Establishment of a cell line carrying single copy of an exogenous mutant reporter gene for assaying the biological activity of ZFNs

Weifeng Zhang, Xiaojing Zheng, Yueying Wang, Qinwen Mao, Haibin Xia*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

In this study, in order to detect the genome-editing activities of ZFNs, a cell line carrying a single copy of mutant reporter eGFP or luciferase gene, with a ZFN target sequence inserted in the middle of the coding region, was built through AAVS1 ZFN mediated knock-in technique. Briefly, AAVS1 ZFN expression vector and donor vector expressing mutant eGFP or luciferase were co-transfected into HEK 293 cells followed by positive/negative selection and cloning procedure. The targeted insertion of a single copy of the exogenous gene was confirmed by PCR, sequencing and southern blot. To prove the principle, hVEGF ZFN was used to test this system. hVEGF ZFN expression vector and donor vector carrying a fragment of wild-type reporter gene corresponding to the mutation-disabled stretch were co-transfected into 293-eGFP-hVEGF-TSF or 293-luci-hVEGF-TSF cell lines. 4 days post transfection, 293-eGFP-hVEGF-TSF group showed increased eGFP positive clones with a correction efficiency of 0.11%, which was significantly higher than that of the control. Similar results were obtained for the 293-luci-hVEGF-TSF group. The results indicated that the novel system, established by taking advantage of AAVS1 ZFN mediated knock-in technique, was useful for detecting the genome-editing activities of ZFNs. AAVS1 ZFN mediated knock-in was much easier to use than the current existing FLP-in technique. In addition, our donor vector system, featuring both positive and negative selection mechanisms, made it even more efficient to set up a system for assaying the biological activity of a new assembled ZFN.

Original languageEnglish (US)
Pages (from-to)191-196
Number of pages6
JournalJournal of Biotechnology
Volume162
Issue number2-3
DOIs
StatePublished - Dec 31 2012

Keywords

  • AAVS1
  • Luciferase correction
  • Single copy cell line
  • ZFN activity assay
  • Zinc finger nucleases
  • eGFP correction

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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