title: Design, Assembly, and Characterization of TALE-Based Transcriptional Activators and Repressors.
abstract: Transcription activator-like effectors (TALEs) are modular DNA-binding proteins that can be fused to a variety of effector domains to regulate the epigenome. Nucleotide recognition by TALE monomers follows a simple cipher, making this a powerful and versatile method to activate or repress gene expression. Described here are methods to design, assemble, and test TALE transcription factors (TALE-TFs) for control of endogenous gene expression. In this protocol, TALE arrays are constructed by Golden Gate cloning and tested for activity by transfection and quantitative RT-PCR. These methods for engineering TALE-TFs are useful for studies in reverse genetics and genomics, synthetic biology, and gene therapy.
authors.affiliation: Department of Biomedical Engineering, Duke University, Durham, NC, 27708, USA.
authors.forename: Pratiksha I
authors.initials: PI
authors.lastname: Thakore
authors.affiliation: Department of Biomedical Engineering, Duke University, Room 136 Hudson Hall, Box 90281, Durham, NC, 27708-0281, USA. charles.gersbach@duke.edu.|Center for Genomic and Computational Biology, Duke University, Durham, NC, 27708, USA. charles.gersbach@duke.edu.|Department of Orthopaedic Surgery, Duke University Medical Center, Durham, NC, 27710, USA. charles.gersbach@duke.edu.
authors.forename: Charles A
authors.initials: CA
authors.lastname: Gersbach
mesh_terms: D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D023281:Genomics / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D011401:Promoter Regions, Genetic; D058615:Synthetic Biology; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics*
chemical_list: D004268:DNA-Binding Proteins; D015534:Trans-Activators
keywords: Gene activation; Gene regulation; Gene repression; Genome engineering; Protein engineering; Synthetic biology; Transcription factors