Bacterial artificial chromosomes as analytical basis for gene transcriptional machineries.
Bacterial artificial chromosomes as analytical basis for gene transcriptional machineries.
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DOI：
10.1007/s11248-010-9469-3
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发表时间：
2011-08
影响因子：
3
通讯作者：
Inoue T
中科院分区：
文献类型：
--
作者：
Asami J;Inoue YU;Terakawa YW;Egusa SF;Inoue T
细菌人工染色体(BAC)一直是各种基因组测序项目的最小组成部分，构成了功能基因组学的完美分析基础。在这里，我们描述了一种增强子筛选策略，即通过转座子标签将覆盖任何感兴趣基因组片段的BAC克隆修饰为携带报告盒，然后通过同源重组介导的系统删除来处理以携带选定的基因调控模块组合。这种细菌细胞中的工程化BAC报告结构已经准备好在小鼠身上有效地转基因，以评估构建中完整或缺失的基因调控模块的活性。通过利用该策略，我们可以快速地确定一个关键的基因组片段，用于时空调控小鼠钙粘附素基因的表达，该基因的结构非常巨大和复杂。因此，这种基于BAC的方法将为在发育、发病和/或进化过程中动态波动的基因转录机制提供一个新的筛选平台。
Bacterial Artificial Chromosomes (BACs) had been minimal components of various genome-sequencing projects, constituting perfect analytical basis for functional genomics. Here we describe an enhancer screening strategy in which BAC clones that cover any genomic segments of interest are modified to harbor a reporter cassette by transposon tagging, then processed to carry selected combinations of gene regulatory modules by homologous recombination mediated systematic deletions. Such engineered BAC-reporter constructs in bacterial cells are ready for efficient transgenesis in mice to evaluate activities of gene regulatory modules intact or absent in the constructs. By utilizing the strategy, we could speedily identify a critical genomic fragment for spatio-temporally regulated expression of a mouse cadherin gene whose structure is extraordinarily huge and intricate. This BAC-based methodology would hence provide a novel screening platform for gene transcriptional machineries that dynamically fluctuate during development, pathogenesis and/or evolution.
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