A recombineering pipeline for functional genomics applied to Caenorhabditis elegans
A recombineering pipeline for functional genomics applied to Caenorhabditis elegans
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DOI：
10.1038/nmeth933
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发表时间：
2006-10-01
期刊：
影响因子：
48
通讯作者：
Stewart, A. Francis
中科院分区：
文献类型：
--
作者：
Sarov, Mihail;Schneider, Susan;Stewart, A. Francis
我们提出了一种DNA工程的新概念，它基于液体培养中的一系列连续重组工程步骤。这种方法快速、直接，并且便于同时并行处理多个样本。我们通过在一个仅需4天的多步骤流程中从秀丽隐杆线虫（Caenorhabditis briggsae）基因组克隆产生绿色荧光蛋白（GFP）标记的转基因来验证了该方法。这些转基因在对天然基因组环境干扰最小的情况下进行了改造，以便在转基因后能够确保正确的表达水平和模式。一个用于秀丽隐杆线虫lin - 59直系同源基因的转基因示例被用于秀丽隐杆线虫的基因枪转化。我们表明，这种跨物种转基因能够正确表达，并挽救由RNA干扰（RNAi）介导的秀丽隐杆线虫内源基因的敲低。我们所描述的策略将大肠杆菌中重组工程的强大功能应用于流畅的DNA工程，并使其能够直接扩展用于基因组项目。
We present a new concept in DNA engineering based on a pipeline of serial recombineering steps in liquid culture. This approach is fast, straightforward and facilitates simultaneous processing of multiple samples in parallel. We validated the approach by generating green fluorescent protein (GFP)-tagged transgenes from Caenorhabditis briggsae genomic clones in a multistep pipeline that takes only 4 d. The transgenes were engineered with minimal disturbance to the natural genomic context so that the correct level and pattern of expression will be secured after transgenesis. An example transgene for the C. briggsae ortholog of lin-59 was used for ballistic transformation in Caenorhabditis elegans. We show that the cross-species transgene is correctly expressed and rescues RNA interference (RNAi)-mediated knockdown of the endogenous C. elegans gene. The strategy that we describe adapts the power of recombineering in Escherichia coli for fluent DNA engineering to a format that can be directly scaled up for genomic projects.