The Skaggs Institute for Chemical Biology, La Jolla, California, USA.
Nat Protoc. 2010 Apr;5(4):791-810. doi: 10.1038/nprot.2010.34. Epub 2010 Apr 1.
Engineered zinc-finger transcription factors (ZF-TF) are powerful tools to modulate the expression of specific genes. Complex libraries of ZF-TF can be delivered into cells to scan the genome for genes responsible for a particular phenotype or to select the most effective ZF-TF to regulate an individual gene. In both cases, the construction of highly representative and unbiased libraries is critical. In this protocol, we describe a user-friendly ZF technology suitable for the creation of complex libraries and the construction of customized ZF-TFs. The new technology described here simplifies the building of ZF libraries, avoids PCR-introduced bias and ensures equal representation of every module. We also describe the construction of a customized ZF-TF that can be transferred to a number of expression vectors. This protocol can be completed in 9-11 d.
基因工程锌指转录因子（ZF-TF）是调节特定基因表达的有力工具。复杂的 ZF-TF 文库可以递送到细胞中，以扫描基因组中负责特定表型的基因，或选择最有效的 ZF-TF 来调节单个基因。在这两种情况下，构建高度有代表性和无偏倚的文库都是至关重要的。在本方案中，我们描述了一种用户友好的 ZF 技术，适用于复杂文库的构建和定制 ZF-TF 的构建。这里描述的新技术简化了 ZF 文库的构建，避免了 PCR 引入的偏差，并确保每个模块的均等表达。我们还描述了定制 ZF-TF 的构建，该 TF 可以转移到多个表达载体中。这个方案可以在 9-11 天内完成。
