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

畜牧兽医学报 ›› 2014, Vol. 45 ›› Issue (6): 879-884.doi: 10.11843/j.issn.0366-6964.2014.06.004

基于GFP的锌指核酸酶真核细胞筛选体系的构建与验证

李静心^1,2,3，王慧利^1,3，孟春花^1,3，张庆晓^1,2，蒋进^1,2，王婧^1,2，曹少先^1,3*

(1.江苏省农业科学院畜牧研究所,南京 210014； 2.南京农业大学动物科技学院，南京 210095；3.江苏省农业科学院动物品种改良和繁育重点实验室,南京 210014)

收稿日期:2013-11-26 出版日期:2014-06-23 发布日期:2014-06-23
通讯作者: 曹少先，博士，研究员，Tel：025-84390352，E-mail：caoshaoxian@163.com
作者简介:李静心（1990-），男，安徽亳州人，硕士，主要从事动物分子育种研究，E-mail：xx1516217@163.com
基金资助:
转基因生物新品种培育科技重大专项（2009ZX08008-009B）；国家自然科学基金（31101715）

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

LI Jing-xin^1,2,3，WANG Hui-li^1,3，MENG Chun-hua^1,3，ZHANG Qing-xiao^1,2，JIANG Jin^1,2，WANG Jing^1,2，CAO Shao-xian^1,3*

(1.Institute of Animal Science，Jiangsu Academy of Agricultural Sciences，Nanjing 210014， China；2.College of Animal Science and Technology，Nanjing Agricultural University，Nanjing 210095， China；3.Key Laboratory of Animal Breeding and Reproduction，Jiangsu Academy of Agricultural Sciences，Nanjing 210014， China)

Received:2013-11-26 Online:2014-06-23 Published:2014-06-23

摘要/Abstract

摘要：

旨在构建基于GFP报告基因的ZFN真核细胞筛选体系，为ZFN的筛选提供一种直观、准确、灵敏的方法。以pEGFP-N1为基础，去除EGFP基因的起始密码子后在多克隆位点上游插入ATG，构建筛选体系的通用载体pEGFP-ATG。将ZFN的靶序列插入到通用载体EGFP基因上游，且使起始密码子和EGFP基因处于不同ORF中，构建筛选载体pEGFP-728。将pEGFP-728转入Hela细胞中，G418筛选Hela-728细胞系，转染ZFN到Hela-728，48 h内通过观察荧光判断ZFN是否有效，挑取荧光细胞进行PCR、测序验证靶序列的敲除。测序分析表明，通用载体pEGFP-ATG和含靶序列的筛选载体pEGFP-728序列完全正确；pEGFP-728转入Hela细胞中观察不到绿色荧光，说明靶序列插入后使EGFP发生移框；转染靶序列对应的ZFN到Hela-728中，48 h观察到荧光的表达，说明ZFN有效；挑取6个荧光细胞单克隆，PCR、测序表明4个克隆靶序列发生缺失突变。锌指核酸酶真核细胞筛选体系构建成功。相对于其他方法本筛选体系更加准确、直观、灵敏，更适用于动植物等真核细胞的ZFN筛选，同时还可应用于TALEN和CAS9的筛选。

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.

中图分类号:

S813.1

李静心，王慧利，孟春花，张庆晓，蒋进，王婧，曹少先. 基于GFP的锌指核酸酶真核细胞筛选体系的构建与验证[J]. 畜牧兽医学报, 2014, 45(6): 879-884.

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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参考文献

［1］PORTEUS M H，CARROLL D.Gene targeting using zinc finger nucleases［J］.Nat Biotechnol，2005，23(8)：967-973.
［2］WU J，KANDAVELOU K，CHANDRASEGARAN S.Custom-designed zinc finger nucleases：What is next［J］.Cell Mol Life Sci，2007，64(22)：3933-3944.
［3］BIBIKOVA M，GOLIC M，GOLIC K G，et al.Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases［J］.Genetics，2002，161：1169-1175.
［4］CARROLL D，BEUMER K J，MORTON J J，et al.Gene targeting in Drosophila and caenorhabditis elegans with zinc finger nucleases［J］. Methods Mol Biol，2008，435：63-77.
［5］LLOYD A，PLAISIER C L，CARROLL D，et al.Targeted mutagenesis using zinc-finger nucleases in arabidopsis［J］.Proc Natl Acad Sci USA，2005，102：2232-2237.
［6］DURAI S，MANI M，KANDAVELOU K，et al.Zinc finger nucleases：Custom-designed molecular scissors for genome engineering of plant and mammalian cells［J］.Nucleic Acids Res，2005，33(18)：5978-5990.
［7］WATANABE M，NAKANO K，MATSUNARI H，et al.Generation of interleukin-2 receptor gamma gene knockout pigs from somatic cells genetically modified by zinc finger nuclease-encoding mRNA［J］. PLoS ONE，2013，8(10):e76478.
［8］MORI T，MORI K，TOBIMASTU T，et al.Sandwiched zinc-finger nucleases demonstrating higher homologous recombination rates than conventional zinc-finger nucleases in mammalian cells［J］. Bioorg Med Chem Lett，2014，24(3):813-816.
［9］CURTIN S J，ANDERSON J E，STARKER C G，et al.Targeted mutagenesis for functional analysis of gene duplication in legumes［J］.Methods Mol Biol，2013，1069:25-42.
［10］ZHANG W，WANG D，LIU S，et al.Multiple copies of a linear donor fragment released in situ from a vector improve the efficiency of zinc-finger nuclease-mediated genome editing［J］.Gene Therapy，2014，21：282-288.
［11］MAEDER M L，BEGANNY S，OSIAK A，et al.Rapid ‘‘open-source’’ engineering of customized zinc-finger nucleases for highly efficient gene modification［J］.Mol Cell，2008，31：294-301.
［12］PORTEUS M H，BALTIMORE D.Chimeric nucleases stimulate gene targeting in human cells［J］.Nature，2005，435(7043)：646-651.
［13］MILLER S M，CONNELLY J P，MAEDER M L，et al.Comparison of zinc finger nucleases for use in gene targeting in mammalian cells［J］.Mol Therapy，2008，16(4)：707-717.
［14］DOYON Y，MCCAMMON J M，MILLER J C，et al.Heritable targeted gene disruption in zebrafish using designed zinc finger nucleases［J］.Nat Biotechnol，2008，26(6)：702-708.
［15］WEST S C，CHAPPELL C，HANAKAHI L A，et al.Double-strand break repair in human cells［J］.Cold Spring Harb Symp Quant Biol，2000，65：315-321.
［16］VASQUEZ K M，MARBURGER K，INTODY Z，et al.Manipulating the mammalian genome by homologous recombination［J］.Proc Natl Acad Sci USA，2001，98：8403-8410.
［17］张开慧.酵母单杂交技术的原理及应用［J］.宁夏农林科技，2012，53(2)：31-32.
［18］SWARTZ J R.Advances in Escherichia coli production of therapeutic proteins［J］.Current Opinion Biotechnol，2001，12(2)：195-201.
［19］LI T J，GUSTIN J，DODLA M，et al.Chapter 32-genetic manipulation of human pluripotent stem cells using zinc finger nucleases［M］.Human Stem Cell Manual，2012：499-515.

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基于GFP的锌指核酸酶真核细胞筛选体系的构建与验证

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

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