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.
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References
Lee, D.K., Seol, W., and Kim, J.S. (2003) Custom DNA-binding proteins and artificial transcription factors. Curr Topics Med Chem. 3, 645–657.
Lindhout, B.I., Pinas, J.E., Hooykaas, P.J., and van der Zaal, B.J. (2006) Employing libraries of zinc finger artificial transcription factors to screen for homologous recombination mutants in Arabidopsis. Plant J. 48, 475–483.
Park, K.S., Jang, Y.S., Lee, H., and Kim, J.S. (2005) Phenotypic alteration and target gene identification using combinatorial libraries of zinc finger proteins in prokaryotic cells. J Bacteriol. 187, 5496–5499.
Lee, J.Y., Sung, B.H., Yu, B.J., Lee, J.H., Lee, S.H., Kim, M.S., Koob, M.D., and Kim, S.C. (2008) Phenotypic engineering by reprogramming gene transcription using novel artificial transcription factors in Escherichia coli. Nucleic Acids Res. 36, e102.
Park, K.S. (2005) Identification and use of zinc finger transcription factors that increase production of recombinant proteins in yeast and mammalian cells. Biotechnol Prog. 21, 664–670.
Park, K.S., Lee, D.K., Lee, H., Lee, Y., Jang, Y.S., Kim, Y.H., Yang, H.Y., Lee, S.I., Seol, W., and Kim, J.S. (2003) Phenotypic alteration of eukaryotic cells using randomized libraries of artificial transcription factors. Nat Biotechnol. 21, 1208–1214.
Lee, D.K., Kim, Y.H., Kim, J.S., and Seol, W. (2004) Induction and characterization of taxol-resistance phenotypes with a transiently expressed artificial transcriptional activator library. Nucleic Acids Res. 32, e116.
Blancafort, P., Chen, E.I., Gonzalez, B., Bergquist, S., Zijlstra, A., Guthy, D., Brachat, A., Brakenhoff, R.H., Quigley, J.P., Erdmann, D., and Barbas, C.F., 3rd (2005) Genetic reprogramming of tumor cells by zinc finger transcription factors. Proc Natl Acad Sci USA. 102, 11716–11721.
Blancafort, P., Tschan, M.P., Bergquist, S., Guthy, D., Brachat, A., Sheeter, D.A., Torbett, B.E., Erdmann, D., and Barbas, C.F., 3rd (2008) Modulation of drug resistance by artificial transcription factors. Mol Cancer Ther. 7, 688–697.
Blancafort, P., Magnenat, L., and Barbas, C.F., 3rd (2003) Scanning the human genome with combinatorial transcription factor libraries. Nat Biotechnol. 21, 269–274.
Kwon, R.J., Kim, S.K., Lee, S.I., Hwang, S.J., Lee, G.M., Kim, J.S., and Seol, W. (2006) Artificial transcription factors increase production of recombinant antibodies in Chinese hamster ovary cells. Biotechnol Lett. 28, 9–15.
Santos, C.N. and Stephanopoulos, G. (2008) Combinatorial engineering of microbes for optimizing cellular phenotype. Curr Opin Chem Biol. 12, 168–176.
Yamanaka, S. (2008) Pluripotency and nuclear reprogramming. Philos Trans R Soc Lond B Biol Sci. 363, 2079–2087.
Takahashi, K. and Yamanaka, S. (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 126, 663–676.
Bae, K.H., Kwon, Y.D., Shin, H.C., Hwang, M.S., Ryu, E.H., Park, K.S., Yang, H.Y., Lee, D.K., Lee, Y., Park, J., Kwon, H.S., Kim, H.W., Yeh, B.I., Lee, H.W., Sohn, S.H., Yoon, J., Seol, W., and Kim, J.S. (2003) Human zinc fingers as building blocks in the construction of artificial transcription factors. Nat Biotechnol. 21, 275–280.
Roederer, M. and Murphy, R.F. (1986) Cell-by-cell autofluorescence correction for low signal-to-noise systems: application to epidermal growth factor endocytosis by 3T3 fibroblasts. Cytometry. 7, 558–565.
Acknowledgments
This work was supported by a grant from the Korea Science and Engineering Foundation (R17-2007-019-01001-0).
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Kim, S., Kim, E.J., Kim, JS. (2010). Construction of Combinatorial Libraries that Encode Zinc Finger-Based Transcription Factors. In: Mackay, J., Segal, D. (eds) Engineered Zinc Finger Proteins. Methods in Molecular Biology, vol 649. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-753-2_8
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DOI: https://doi.org/10.1007/978-1-60761-753-2_8
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