A robust dual reporter system to visualize and quantify gene expression mediated by transcription activator-like effecto
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SHORT REPORT
Biological Procedures Online Open Access
A robust dual reporter system to visualize and quantify gene expression mediated by transcription activator-like effectors Claudia Uhde-Stone1, Joseph Huang2 and Biao Lu2*
Abstract Background: Transcription activator-like effectors (TALEs) are a class of naturally occurring transcription effectors that recognize specific DNA sequences and modulate gene expression. The modularity of TALEs DNA binding domain enables sequence-specific perturbation and offers broad applications in genetic and epigenetic studies. Although the efficient construction of TALEs has been established, robust functional tools to assess their functions remain lacking. Results: We established a dual reporter system that was specifically designed for real-time monitoring and quantifying gene expression mediated by TALEs. We validated both sensitivity and specificity of this dual-reporter system in mammalian cells, and demonstrated that this dual reporter system is robust and potentially amenable to high throughput (HTP) applications. Conclusion: We have designed, constructed and validated a novel dual reporter system for assessing TALE mediated gene regulations. This system offers a robust and easy-to- use tool for real-time monitoring and quantifying gene expression in mammalian cells. Keywords: Dual reporter, Gene editing, Transcription activator-like effector, Green fluorescent protein, Firefly luciferase
Background Zinc-finger nucleases (ZFNs) and meganucleases have been well-established as tools to edit specific sites in complex genomes [1,2]. However, the DNA-binding domain of zinc-finger nucleases are challenging to design and require experimental optimization, and the target sequences of meganucleases are limited. Transciption activator-like effector (TALE) technology has recently emerged as an alternative robust and efficient genome editing tool [3,4]. Natural TALEs are transcription factors used by plant-pathogenic bacteria in the genus Xanthomonas. The native function of TALEs is to modulate host gene expression by binding to specific sequences in host gene promoters and activate transcription [5,6]. The DNA binding domain of TALEs consist of a central domain of 33–35 amino acid repeats * Correspondence: [email protected] 2 System Biosciences (SBI), 265 North Whisman Road, Mountain View CA 94043, USA Full list of author information is available at the end of the article
arranged in tandem, followed by a single truncated repeat of 20 amino acids. The tandem repeats are nearly identical, except for two variable amino acids in position 12 and 13, referred to as “repeat-variable di-residue” (RVD), with the four most common RVDs each specifying binding of one of the four DNA bases [7,8]. The modular nature of the TALE DNA-binding domain and the straightforward sequence relationships enables efficient customization of TAL effector repeat domains. The simplicity of TALE design and construction to target nearly any DNA sequence within the genome has been an important advantage of the t
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