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Rapid assembly of customized TALENs into multiple

Zhang, Z and Zhang, S and Huang, X and Orwig, KE and Sheng, Y (2013) Rapid assembly of customized TALENs into multiple. PLoS ONE, 8 (11).

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Abstract

Transcriptional activator-like effector nucleases (TALENs) have become a powerful tool for genome editing. Here we present an efficient TALEN assembly approach in which TALENs are assembled by direct Golden Gate ligation into Gateway® Entry vectors from a repeat variable di-residue (RVD) plasmid array. We constructed TALEN pairs targeted to mouse Ddx3 subfamily genes, and demonstrated that our modified TALEN assembly approach efficiently generates accurate TALEN moieties that effectively introduce mutations into target genes. We generated "user friendly" TALEN Entry vectors containing TALEN expression cassettes with fluorescent reporter genes that can be efficiently transferred via Gateway (LR) recombination into different delivery systems. We demonstrated that the TALEN Entry vectors can be easily transferred to an adenoviral delivery system to expand application to cells that are difficult to transfect. Since TALENs work in pairs, we also generated a TALEN Entry vector set that combines a TALEN pair into one PiggyBac transposon-based destination vector. The approach described here can also be modified for construction of TALE transcriptional activators, repressors or other functional domains. © 2013 Zhang et al.


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Details

Item Type: Article
Status: Published
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Zhang, Z
Zhang, S
Huang, Xxih20@pitt.eduXIH20
Orwig, KEkeo6@pitt.eduKEO6
Sheng, Yyis14@pitt.eduYIS14
Contributors:
ContributionContributors NameEmailPitt UsernameORCID
EditorChan, ChristinaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Centers: Other Centers, Institutes, or Units > Pittsburgh Cancer Institute
Date: 7 November 2013
Date Type: Publication
Journal or Publication Title: PLoS ONE
Volume: 8
Number: 11
DOI or Unique Handle: 10.1371/journal.pone.0080281
Schools and Programs: School of Medicine > Obstetrics, Gynecology, and Reproductive Sciences
Refereed: Yes
Date Deposited: 30 Jan 2014 17:44
Last Modified: 04 Feb 2019 22:55
URI: http://d-scholarship.pitt.edu/id/eprint/20375

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