Protocol abstract
Nature Protocols 1, - 1637 - 1652 (2006)
Published online: 16 November 2006 | doi:10.1038/nprot.2006.259
Subject Categories: Biochemistry and protein analysis | Computational and theoretical biology | Genetic modification | Nucleic acid based molecular biology
Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly
David A Wright1, Stacey Thibodeau-Beganny2, Jeffry D Sander1,3, Ronnie J Winfrey1, Andrew S Hirsh2,4, Magdalena Eichtinger2,4, Fengli Fu1,3, Matthew H Porteus5, Drena Dobbs1,3, Daniel F Voytas1 & J Keith Joung2,4
Abstract
Engineered zinc finger nucleases can stimulate gene targeting at specific genomic loci in insect, plant and human cells. Although several platforms for constructing artificial zinc finger arrays using "modular assembly" have been described, standardized reagents and protocols that permit rapid, cross-platform "mixing-and-matching" of the various zinc finger modules are not available. Here we describe a comprehensive, publicly available archive of plasmids encoding more than 140 well-characterized zinc finger modules together with complementary web-based software (termed ZiFiT) for identifying potential zinc finger target sites in a gene of interest. Our reagents have been standardized on a single platform, enabling facile mixing-and-matching of modules and transfer of assembled arrays to expression vectors without the need for specialized knowledge of zinc finger sequences or complicated oligonucleotide design. We also describe a bacterial cell-based reporter assay for rapidly screening the DNA-binding activities of assembled multi-finger arrays. This protocol can be completed in approximately 24–26 d.
- Department of Genetics, Development & Cell Biology, Iowa State University, 1035A Roy J. Carver Co-Lab, Ames, Iowa 50011, USA.
- Molecular Pathology Unit and Center for Cancer Research, Massachusetts General Hospital, 149 13th Street, Charlestown, Massachusetts 02129, USA.
- Interdepartmental Graduate Program in Bioinformatics & Computational Biology, Iowa State University, 2114 Molecular Biology Building, Ames, Iowa 50011, USA.
- Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.
- Departments of Pediatrics & Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390, USA.
Correspondence to: J Keith Joung2,4 e-mail: jjoung@partners.org
nature-products
A-Z product listing
- 10
restriction enzyme buffers(New England Biolabs) - 32 mM S-adenosyl methionine (SAM)(New England Biolabs)
- 96-well microtiter plates(Corning-Costar)
- Calf intestinal phosphatase, CIP(Promega)
- Cloned Pfu polymerase(Stratagene)
- Deep-well 96-well blocks(Greiner)
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