ArticlePloS one2013
Repeatable construction method for engineered zinc finger nuclease based on overlap extension PCR and TA-cloning.
Article in PloS one, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 8 papers.
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Who cites it
8 citing papers in PubMed.
- Pathological evaluation of rats carrying in-frame mutations in the dystrophin gene: a new model of Becker muscular dystrophy.Disease models & mechanisms · 2020Article
- Review
- Efficient Generation of Genome-Modified Mice Using Campylobacter jejuni-Derived CRISPR/Cas.International journal of molecular sciences · 2017Article
- Harnessing the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated Cas9 system to disrupt the hepatitis B virus.Gene therapy · 2015Article
- Finding of a highly efficient ZFN pair for Aqpep gene functioning in murine zygotes.The Journal of reproduction and development · 2015Article
- Generation of muscular dystrophy model rats with a CRISPR/Cas system.Scientific reports · 2014Article
- Efficient generation of large-scale genome-modified mice using gRNA and CAS9 endonuclease.Nucleic acids research · 2013Article
- Simultaneous splicing of multiple DNA fragments in one PCR reaction.Biological procedures online · 2013Article
Corrections and comments
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Authors and funding
4 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Zinc finger nuclease (ZFN) is a useful tool for endogenous site-directed genome modification. The development of an easier, less expensive and repeatedly usable construction method for various sequences of ZFNs should contribute to the further widespread use of this technology. Here, we establish a novel construction method for ZFNs. Zinc finger (ZF) fragments were synthesized by PCR using short primers coding DNA recognition helices of the ZF domain. DNA-binding domains composed of 4 to 6 ZFs were synthesized by overlap extension PCR of these PCR products, and the DNA-binding domains were joined with a nuclease vector by TA cloning. The short primers coding unique DNA recognition helices can be used repeatedly for other ZFN constructions. By using this novel OLTA (OverLap extension PCR and TA-cloning) method, arbitrary ZFN vectors were synthesized within 3 days, from the designing to the sequencing of the vector. Four different ZFN sets synthesized by OLTA showed nuclease activities at endogenous target loci. Genetically modified mice were successfully generated using ZFN vectors constructed by OLTA. This method, which enables the construction of intended ZFNs repeatedly and inexpensively in a short period of time, should contribute to the advancement of ZFN technology.
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