A transposon-based chromosomal engineering method to survey a large cis-regulatory landscape in mice
A transposon-based chromosomal engineering method to survey a large cis-regulatory landscape in mice
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DOI：
10.1038/ng.397
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发表时间：
2009-08-01
期刊：
影响因子：
30.8
通讯作者：
Takeda, Junji
中科院分区：
文献类型：
--
作者：
Kokubu, Chikara;Horie, Kyoji;Takeda, Junji
大的顺式调控格局是脊椎动物基因组的共同特征，特别是在具有精细调控表达模式的关键发育基因座上。现有的基因工具用于调查跨越数百个碱基的大片感兴趣的基因组区域是有限的。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。我们通过定向整合转座子载体，将携带增强子检测LacZ报告基因和loxP盒的转座子载体整合到发育关键的Pax1基因座上，然后进行有效的局部转座、嵌套缺失形成和四倍体互补获得胚胎。不同插入/缺失胚胎之间的比较报告表达分析大大促进了基因组邻近区域的长距离顺式调控元件定位，并证明了基于转座子的方法作为一种通用工具的潜力，用于探索确定的具有功能或临床相关性的基因组间隔，例如疾病相关的微缺失。
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.