Repeatable construction method for engineered zinc finger nuclease based on overlap extension PCR and TA-cloning

PLoS One. 2013;8(3):e59801. doi: 10.1371/journal.pone.0059801. Epub 2013 Mar 25.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cloning, Molecular
  • Endonucleases / genetics*
  • Endonucleases / metabolism*
  • Female
  • Gene Expression
  • Gene Order
  • Genetic Vectors
  • Male
  • Mice
  • Mice, Transgenic
  • Mutagenesis, Site-Directed
  • Polymerase Chain Reaction
  • Protein Engineering / methods*
  • Zinc Fingers / genetics*

Substances

  • Endonucleases

Grant support

This work was supported by a Grant-in-Aid for JSPS Fellows (11J02886 to WF), Grant-in-Aid for Scientific Research (no. 22380147 to KN) and Grant-in-Aid for Challenging Exploratory Research (no. 24658232 to KN) from the Japan Society for the Promotion of Science (http://www.jsps.go.jp/english/index.html). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.