Engineering and analysis of mouse gene clusters by exploiting local hopping characteristic of transposon

大型顺调控景观是脊椎动物基因组的一个显著特征，特别是在具有精细调节表达模式的关键发育基因位点上。现有的基因工具用于测量如此大的基因组区域，范围从几kb到几Mb，是有限的。本文提出了一种利用小鼠基因组中睡美人（SB）转座子的局部跳跃特性的染色体工程策略。我们设计了基于转座子的局部跳跃增强子检测器（LHED）载体，携带增强子检测LacZ报告子和loxP盒式，可以监测本地基因组背景下的转录增强活性和转座子供体和受体位点之间cre介导的缺失/倒置重排。作为原理证明，我们将LHED载体靶向整合到发育和进化关键的Paxl基因位点，然后进行有效的局部转位，巢式缺失形成，并通过四倍体互补快速衍生胚胎，从而产生胚胎干细胞。工程胚胎的全安装报告基因表达分析显示，每个插入/删除配置对应不同的表达模式，并显著促进了该基因组邻域的远程顺式调控元件定位，证明了该实验平台在小鼠基因组非常集中区域的功能探索方面的巨大潜力。

Large cisregulatory landscapes are a remarkable feature of vertebrate genomes, particularly seen at key developmental gene loci with finely tuned expression patterns. Existing genetic tools for surveying such a large genomic region of interest, ranging from a few kb to a few Mb, are limited. Here we propose a chromosomal engineering strategy exploiting the local hopping property of the Sleeping Beauty (SB) transposon in the mouse genome. We devised the transposon-based Local Hopping Enhancer Detector (LHED) vector carrying an enhancer-detection LacZ reporter and loxP cassette, which allows monitoring of transcription-enhancing activities in the native genomic context and Cre-mediated deletion/inversion rearrangement between the transposon donor and acceptor sites.As a proof of principle, we generated embryonic stem (ES) cells with a targeted integration of the LHED vector into the developmentally and evolutionally critical Paxl gene locus, followed by efficient local transposition, nested deletion formation, and rapid derivation of embryos by tetraploid complementation. Whole-mount reporter expression analysis of the engineered embryos showed different expression patterns corresponding to each insertion/deletion configuration and markedly facilitated long-range cis-regulatory element mapping in this genomic neighborhood, demonstrating the great potential of this experimental platform for functional exploration of very focused areas in the mouse genome (manuscript in revision).