A do-it-yourself protocol for simple transcription activator-like effector assembly

Biological Procedures Online

Methodology

Claudia Uhde-Stone¹  Nilang Gor²  Biao Lu³  Joseph Huang³  Tiffany Chin³ 

[1] Department of Biological Sciences, California State University, East Bay, 25800 Carlos Bee Blvd, 94542, Hayward, CA, USA;Department of Biological Sciences, California State University, East Bay, 25800 Carlos Bee Blvd, 94542, Hayward, CA, USA;System Biosciences (SBI), 265 North Whisman Rd, 94043, Mountain View, CA, USA;System Biosciences (SBI), 265 North Whisman Rd, 94043, Mountain View, CA, USA;

关键词: TALEN;    TALE-TF;    Golden Gate;    Transcription-activator-like effector;   

DOI  :  10.1186/1480-9222-15-3

 received in 2013-01-03, accepted in 2013-01-05,  发布年份 2013

来源: Springer

【 摘 要 】

BackgroundTALEs (transcription activator-like effectors) are powerful molecules that have broad applications in genetic and epigenetic manipulations. The simple design of TALEs, coupled with high binding predictability and specificity, is bringing genome engineering power to the standard molecular laboratory. Currently, however, custom TALE assembly is either costly or limited to few research centers, due to complicated assembly protocols, long set-up time and specific training requirements.ResultsWe streamlined a Golden Gate-based method for custom TALE assembly. First, by providing ready-made, quality-controlled monomers, we eliminated the procedures for error-prone and time-consuming set-up. Second, we optimized the protocol toward a fast, two-day assembly of custom TALEs, based on four thermocycling reactions. Third, we increased the versatility for diverse downstream applications by providing series of vector sets to generate both TALENs (TALE nucleases) and TALE-TFs (TALE-transcription factors) under the control of different promoters. Finally, we validated our system by assembling a number of TALENs and TALE-TFs with DNA sequencing confirmation. We further demonstrated that an assembled TALE-TF was able to transactivate a luciferase reporter gene and a TALEN pair was able to cut its target.ConclusionsWe established and validated a do-it-yourself system that enables individual researchers to assemble TALENs and TALE-TFs within 2 days. The simplified TALE assembly combined with multiple choices of vectors will facilitate the broad use of TALE technology.

【 授权许可】

Unknown   
© Uhde-Stone et al.; licensee BioMed Central Ltd. 2013. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.