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

Paper ID Volume ID Publish Year Pages File Format Full-Text
23622 43459 2012 6 PDF Available
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.

► We established a novel system to detect the in vivo genome-editing activities of ZFNs. ► hVEGF ZFN was used to test this system. ► hVEGF ZFN-mediated gene targeting was observed and then quantified by correction frequency of mutant reporter gene. ► The novel system would greatly facilitate the application of custom-designed zinc-finger nucleases (ZFNs) in basic research and disease therapy.

Keywords
Zinc finger nucleases; AAVS1; ZFN activity assay; eGFP correction; Luciferase correction; Single copy cell line
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Establishment of a cell line carrying single copy of an exogenous mutant reporter gene for assaying the biological activity of ZFNs
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 162, Issues 2–3, 31 December 2012, Pages 191–196
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us