利用锌指核酸酶的高效多位点基因打靶研究
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摘要
基因打靶技术已经越来越受到研究工作者的青睐,该技术也逐渐走向成熟。但是,传统的基因打靶所依赖的同源重组仅仅是个10~(-6)-10~(-5)之间的低概率事件。近几年以来,本课题组已经建立起了以基因组中度重复序列rDNA的间隔序列作为靶位点的多位点基因打靶技术,成功得到转基因鳜鱼和重组奶牛细胞,将打靶效率提高了200-300倍。但是,该技术离高效打靶还差很远。依据近几年的研究进展,国内外有很多卓有成效的基因打靶技术应用于动植物体内转基因。其中,最有效的就是锌指核酸酶(Zinc Finger Nuclease,即ZFN)的应用,它将基因定点整合率大大提高。
本课题将锌指核酸酶的应用与多位点打靶相结合,理论上可将打靶效率提高到18%以上。本文经过筛选将人基因组上的中度重复序列rDNA的18S和5.8S之间的间隔序列ITS1作为靶位点,设计针对靶标的一对三联体锌指蛋白。分别与FokⅠ切割结构域连接,合成了一对锌指嵌合核酸酶。接着,将该对锌指核酸酶分别插入到真核表达核载体pcDNA3.1-NLS中,构建了一对真核表达载体pcS-ZFNs。利用PCR方法从pEGFP-N1中扩增pCMV-EGFP,从人基因组rDNA基因家族中靶基因插入位点两侧分别扩增两条基因打靶同源重组引导序列DS1和DS2,将DS1、DS2片段插入pMD19-pCMV-EGFP中,构建成功多位点基因打靶载体pMD19-DS1-pCMV-EGFP-DS2。
本课题选取HEK293细胞作为研究对象,通过共转染一对pcS-ZFNs和基因打靶载体,得到了生长增殖稳定、绿色荧光持续表达的基因整合细胞。经提取基因组,进行靶位点验证性PCR试验,证实了基因的定点整合,并将定点整合效率提高到了20%以上。本课题的研究避免了药物的毒副作用,不经药物筛选就明显提高了基因打靶效率,为动物定点转基因技术的建立和人类遗传疾病的基因治疗提供了技术方法。
The researchers have been more and more caring about the technique of gene target which is gradually coming to be mature.However,the traditional gene target is merely relied on homologous recombination,whose efficiency is just between 10~(-6) and 10~(-5).Recently,we have managed to establish a technique of multiple locus on gene targeting using the middle repeat of the interval sequence of rDNA as a target,and succeeded to produce the transgenic mandarin fishes and the recombination cow fertilized egg cell.Although in these researches the efficiency of gene target has been up to 200-300 folds,it is still a long way to real high efficiency. According to recent research,so many techniques about gene target with high efficiency have been put into use at home and abroad,in which a famous method is the application of zinc finger nuclease that can high improved the efficiency of gene target.
Our research has combined the application of zinc finger nuclease with the multiple loci gene target,which can improved the efficiency of gene target up to 18%in theory.This article has used the ITS1 between 18S and 5.8S of rDNA as a target,and got a pair of three-zinc finger gene cutting the target sequence which combined with the coding sequence of Fok I slicing domain,thus we got a pair of zinc finger nucleases gene.And then,we inserted each zinc finger nuclease gene into the eukaryotic expression vector of pcDNA3.1-NLS,and got a pair of pcS-ZFNs.It was inserted into the pMD19-T vector for the construction of the recombinant vector pMD19-DS1-pCMV-EGFP-DS2 that the pCMV-EGFP gene which amplified by PCR using pEGFP-N1 out of MCS as template and two homogenous recombination arms of DS1 and DS2 which were amplified from human genome by PCR.
By cotransfecting a pair of pcS-ZFNs and recombinant vector into HEK293 cells,we got the gene targeting cells with stable growth and reproduction,and persistent expression of the green fluorescence.After extracting the genome DNA of former HEK293 cells and testing the gene target by PCR,we managed to test the recombination efficiency of the gene target which is up to 20%.This research can avoid the bad reaction of the drug,improve the efficiency of gene target without drug selection and provide a transgenic technique for the transgenic animal and the gene therapy and diagnostics of human.
本课题将锌指核酸酶的应用与多位点打靶相结合,理论上可将打靶效率提高到18%以上。本文经过筛选将人基因组上的中度重复序列rDNA的18S和5.8S之间的间隔序列ITS1作为靶位点,设计针对靶标的一对三联体锌指蛋白。分别与FokⅠ切割结构域连接,合成了一对锌指嵌合核酸酶。接着,将该对锌指核酸酶分别插入到真核表达核载体pcDNA3.1-NLS中,构建了一对真核表达载体pcS-ZFNs。利用PCR方法从pEGFP-N1中扩增pCMV-EGFP,从人基因组rDNA基因家族中靶基因插入位点两侧分别扩增两条基因打靶同源重组引导序列DS1和DS2,将DS1、DS2片段插入pMD19-pCMV-EGFP中,构建成功多位点基因打靶载体pMD19-DS1-pCMV-EGFP-DS2。
本课题选取HEK293细胞作为研究对象,通过共转染一对pcS-ZFNs和基因打靶载体,得到了生长增殖稳定、绿色荧光持续表达的基因整合细胞。经提取基因组,进行靶位点验证性PCR试验,证实了基因的定点整合,并将定点整合效率提高到了20%以上。本课题的研究避免了药物的毒副作用,不经药物筛选就明显提高了基因打靶效率,为动物定点转基因技术的建立和人类遗传疾病的基因治疗提供了技术方法。
The researchers have been more and more caring about the technique of gene target which is gradually coming to be mature.However,the traditional gene target is merely relied on homologous recombination,whose efficiency is just between 10~(-6) and 10~(-5).Recently,we have managed to establish a technique of multiple locus on gene targeting using the middle repeat of the interval sequence of rDNA as a target,and succeeded to produce the transgenic mandarin fishes and the recombination cow fertilized egg cell.Although in these researches the efficiency of gene target has been up to 200-300 folds,it is still a long way to real high efficiency. According to recent research,so many techniques about gene target with high efficiency have been put into use at home and abroad,in which a famous method is the application of zinc finger nuclease that can high improved the efficiency of gene target.
Our research has combined the application of zinc finger nuclease with the multiple loci gene target,which can improved the efficiency of gene target up to 18%in theory.This article has used the ITS1 between 18S and 5.8S of rDNA as a target,and got a pair of three-zinc finger gene cutting the target sequence which combined with the coding sequence of Fok I slicing domain,thus we got a pair of zinc finger nucleases gene.And then,we inserted each zinc finger nuclease gene into the eukaryotic expression vector of pcDNA3.1-NLS,and got a pair of pcS-ZFNs.It was inserted into the pMD19-T vector for the construction of the recombinant vector pMD19-DS1-pCMV-EGFP-DS2 that the pCMV-EGFP gene which amplified by PCR using pEGFP-N1 out of MCS as template and two homogenous recombination arms of DS1 and DS2 which were amplified from human genome by PCR.
By cotransfecting a pair of pcS-ZFNs and recombinant vector into HEK293 cells,we got the gene targeting cells with stable growth and reproduction,and persistent expression of the green fluorescence.After extracting the genome DNA of former HEK293 cells and testing the gene target by PCR,we managed to test the recombination efficiency of the gene target which is up to 20%.This research can avoid the bad reaction of the drug,improve the efficiency of gene target without drug selection and provide a transgenic technique for the transgenic animal and the gene therapy and diagnostics of human.
引文
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[32]Lei Zhang,S.Kaye Spratt,Qiang Liu.Synthetic Zinc Finger Transcription Factor Action at an Endogenous Chromosomal Site.The Journal of Biological Chemistry.2000,275(43):33850-33860
[33]Rodier,J.F.,Janser,J.C.Routiot,T.,et al.Sentinel lymph node procedure-a valid selection criterion? Swiss Surg,1999,5(5):214-216
[34]DAVID J.SEGAL,BIRGIT DREIER.Toward controlling gene expression at will:Selection and design of zinc finger domains recognizing each of the 5'-GNN-3' DNA target sequences.Proc.Natl.Acad.Sci.USA,1999,96:2758-2763
[35]王波,唐冬生,蒋泓,等.通过PCR技术简单扩增奶牛高GC含量的rDNA重复序列[J].湖南农业大学学报(自然科学版),2005,31(6):602-604.
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[37]汤熙翔,夏家辉.提高GC富集区的扩增效率[J].国外医学遗传学分册,1999,22(3):120-123
[38]郑以漫,李小英,朱宏达,等.高GC含量靶基因的PCR扩增方法[J].中华医学遗传学杂志,1999,16(3):197-198
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[2]Schnieke A S,Kind A J,Ritchie W A,et al.Human factor Ⅸ transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts[J].Science,1997,278:2130-2133
[3]Baquisi A,Behboodi E,Melican DT,et al.Production of goats by somatic cell nuclear tranfer[J].Nat biotechnol,1999,17:456-461
[4]Cibelli J B,Stice S L,Golueke P J,et al.Cloned transgenic calved produced from nonquiescent fetal fibroblasts[J].Science,1998,280:1256-1258
[5]Li C,Chen L,Iwata T,et al.A Lys644Glu substitution in fibroblast growth factor receptor3(FGFR3) causes dwarfism in mice by activation of STAT3 and ink4 cell cycle inhibitors[J].Hum Mol Genet,1999,8(1):35-44
[6]龚国春,戴蕴平,樊宝良,等.利用体细胞核移植技术生产转基因牛[J].科学通报,2003,48(24):2528-2533
[7]唐冬生,施家琦,田允波,等.体细胞多位点基因打靶技术的研究[J].佛山科学技术学院学报(自然科学版)2002,20(2):64-68
[8]Kim,Y.G.,Cha,J,Chandrasegaran,s.Hybrid restriction enzymes:Zinc finger fusions to Fok I cleavage domain.Proc Natl Acad Sci USA,1996,93:1156-1160
[9]刘广振.奶牛胎儿成纤维细胞系的建立及多位点基因打靶的研究.华南农业大学硕士学位论文,2006
[10]唐冬生,高东,蒋泓,严霞.多位点基因打靶技术的鳜鱼体内实验研究[J].广西农业生物科学,2006,25:205-206
[11]Brown,R.S.,Sander,C.Argos,P.The primary structure of transcription factor TFⅢA has 12 consecutive repeats.FEBS Lett,1985,186(2):271-274
[12]Miller,J.,McLachlan,A.D.Klug,A.Repetitive zinc-binding domains in the protein transcription factor ⅢA from Xenopus oocyteso EMBO J,1985,4(6):1609-1614
[13]Wolfe,S.A.,Greisman,H.A.Ramm,E.I.,et al.Analysis of zinc fingers optimized via phage display:evaluating the utility of a recognition code.J Mol Biol,1999,285(5):1917-1934
[14]Pavletich,N.P.Pabo,C.O.Zinc finger-DNA recognition:crystal structure of a Zif268-DNA complex at 2.1 A.Science,1991,252(5007):809-817
[15]Nagaoka,M.,Nomura,W.Shiraishi,Y.,et al.Significant effect of linker sequence on DNA recognition by multi-zinc finger protein.Biochem Biophys Res Commun,2001,282(4):1001-1007
[16]Dreier,B.,Beerli,R.R.Segal,D.J.,et al.Development of zinc finger domains for recognition of the 5'-ANN-3' family of DNA sequences and their use in the construction of artificial transcription factors.J Biol Chem,2001,276(31):29466-29478
[17]Mark Isalan,Aaron Klug,A.Y.C.A rapid,generally applicable method to engineer zinc fingers illustrated by targeting the HIV-1 promoter Nat Biotechnol,2001,19:656-660
[18]Pabo,C.O.,Peisach,E.Grant,R.A.Design and selection of novel Cys2His2 zinc finger proteins.Annu Rev Biochem,2001,70:313-340
[19]Desjarlais,J.R.Berg,J.M.Use of a zinc-finger consensus sequence framework and specificity rules to design specific DNA binding proteins.Proc Natl Acad Sci U S A,1993,90(6):2256-2260
[20]曾芳.锌指核酸酶基因的人工合成与表达.华南农业大学硕士学位论文,2008.5
[21]DAVID A.WAH,JURATE BITINAITE.Structure of Fok I has implications for DNA cleavage.Proc.Natl.Acad.Sci.USA,1998,95:10564-10569
[22]Smith,J.,Berg,J.M.,Chandrasegaran,S.A detailed study of the substrate specificity of a chimeric restriction enzyme.Nucleic Acids Res.1999,27:674-681
[23]MARINA BIBIKOVA,DANA CARROLL.Stimulation of Homologous Recombination through Targeted Cleavage by Chimeric Nucleases.MOLECULAR AND CELLULAR BIOLOGY,2001,21(1):289-297
[24]Mala Mani,Jeff Smith.Binding of two zinc finger nuclease monomers to two specific sites is required for effective double-strand DNA cleavage.Biochemical and Biophysical Research Communications.2005,334:1191-1197
[25]Moore M,Klug A,Choo Y.Improved DNA binding specificity from polyzinc finger peptides by using strings of two-finger units.Proc Natl Acad Sci.USA,2001,98:1437-1441
[26]Jeffry D.Sander,Peter Zaback.Zinc Finger Targeter(ZiFiT):an engineered zinc finger/target site design tool.Nucleic Acids Research,2007(35):W599-W605
[27]Paques,F.Haber,J.E.Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae.Microbiol Mol Biol Rev,1999,63(2):349-404
[28]Symington,L.S.Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair.Microbiol Mol Biol Rev,2002,66(4):630-670
[29]West,S.C.Molecular views of recombination proteins and their control.Nat Rev Mol Cell Biol,2003,4(6):435-445
[30]Urnov,F.D.,Miller,J.C.Lee,Y.L.,et al.Highly efficient endogenous human gene correction using designed zinc-finger nucleases.Nature,2005,435(7042):646-651
[31]J.C.Miller,M.C.Holmes J.Wang,et al.An improved zinc-finger nuclease architecture for highly specific genome editing.Nat Biotechnol,2007,25(7):778-85
[32]Lei Zhang,S.Kaye Spratt,Qiang Liu.Synthetic Zinc Finger Transcription Factor Action at an Endogenous Chromosomal Site.The Journal of Biological Chemistry.2000,275(43):33850-33860
[33]Rodier,J.F.,Janser,J.C.Routiot,T.,et al.Sentinel lymph node procedure-a valid selection criterion? Swiss Surg,1999,5(5):214-216
[34]DAVID J.SEGAL,BIRGIT DREIER.Toward controlling gene expression at will:Selection and design of zinc finger domains recognizing each of the 5'-GNN-3' DNA target sequences.Proc.Natl.Acad.Sci.USA,1999,96:2758-2763
[35]王波,唐冬生,蒋泓,等.通过PCR技术简单扩增奶牛高GC含量的rDNA重复序列[J].湖南农业大学学报(自然科学版),2005,31(6):602-604.
[36]邢小黑,姜卫红.提高富含GC碱基DNA模板PCR扩增效率的方法[J].南京农业大学学报,1999,22(3):112-114
[37]汤熙翔,夏家辉.提高GC富集区的扩增效率[J].国外医学遗传学分册,1999,22(3):120-123
[38]郑以漫,李小英,朱宏达,等.高GC含量靶基因的PCR扩增方法[J].中华医学遗传学杂志,1999,16(3):197-198
[39]李爱丽,姜涛,马峙英,等.提高PCR产物的几种有效方法[J].生物技术通报,2002,(6):33-35
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