title: Establishment of a cell line carrying single copy of an exogenous mutant reporter gene for assaying the biological activity of ZFNs.
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.
authors.affiliation: Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University, Xi'an 710062 Shaanxi, PR China.
authors.forename: Weifeng
authors.initials: W
authors.lastname: Zhang
authors.forename: Xiaojing
authors.initials: X
authors.lastname: Zheng
authors.forename: Yueying
authors.initials: Y
authors.lastname: Wang
authors.forename: Qinwen
authors.initials: Q
authors.lastname: Mao
authors.forename: Haibin
authors.initials: H
authors.lastname: Xia
mesh_terms: D001483:Base Sequence; D001709:Biotechnology / Q000379:methods*; D003001:Cloning, Molecular / Q000379:methods; D004262:DNA Restriction Enzymes / Q000235:genetics / Q000378:metabolism*; D000229:Dependovirus / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016335:Zinc Fingers / Q000235:genetics*
chemical_list: D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004262:DNA Restriction Enzymes