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Modular system for the construction of zinc-finger libraries and proteins

Nature Protocols volume 5, pages 791–810 (2010)Cite this article

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Abstract

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.

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Figure 1: Plasmid map of the SuperZiF plasmids GNN, ANN and CNN.
Figure 2: Library cloning scheme.
Figure 3: Agarose gel electrophoresis (1.5% in 1× TAE with EtBr) of individual clones from the 5ZF GNH–ANN library.
Figure 4: Western blot of 4ZF and 5ZF GNH–ANN library expression with detection by an HA tag with anti-HA-peroxidase antibody (Roche).

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Acknowledgements

We thank S. Juraja and S. Alonso for critical reading of the manuscript and members of our group for helpful suggestions. L.J.S. is supported by The American Cancer Society Illinois Division—Linda M. Campbell Postdoctoral Fellowship. Funding was provided by grants from the US National Institutes of Health.

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  1. Beatriz Gonzalez and Lauren J Schwimmer: These authors contributed equally to this work.

Authors and Affiliations

  1. The Skaggs Institute for Chemical Biology and the Departments of Molecular Biology and Chemistry, The Scripps Research Institute, La Jolla, California, USA

    Beatriz Gonzalez, Lauren J Schwimmer, Roberta P Fuller, Yongjun Ye, Lily Asawapornmongkol & Carlos F Barbas III

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Contributions

B.G. and L.J.S. contributed equally to this work. C.F.B. III conceived of the SuperZif library construction concept and directed the research. Y.Y., B.G. and C.F.B. III designed the SuperZif vectors. R.P.F. designed and constructed the pSCV vector, modified the SuperZiFCNN vector and aided in library construction. B.G., L.J.S. and L.A. constructed and tested the libraries. The paper was written by L.J.S. with assistance from B.G., C.F.B. III and R.P.F.

Note: Supplementary information is available via the HTML version of this article.

Corresponding author

Correspondence to Carlos F Barbas III.

Supplementary information

Supplementary Information

Supplementary Sequence Archive (DOC 323 kb)

Supplementary Figure 1

Vector maps of SuperZiF vectors and pSCV. (PDF 262 kb)

Supplementary Figure 2

Vector maps of expression vectors in Table 1. (PDF 597 kb)

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Gonzalez, B., Schwimmer, L., Fuller, R. et al. Modular system for the construction of zinc-finger libraries and proteins. Nat Protoc 5, 791–810 (2010). https://doi.org/10.1038/nprot.2010.34

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