Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly
Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly
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DOI：
10.1038/nprot.2006.259
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Joung, J. Keith
中科院分区：
文献类型：
--
作者：
Wright, David A.;Thibodeau-Beganny, Stacey;Joung, J. Keith
基因工程锌指核酸酶可以在昆虫、植物和人类细胞中刺激针对特定基因组位点的基因靶向。尽管已经描述了几个使用“模块化组装”来构建人造锌指阵列的平台，但是还没有标准化试剂和方案来允许快速、跨平台地“混合和匹配”各种锌指模块。在这里，我们描述了一个全面的、可公开获得的、编码140多个特征良好的锌指模块的质粒库，以及用于识别目标基因中潜在的锌指靶点的互补的基于网络的软件(称为ZiFiT)。我们的试剂已经在单一平台上实现了标准化，能够方便地混合和匹配模块，并将组装的阵列转移到表达载体，而不需要锌指序列或复杂的寡核苷酸设计的专业知识。我们还描述了一种基于细菌细胞的报告实验，用于快速筛选组装的多指阵列的DNA结合活性。该方案可在大约24-26天内完成。
Engineered zinc finger nucleases can stimulate gene targeting at specific genomic loci in insect, plant and human cells. Although several platforms for constructing artificial zinc finger arrays using "modular assembly" have been described, standardized reagents and protocols that permit rapid, cross-platform "mixing-and-matching" of the various zinc finger modules are not available. Here we describe a comprehensive, publicly available archive of plasmids encoding more than 140 well-characterized zinc finger modules together with complementary web-based software (termed ZiFiT) for identifying potential zinc finger target sites in a gene of interest. Our reagents have been standardized on a single platform, enabling facile mixing-and-matching of modules and transfer of assembled arrays to expression vectors without the need for specialized knowledge of zinc finger sequences or complicated oligonucleotide design. We also describe a bacterial cell-based reporter assay for rapidly screening the DNA-binding activities of assembled multi-finger arrays. This protocol can be completed in approximately 24-26 d.