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Dreier, B., R. P. Fuller, et al. (2005). "Development of zinc finger domains for recognition of the 5'-CNN-3' family DNA sequences and their use in the construction of artificial transcription factors." J Biol Chem. Considerable progress has been made in recent years in the design of transcription factors for the directed regulation of endogenous genes. While many strategies involve selection methods that must be applied for each new target sequence, we have developed an approach based on linkage of predefined zinc finger domains that each recognize a 3 base pair DNA sequence to construct artificial transcription factors that bind to a desired sequence. These domains can be assembled to recognize unique 18 base pair DNA sequences with high specificity. Here we report the development and characterization of zinc finger domains that bind to 15 out of the 16 5'-CNN-3' subsites. These domains were created through a combination of phage display selection, site-directed mutagenesis and de novo design. Further, these domains were used to generate a highly specific 6-finger protein targeting the ERBB-2 promoter. When fused to regulatory domains, this protein was capable of up- and down-regulating the expression of the endogenous ERBB-2 gene. With the addition of this collection of predefined zinc finger domains, most 5'-CNN-3', 5'-GNN-3', and 5'-ANN-3' containing sequences can now be rapidly targeted for directed gene regulation and nuclease cleavage.


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