Protocol

Engineered Zinc Finger Proteins

Volume 649 of the series Methods in Molecular Biology pp 133-147

Date:

Construction of Combinatorial Libraries that Encode Zinc Finger-Based Transcription Factors

  • Seokjoong KimAffiliated withDepartment of Chemistry, Seoul National University
  • , Eun Ji KimAffiliated withDepartment of Chemistry, Seoul National University
  • , Jin-Soo KimAffiliated withDepartment of Chemistry, Seoul National University

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Abstract

Combinatorial retroviral libraries of zinc finger transcription factors (ZF-TFs) can be constructed that encode tens of thousands of different, multi-finger zinc finger proteins (ZFPs) with distinct DNA-binding specificities, each of which is fused to a transcriptional activation or repression domain. Individual zinc fingers (ZFs) recognize their target DNA subsites and retain their binding specificities in the context of artificially constructed multi-finger ZFPs. Because of this modular nature, expression libraries that specify diverse multi-finger ZF-TFs can be created by the combinatorial stitching together of individual modules in a pool of single-ZF-encoding DNA segments. When these libraries are introduced into cells, the encoded ZF-TFs are expressed and can then activate or repress the transcription of endogenous target genes. Ideally, the ZF-TF-encoding retroviral vectors in the library enter cells randomly at a ratio of ~1 per cell. As a result, individual cells express different ZF-TFs and thus display distinct phenotypical changes. Using an appropriate screening or selection method, one can isolate clonal cells that display phenotypes of interest. One can then identify the ZF-TFs responsible for the phenotypes and, ultimately, the genes that are targeted by the selected ZF-TFs. Here, we provide protocols for the preparation of retroviral libraries that encode ZF-TFs for use in mammalian cells.

Key words

Cellular reprogramming artificial transcription factor zinc finger metabolic engineering stem cell strain improvement