CRISPR/Cas9介导的外源基因靶向插入鸡EAV-HP基因组
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摘要
本研究旨在通过CRISPR/Cas9介导外源基因靶向插入鸡EAV-HP基因组。首先设计特异性引物并扩增鸡内源性病毒(EAV-HP)左右同源臂和增强型绿色荧光蛋白(eGFP)基因表达盒,然后通过重叠延伸PCR技术将两个同源臂DNA连接至eGFP表达盒两侧,获得全长DNA片段LER,并克隆至pMD19-T载体,获得携带eGFP基因的供体载体pMDT-LER。随后在HEK293T细胞中验证供体载体pMDT-LER能成功表达eGFP后,将EAV-HP打靶载体和供体载体共转染至DF-1细胞,观察绿色荧光阳性细胞,提取细胞基因组,PCR检测外源基因eGFP成功整合至鸡基因组EAV-HP位点。最后,将转基因细胞DF-1传至第7代,用PCR和Western blotting检测eGFP在转基因细胞中稳定表达。文中初步验证外源基因eGFP能整合至鸡EAV-HP位点并稳定表达,为转基因鸡的研究提供新整合位点。
The study aims to use CRISPR/Cas9 introducing foreign gene targeted knock-in into chicken EAV-HP genome. First, specific primers were designed for amplification of EAV-HP left, right homologous arms and enhanced green fluorescent protein(eGFP) expression cassette. PCR products of homologous arms were ligated to both sides of eGFP by overlap extension PCR, resulting in full-length donor DNA fragment designated as LER. Then LER fragments were cloned into pMD19-T to obtain donor vector pMDT-LER. Subsequently, the donor vector pMDT-LER was transfected into HEK293T cells to verify the expression of eGFP gene. Furthermore, co-transfection of CRISPR/Cas9 expression vector and pMDT-LER into chicken DF-1 cells was performed to achieve eGFP transgenic cells. Meanwhile, eGFP expression was observed in cells, and the event of eGFP integration into EAV-HP genome was detectable by amplification of target DNA. Finally, the transgenic DF-1 cells were passaged seven times, and the stable integration and expression of eGFP was checked by PCR and Western blotting. These results demonstrated that eGFP gene was knocked into the EAV-HP genome successfully, which provides a new integration site for research of transgenic chicken.
The study aims to use CRISPR/Cas9 introducing foreign gene targeted knock-in into chicken EAV-HP genome. First, specific primers were designed for amplification of EAV-HP left, right homologous arms and enhanced green fluorescent protein(eGFP) expression cassette. PCR products of homologous arms were ligated to both sides of eGFP by overlap extension PCR, resulting in full-length donor DNA fragment designated as LER. Then LER fragments were cloned into pMD19-T to obtain donor vector pMDT-LER. Subsequently, the donor vector pMDT-LER was transfected into HEK293T cells to verify the expression of eGFP gene. Furthermore, co-transfection of CRISPR/Cas9 expression vector and pMDT-LER into chicken DF-1 cells was performed to achieve eGFP transgenic cells. Meanwhile, eGFP expression was observed in cells, and the event of eGFP integration into EAV-HP genome was detectable by amplification of target DNA. Finally, the transgenic DF-1 cells were passaged seven times, and the stable integration and expression of eGFP was checked by PCR and Western blotting. These results demonstrated that eGFP gene was knocked into the EAV-HP genome successfully, which provides a new integration site for research of transgenic chicken.
引文
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[15]Wang L,Zhang T,Yin YJ.Research progress of retroviral vector-mediated generation of transgenic chicken.China Poultry,2015,37(14):44-48(in Chinese).王令,张涛,尹亚军.逆转录病毒载体介导的转基因鸡研究进展.中国家禽,2015,37(14):44-48.
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[19]Li N,Sun YM,Zhao BH.Cloning of F gene of newcastle diseasa virus HeB02 isolate and the study of its DNA vaccine.Chin J Biotech,2006,22(3):445-450(in Chinese).李楠,孙一敏,赵宝华.鸡新城疫病毒HeB02分离株F基因的克隆及其DNA疫苗的研究.生物工程学报,2006,22(3):445-450.
[20]Liu TX.Oviduct-specific expression of human neutrophil defensin 4 in transgenic chickens[D].Beijing:China Agricultural University,2015(in Chinese).刘同欣.输卵管特异性表达人防御素4基因转基因鸡的制备[D].北京:中国农业大学,2015.
[21]Sacco MA,Flannery DMJ,Howes K,et al.Avian endogenous retrovirus EAV-HP shares regions of identity with avian leukosis virus subgroup J and the avian retrotransposon ART-CH.J Virol,2000,74(3):1296-1306.
[22]Sacco MA,Howes K,Venugopal K.Intact EAV-HPendogenous retrovirus in Sonnerat’s jungle fowl.J Virol,2001,75(4):2029-2032.
[23]Sacco MA,Howes K,Smith LP,et al.Assessing the roles of endogenous retrovirus EAV-HP in avian leukosis virus subgroup J emergence and tolerance.JVirol,2004,78(19):10525-10535.
[24]Wang ZP,Qu LJ,Yao JF,et al.An EAV-HP insertion in5?flanking region of SLCO1B3 causes blue eggshell in the chicken.PLoS Genet,2013,9(1):e1003183.
[25]Zhou CC,Liu XX,Xie HH,et al.The new member of CRISPR family,CRISPR-Cpf1.Progr Biochem Biophys,2018,45(6):582-592(in Chinese).周晨晨,刘写写,谢海华,等.CRISPR家族新成员:CRISPR-Cpf1.生物化学与生物物理进展,2018,45(6):582-592.
[26]Wang C,Wang KJ.Advances in CRISPR-Cas-mediated genome editing system in plants.Chin J Biotech,2017,33(10):1712-1722(in Chinese).王春,王克剑.CRISPR-Cas系统在植物基因组编辑中的研究进展.生物工程学报,2017,33(10):1712-1722.
[27]Hasty P,Rivera-Pérez J,Chang C,et al.Target frequency and integration pattern for insertion and replacement vectors in embryonic stem cells.Mol Cellular Biol,1991,11(9):4509-4517.
[2]Liu B,Wei W,Wang LH.Progress in new techniques of gene editing.Subtrop Agric Res,2013,9(4):262-269(in Chinese).刘蓓,尉玮,王丽华.基因编辑新技术研究进展.亚热带农业研究,2013,9(4):262-269.
[3]Ji HY,Zhu HZ.Progress of genome editing approaches towards gene therapy.Chin Bull Life Sci,2015,27(1):71-82(in Chinese).季海艳,朱焕章.基因编辑技术在基因治疗中的应用进展.生命科学,2015,27(1):71-82.
[4]Men K,Duan XM,He ZY,et al.CRISPR/Cas9-mediated correction of human genetic disease.Sci China Life Sci,2017,60(5):447-457.
[5]Zhou WL,Tang L,Cheng K,et al.Construction of prototrophic glutathione-high-producing yeast strain mediated by CRISPR/Cas9.Chin J Biotech,2017,33(12):1999-2008(in Chinese).周文龙,唐亮,成凯,等.CRISPR/Cas9介导的高产谷胱甘肽原养型酵母工程菌的构建.生物工程学报,2017,33(12):1999-2008.
[6]Li JH,Shou J,Wu Q.DNA fragment editing of genomes by CRISPR/Cas9.Hereditas(Beijing),2015,37(10):992-1002(in Chinese).李金环,寿佳,吴强.CRISPR/Cas9系统在基因组DNA片段编辑中的应用.遗传,2015,37(10):992-1002.
[7]Hsu PD,Lander ES,Zhang F.Development and applications of CRISPR-Cas9 for genome engineering.Cell,2014,157(6):1262-1278.
[8]Jao LE,Wente SR,Chen WB.Efficient multiplex biallelic zebrafish genome editing using a CRISPRnuclease system.Proc Natl Acad Sci USA,2013,110(34):13904-13909.
[9]Xu JX,Li N.Biosafety assessment of genetically engineered animals.Chin J Biotech,2012,28(3):267-281(in Chinese).许建香,李宁.转基因动物生物安全研究与评价.生物工程学报,2012,28(3):267-281.
[10]Wu B,Zhu ZY.Integration sites of transgenes in transgenic animals.Hereditas(Beijing),2003,25(1):77-80(in Chinese).吴波,朱作言.转基因动物整合位点的研究进展.遗传,2003,25(1):77-80.
[11]Ma LY,Wang YZ,Wang HT,et al.Screen and verification for transgene integration sites in pigs.Sci Rep,2018,8(1):7433.
[12]Liu WL,Li H.Production and research prospect of transgenic chickens.China Poult,2013,35(18):2-5(in Chinese).刘文利,李辉.转基因鸡的制作及研究展望.中国家禽,2013,35(18):2-5.
[13]Woodcock ME,Idoko-Akoh A,McGrew MJ.Gene editing in birds takes flight.Mamm Genome,2017,28(7/8):315-323.
[14]Liu TB,Hu Y,Guo SY,et al.Identification and characterization of MYH9 locus for high efficient gene knock-in and stable expression in mouse embryonic stem cells.PLoS ONE,2018,13(2):e192641.
[15]Wang L,Zhang T,Yin YJ.Research progress of retroviral vector-mediated generation of transgenic chicken.China Poultry,2015,37(14):44-48(in Chinese).王令,张涛,尹亚军.逆转录病毒载体介导的转基因鸡研究进展.中国家禽,2015,37(14):44-48.
[16]Man CL,Chen Y.Retrovirus and transgenic chicken.China Biotechnol,2007,27(7):117-121(in Chinese).满朝来,陈岩.逆转录病毒与转基因鸡.中国生物工程杂志,2007,27(7):117-121.
[17]Wang L,Zhang T,Lu HZ,et al.Rapid construction of CRISPR/Cas9 system for target genomic editing.Chin JBiochem Mol Biol,2015,31(10):1117-1124(in Chinese).王令,张涛,路宏朝,等.快速构建CRISPR/Cas9基因组靶向编辑系统.中国生物化学与分子生物学报,2015,31(10):1117-1124.
[18]Andrew L.Why Did the Chicken Cross the World?Translated by Xiao Aoran.China:CITIC Press Group,2017:1-11(in Chinese).安德鲁·劳勒.鸡征服世界.萧傲然,译.中国:中信出版集团股份有限公司,2017:1-11.
[19]Li N,Sun YM,Zhao BH.Cloning of F gene of newcastle diseasa virus HeB02 isolate and the study of its DNA vaccine.Chin J Biotech,2006,22(3):445-450(in Chinese).李楠,孙一敏,赵宝华.鸡新城疫病毒HeB02分离株F基因的克隆及其DNA疫苗的研究.生物工程学报,2006,22(3):445-450.
[20]Liu TX.Oviduct-specific expression of human neutrophil defensin 4 in transgenic chickens[D].Beijing:China Agricultural University,2015(in Chinese).刘同欣.输卵管特异性表达人防御素4基因转基因鸡的制备[D].北京:中国农业大学,2015.
[21]Sacco MA,Flannery DMJ,Howes K,et al.Avian endogenous retrovirus EAV-HP shares regions of identity with avian leukosis virus subgroup J and the avian retrotransposon ART-CH.J Virol,2000,74(3):1296-1306.
[22]Sacco MA,Howes K,Venugopal K.Intact EAV-HPendogenous retrovirus in Sonnerat’s jungle fowl.J Virol,2001,75(4):2029-2032.
[23]Sacco MA,Howes K,Smith LP,et al.Assessing the roles of endogenous retrovirus EAV-HP in avian leukosis virus subgroup J emergence and tolerance.JVirol,2004,78(19):10525-10535.
[24]Wang ZP,Qu LJ,Yao JF,et al.An EAV-HP insertion in5?flanking region of SLCO1B3 causes blue eggshell in the chicken.PLoS Genet,2013,9(1):e1003183.
[25]Zhou CC,Liu XX,Xie HH,et al.The new member of CRISPR family,CRISPR-Cpf1.Progr Biochem Biophys,2018,45(6):582-592(in Chinese).周晨晨,刘写写,谢海华,等.CRISPR家族新成员:CRISPR-Cpf1.生物化学与生物物理进展,2018,45(6):582-592.
[26]Wang C,Wang KJ.Advances in CRISPR-Cas-mediated genome editing system in plants.Chin J Biotech,2017,33(10):1712-1722(in Chinese).王春,王克剑.CRISPR-Cas系统在植物基因组编辑中的研究进展.生物工程学报,2017,33(10):1712-1722.
[27]Hasty P,Rivera-Pérez J,Chang C,et al.Target frequency and integration pattern for insertion and replacement vectors in embryonic stem cells.Mol Cellular Biol,1991,11(9):4509-4517.
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