxford Nanopore Technologies) to overcome this limitation. We obtained 922,210 reads using MinION with genomic DNA from a transgenic mouse strain (4C30, Proc. Jpn. Acad. Ser. B. Phys. Biol. Sci. 87: 550-562, 2011). Among the reads, we found one 21,457-bp read containing the transgene using a local BLAST search. Nucleotide dot plot analysis revealed that the transgene was inserted in the genome as a tandem concatemer with an almost entire construct (15-3,508 of 3,508 bp) and a partial fragment (4-660, 657 bp). Ensembl's BLAST search against the C57BL/6N genome revealed a 9,388-bp deletion at the insertion position in the intron of the Sgcd gene, confirming that mutations such as a large genomic deletion could occur at the time of transgene insertion. Thus, long-read sequencers are useful tools for the analysis of transgene insertion patterns.
转基因插入模式对于转基因动物的分析至关重要，因为转基因的影响可能会因插入模式（如串联的拷贝数和方向）和在基因组中的插入位置而改变。我们之前报道了一种基因组步行策略，用于定位转基因小鼠基因组中的转基因（Exp. Anim. 53: 103-111, 2004）并分析转基因插入模式（Exp. Anim. 55: 65-69, 2006）。然而，由于读取长度的限制，我们无法确定转基因的拷贝数或转基因插入引起的全基因组修饰。在这项研究中，我们使用长读测序仪（MinION，Oxford Nanopore Technologies）来克服这一限制。我们使用 MinION 从转基因小鼠品系（4C30，Proc. Jpn. Acad. Ser. B. Phys. Biol. Sci. 87: 550-562, 2011）的基因组 DNA 获得了 922,210 个读取。在这些读取中，我们使用局部 BLAST 搜索找到了一个包含转基因的 21,457-bp 读取。核苷酸点图分析表明，转基因作为串联串联体插入基因组中，具有几乎完整的构建体（15-3,508 个碱基对，3,508 个碱基对）和一个部分片段（4-660 个碱基对，657 个碱基对）。Ensembl 对 C57BL/6N 基因组的 BLAST 搜索揭示了插入位置处 Sgcd 基因内含子中的 9,388-bp 缺失，证实了在转基因插入时可能发生诸如大片段基因组缺失等突变。因此，长读测序仪是分析转基因插入模式的有用工具。
