Department of Genetic Medicine and Development, University of Geneva Medical School and University Hospitals of Geneva, Geneva, Switzerland.
PLoS One. 2010 Dec 29;5(12):e15741. doi: 10.1371/journal.pone.0015741.
Finding sequences that control expression of genes is central to understanding genome function. Previous studies have used evolutionary conservation as an indicator of regulatory potential. Here, we present a method for the unbiased in vivo screen of putative enhancers in large DNA regions, using the mouse as a model. We cloned a library of 142 overlapping fragments from a 200 kb-long murine BAC in a lentiviral vector expressing LacZ from a minimal promoter, and used the resulting vectors to infect fertilized murine oocytes. LacZ staining of E11 embryos obtained by first using the vectors in pools and then testing individual candidates led to the identification of 3 enhancers, only one of which shows significant evolutionary conservation. In situ hybridization and 3C/4C experiments suggest that this enhancer, which is active in the neural tube and posterior diencephalon, influences the expression of the Olig1 and/or Olig2 genes. This work provides a new approach for the large-scale in vivo screening of transcriptional regulatory sequences, and further demonstrates that evolutionary conservation alone seems too limiting a criterion for the identification of enhancers.
发现控制基因表达的序列是理解基因组功能的核心。先前的研究已经将进化保守性作为调控潜力的指标。在这里，我们提出了一种在大型 DNA 区域中进行无偏倚体内筛选潜在增强子的方法，以小鼠作为模型。我们从一个 200kb 长的小鼠 BAC 克隆了一个包含 142 个重叠片段的文库，该文库在一个最小启动子的表达 LacZ 的慢病毒载体中，并用得到的载体感染受精的小鼠卵母细胞。首先使用载体进行池化，然后测试单个候选物，对获得的 E11 胚胎进行 LacZ 染色，鉴定出 3 个增强子，其中只有一个表现出显著的进化保守性。原位杂交和 3C/4C 实验表明，这个在神经管和后脑区域活跃的增强子，影响 Olig1 和/或 Olig2 基因的表达。这项工作为大规模的体内转录调控序列筛选提供了一种新方法，并进一步表明，仅通过进化保守性作为鉴定增强子的标准过于局限。
