Kokubu Chikara, Horie Kyoji, Abe Koichiro, Ikeda Ryuji, Mizuno Sumi, Uno Yoshihiro, Ogiwara Sanae, Ohtsuka Masato, Isotani Ayako, Okabe Masaru, Imai Kenji, Takeda Junji
Center for Advanced Science and Innovation, Osaka University, Suita, Osaka, Japan.
Nat Genet. 2009 Aug;41(8):946-52. doi: 10.1038/ng.397. Epub 2009 Jul 26.
A large cis-regulatory landscape is a common feature of vertebrate genomes, particularly at key developmental gene loci with finely tuned expression patterns. Existing genetic tools for surveying large genomic regions of interest spanning over hundreds of kilobases are limited. Here we propose a chromosomal engineering strategy exploiting the local hopping trait of the Sleeping Beauty transposon in the mouse genome. We generated embryonic stem cells with a targeted integration of the transposon vector, carrying an enhancer-detecting lacZ reporter and loxP cassette, into the developmentally critical Pax1 gene locus, followed by efficient local transpositions, nested deletion formation and derivation of embryos by tetraploid complementation. Comparative reporter expression analysis among different insertion/deletion embryos substantially facilitated long-range cis-regulatory element mapping in the genomic neighborhood and demonstrated the potential of the transposon-based approach as a versatile tool for exploration of defined genomic intervals of functional or clinical relevance, such as disease-associated microdeletions.
大型顺式调控景观是脊椎动物基因组的一个共同特征，尤其是在具有精细调控表达模式的关键发育基因位点。现有的用于检测跨越数百千碱基的大型感兴趣基因组区域的遗传工具有限。在这里，我们提出了一种染色体工程策略，利用小鼠基因组中睡美人转座子的局部跳跃特性。我们生成了胚胎干细胞，将携带增强子检测lacZ报告基因和loxP盒的转座子载体靶向整合到发育关键的Pax1基因位点，随后进行高效的局部转座、嵌套缺失形成，并通过四倍体互补获得胚胎。对不同插入/缺失胚胎之间的报告基因表达进行比较分析，极大地促进了基因组邻域中远距离顺式调控元件的定位，并证明了基于转座子的方法作为探索具有功能或临床相关性的特定基因组区间（如疾病相关微缺失）的通用工具的潜力。
