基于GFP的锌指核酸酶真核细胞筛选体系的构建与验证

doi:10.11843/j.issn.0366-6964.2014.06.004

Abstract

Abstract:

To develop a visible，accurate and sensitive method for zinc-finger nucleases (ZFN) screening，a screening system based on GFP (green fluorescence protein) was constructed in eukaryotic cells.A general screening vector pEGFP-ATG was constructed by removing the initiation codon of the GFP gene to the upstream of multiple cloning sites (MCS) in pEGFP-N1 vector.The target sequence of ZFN was then inserted into the upstream of the GFP sequence，the initiation codon and GFP gene were in the different ORF.The screening vector pEGFP-728 was constructed.Hela cells were transfected with pEGFP-728 vector and selected by G418 to produce the stable Hela-728 cell lines，which were thereafter transfected by the ZFNs.The fluorescence in transfected cells within 48 hours was observed to determine the efficiency of the ZFNs.Those cells with fluorescence were cloned in 96-well plate.Their genomic DNA was extracted，and the target sequence amplified by PCR was sequenced.Sequencing analysis showed that the general vector pEGFP-ATG and the screening vector pEGFP-728 were constructed correctly.The green fluorescence was not observed in Hela cells transfected with pEGFP-728，indicating that the frameshift took place in the GFP gene after the target sequence was inserted.However，the green fluorescence was observed in Hela-728 transfected with ZFNs in 48 hours，suggesting that the ZFNs were effective.Six fluorescent cell clones were selected；PCR and sequencing analysis showed that deletion mutations appeared in the target sequences of four clones.A ZFN screening system was constructed successfully in eukaryotic cells.It was more accurate，visible and sensitive than other screening methods previously reported，and more suitable for screening ZFNs in animal and plant cells.Furthermore，this system can also be applied in TALEN and Cas9 selection.

CLC Number:

S813.1

LI Jing-xin，WANG Hui-li，MENG Chun-hua，ZHANG Qing-xiao，JIANG Jin，WANG Jing，CAO Shao-xian. Construction and Verification of A Screening System Based on GFP for Zinc-Finger Nucleases in Eukaryotic Cell[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(6): 879-884.

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Construction and Verification of A Screening System Based on GFP for Zinc-Finger Nucleases in Eukaryotic Cell

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