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Eukaryotic Transcription Activators02:42

Eukaryotic Transcription Activators

Transcription activators are proteins that promote the transcription of genes from DNA to RNA. In most cases, these proteins contain two separate domains ‒ a domain that binds to DNA and a domain for activating transcription; however, in some cases, a single domain is responsible for both binding and activation of transcription, as seen in the glucocorticoid receptor and MyoD.
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A transcription activator-like effector toolbox for genome engineering.

Neville E Sanjana1, Le Cong, Yang Zhou

  • 1Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, USA.

Nature Protocols
|January 7, 2012
PubMed
Summary

Researchers developed a TALE toolbox for fast, affordable genome engineering. This system enables rapid construction of custom transcription activator-like effectors (TALE-TFs) and nucleases (TALENs) for diverse biological applications.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Transcription activator-like effectors (TALEs) are DNA-binding proteins from Xanthomonas sp.
  • TALE DNA-binding domains feature modular repeats that can be reconfigured to target specific DNA sequences.
  • Customized TALEs are valuable tools for genome engineering, including transcriptional modulation and gene editing.

Purpose of the Study:

  • To describe a novel toolbox for the rapid and affordable construction of custom TALE transcription factors (TALE-TFs) and TALE nucleases (TALENs).
  • To facilitate the parallel construction of multiple TALEs for various genomic targets.
  • To provide methods for testing the activity of custom TALE-TFs and TALENs in mammalian cells.

Main Methods:

  • A hierarchical ligation procedure was employed for the efficient assembly of custom TALE constructs.
  • Quantitative reverse-transcription PCR was used to assess TALE-TF activity.
  • Surveyor nuclease assays were utilized to evaluate TALEN activity.

Main Results:

  • The developed toolbox enables the rapid construction of custom TALE-TFs and TALENs within one week.
  • The procedure is cost-effective and scalable for high-throughput TALE construction.
  • Successful testing of custom TALE-TFs and TALENs in mammalian cells was demonstrated.

Conclusions:

  • The TALE toolbox offers a streamlined approach for creating custom TALE-based genome engineering tools.
  • This system significantly reduces the time and cost associated with TALE construction.
  • The toolbox is expected to broaden the accessibility and application of TALE technology in biological research.