An Integrated Chip for the High‐Throughput Synthesis of Transcription Activator‐like Effectors

Wang, Zhao; Li, Juan; Huang, Huang; Wang, Gancheng; Jiang, Mingjun; Yin, Shenyi; Sun, Cheng; Zhang, Hanshuo; Zhuang, Fang‐Dong; Xi, Jianzhong

doi:10.1002/ange.201203597

Cited by 11 publications

(16 citation statements)

References 14 publications

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“…1B). 12 Then, we implemented a solid-phase synthesis strategy, 3 which allows a quick and simple purification of the ligation product to construct TALEN vectors. We performed the TALE production by use of a solidphase gene synthesis approach we reported previously.…”

Section: Design and Construction Of Talen-expressing Vectorsmentioning

confidence: 99%

“…3 If the TALENs worked well, the luciferase activity (firefly/Renilla) of target gene could be recovered through SSA repair, compared with an empty vector in the negative control experiment (NC). The cells were transfected with the plasmids in the following ratio: TALEN-L plasmid/TALEN-R plasmid/ luciferase reporter/Renilla = 1:1:1:0.05, where Renilla was used as the internal control.…”

Section: Examination Of Exogenous or Endogenous Activitymentioning

confidence: 99%

“…3,5 In this work, we exploited the solid-phase gene synthesis of TALENs and presented an efficient method of producing a stable cell line with specific gene mutants. More recently, type II clustered regularly interspaced short palindromic repeats (CRISPRs) have been demonstrated as an alternative approach on genome editing in a variety of mammalian cells and model organisms.…”

Section: Perspectivementioning

confidence: 99%

“…3,5,6 To date, TALENs have successfully been applied to edit target genes in various species, such as Drosophila, zebrafish, mouse, and even human cells. [7][8][9][10] To efficiently generate genome editing products for human cells, DNA double-strand breaks (DSBs) have to be introduced at specific chromosome sites by TALENs.…”

Section: Introductionmentioning

confidence: 99%

“…To illustrate this strategy, we chose mTOR as an editing target because of its critical role in regulating cell autophagy. 11 First, we exploited a magnetic beads-based TALE assembly method to synthesize a group of TALEN vectors to target mTOR 3 and introduced them into 293T cells by transfection. Second, we confirmed the gene editing activity of the TALENs using exogenous or endogenous detection assays.…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

Efficient Production of a Gene Mutant Cell Line through Integrating TALENs and High-Throughput Cell Cloning

Sun

Fan

et al. 2015

SLAS Technology

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Transcription activator-like effectors (TALEs) are becoming powerful DNA-targeting tools in a variety of mammalian cells and model organisms. However, generating a stable cell line with specific gene mutations in a simple and rapid manner remains a challenging task. Here, we report a new method to efficiently produce monoclonal cells using integrated TALE nuclease technology and a series of high-throughput cell cloning approaches. Following this method, we obtained three mTOR mutant 293T cell lines within 2 months, which included one homozygous mutant line.

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Section: Design and Construction Of Talen-expressing Vectorsmentioning

confidence: 99%

Section: Examination Of Exogenous or Endogenous Activitymentioning

confidence: 99%

Section: Perspectivementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Efficient Production of a Gene Mutant Cell Line through Integrating TALENs and High-Throughput Cell Cloning

Sun

Fan

et al. 2015

SLAS Technology

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Engineering Designer Transcription Activator‐‐Like Effector Nucleases (TALENs) by REAL or REAL‐Fast Assembly

Reyon

Khayter

Regan

et al. 2012

CP Molecular Biology

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Engineered transcription activator-like effector nucleases (TALENs) are broadly useful tools for performing targeted genome editing in a wide variety of organisms and cell types including plants, zebrafish, C. elegans, rat, human somatic cells, and human pluripotent stem cells. Here we describe a detailed protocol for the serial, hierarchical assembly of TALENs that requires neither PCR nor specialized multi-fragment ligations and that can be implemented by any laboratory. This restriction enzyme and ligation (REAL) protocol can be practiced using a small plasmid library and user-friendly, web-based software that both identifies target sites in sequences of interest and generates printable graphical guides that facilitate assembly of TALENs. All plasmids required to perform the REAL assembly method are publicly available through the Addgene plasmid distribution service (http://www.addgene.org/talengineering). Alternatively, to decrease the time and effort required, users can practice REAL using a library of plasmids encoding pre-assembled TALE repeats. With the platform of reagents, protocols, and software we describe, researchers can easily engineer multiple TALENs within two weeks or less using standard cloning techniques.

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Engineering Customized TALE Nucleases (TALENs) and TALE Transcription Factors by Fast Ligation‐Based Automatable Solid‐Phase High‐Throughput (FLASH) Assembly

Reyon

Maeder

Khayter

et al. 2013

CP Molecular Biology

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Customized DNA-binding domains made using Transcription Activator-Like Effector (TALE) repeats are rapidly growing in importance as widely applicable research tools. TALE nucleases (TALENs), composed of an engineered array of TALE repeats fused to the FokI nuclease domain, have been used successfully for directed genome editing in multiple different organisms and cell types. TALE transcription factors (TALE-TFs), consisting of engineered TALE repeat arrays linked to a transcriptional regulatory domain, have been used to up- or down-regulate expression of endogenous genes in human cells and plants. Here we describe a detailed protocol for practicing the recently described Fast Ligation-based Automatable Solid-phase High-throughput (FLASH) assembly method. FLASH enables automated high-throughput construction of engineered TALE repeats using an automated liquid handling robot or manually using a multi-channel pipet. With the automated version of FLASH, a single researcher can construct up to 96 DNA fragments encoding various length TALE repeat arrays in one day and then clone these to construct sequence-verified TALEN or TALE-TF expression plasmids in one week or less. Plas-mids required to practice FLASH are available by request from the Joung Lab (http://www.jounglab.org/). We also describe here improvements to the Zinc Finger and TALE Targeter (ZiFiT Targeter) webserver (http://ZiFiTBeta.partners.org) that facilitate the design and construction of FLASH TALE repeat arrays in high-throughput.

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An Integrated Chip for the High‐Throughput Synthesis of Transcription Activator‐like Effectors

Cited by 11 publications

References 14 publications

Efficient Production of a Gene Mutant Cell Line through Integrating TALENs and High-Throughput Cell Cloning

Efficient Production of a Gene Mutant Cell Line through Integrating TALENs and High-Throughput Cell Cloning

Engineering Designer Transcription Activator‐‐Like Effector Nucleases (TALENs) by REAL or REAL‐Fast Assembly

Engineering Customized TALE Nucleases (TALENs) and TALE Transcription Factors by Fast Ligation‐Based Automatable Solid‐Phase High‐Throughput (FLASH) Assembly

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