转人抑癌基因p53鸡的研究
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摘要
【目的】探索MAR调控序列对外源人抑癌基因p53在转基因鸡体内整合稳定性的影响,为转基因禽类输卵管生物反应器的建立奠定基础。【方法】利用脂质体包埋法,将pEGFP-N1-p53/MAR重组真核表达载体转染人肝癌细胞(Hep3B),荧光显微镜和透射电镜检测转染含人野生型p53基因的Hep3B细胞形态及超微观结构的变化情况;显微注射法将脂质体介导的重组载体注入鸡X期胚盘的明区和暗区,制备G0代转基因鸡。以EGFP为报告基因,人工授精法制备G1代转基因鸡,对G0代和G1代鸡体外源基因p53进行PCR检测,并对G1代转基因鸡进行小动物活体成像检测。【结果】转染含人p53基因重组载体的Hep3B细胞明显皱缩变圆;微观结构层面,粗面内质网不明显,线粒体肿胀,细胞核中异染色质边集明显,并出现凋亡小体。G0代转基因鸡暗区组孵化率(45%)显著高于明区组孵化率(0)(P<0.05),但均显著低于对照组孵化率(82.5%)(P<0.05);获得4只基因组中含有外源p53基因的G0代转基因鸡,外源基因转染率为22.2%(4/18);获得2只G1代转基因鸡,转基因世代遗传率为3.08%(2/65)。【结论】重组真核表达载体pEGFP-N1-p53/MAR可以促进人肝癌细胞Hep3B的凋亡;脂质体介导的含MAR序列调控元件的pEGFP-N1-p53/MAR质粒,可以提高p53在转基因鸡基因组中的整合稳定性,并可遗传给后代。
【Objective】 The study was to explore the effect of MAR regulation sequence on the integration stability of exogenous human oncogene p53 in transgenic chickens,and to lay a foundation for the establishment of transgenic poultry oviduct bioreactor.【Method】 Liposome embedding method was used to carry recombinant eukaryotic expression vector pEGFP-N1-p53/MAR into human liver cancer cells(Hep3 B).The change of cellular morphology and microstructure of Hep3 B were detected by fluorescence microscope and transmission electron microscopy.G0 generation of transgenic chicken was preparation using liposome embedding method to deliver the recombinant expression vector pEGFP-N1-p53/MAR into chicken embryos area pellucida and area opaca.Using EGFP as a marker gene,artificial insemination was used to prepare G1 generation transgenic chicken.Screening and identification exogenous p53 were conducted by PCR genetic testing and Xenogen IVIS were used to detect the fluorescence of G1 transgenic chicken.【Result】 Cellular morphology of Hep3 B transfected with people p53 gene became shrinkage and round.Microstructural changes included rough endoplasmic reticulum(ER) inconspicuous,mitochondria(M) swelling,nucleus heterochromatin(HC) side,and accompanied with apoptotic body(AB).G0 generation of transgenic chicken area opaca group had significantly higher hatching rate(45%) than the area pellucida hatching rate(0)(P<0.05),both were lower than that of the control group(82.5%)(P<0.05).The PCR verified that 4 hatched chicks from the G0 generation expressing the human tumor suppressor gene p53 were obtained and the exogenous gene transfection rate was 22.2%(4/18).The fluorescence signal detection and PCR verified that 2 hatched chicks of G1 generation expressing exogenous gene and the genetically modified generation genetic rate was 3.08%(2/65).【Conclusion】 Recombinant eukaryotic expression vector pEGFP-N1-p53/MAR can contribute to the apoptosis of human liver cancer cells Hep3 B.Liposome mediated pEGFP-N1-p53/MAR plasmid with MAR sequence control element could improve the exogenous gene p53 stability of consolidation in transgenic chicken genome and transfer to future generations.
【Objective】 The study was to explore the effect of MAR regulation sequence on the integration stability of exogenous human oncogene p53 in transgenic chickens,and to lay a foundation for the establishment of transgenic poultry oviduct bioreactor.【Method】 Liposome embedding method was used to carry recombinant eukaryotic expression vector pEGFP-N1-p53/MAR into human liver cancer cells(Hep3 B).The change of cellular morphology and microstructure of Hep3 B were detected by fluorescence microscope and transmission electron microscopy.G0 generation of transgenic chicken was preparation using liposome embedding method to deliver the recombinant expression vector pEGFP-N1-p53/MAR into chicken embryos area pellucida and area opaca.Using EGFP as a marker gene,artificial insemination was used to prepare G1 generation transgenic chicken.Screening and identification exogenous p53 were conducted by PCR genetic testing and Xenogen IVIS were used to detect the fluorescence of G1 transgenic chicken.【Result】 Cellular morphology of Hep3 B transfected with people p53 gene became shrinkage and round.Microstructural changes included rough endoplasmic reticulum(ER) inconspicuous,mitochondria(M) swelling,nucleus heterochromatin(HC) side,and accompanied with apoptotic body(AB).G0 generation of transgenic chicken area opaca group had significantly higher hatching rate(45%) than the area pellucida hatching rate(0)(P<0.05),both were lower than that of the control group(82.5%)(P<0.05).The PCR verified that 4 hatched chicks from the G0 generation expressing the human tumor suppressor gene p53 were obtained and the exogenous gene transfection rate was 22.2%(4/18).The fluorescence signal detection and PCR verified that 2 hatched chicks of G1 generation expressing exogenous gene and the genetically modified generation genetic rate was 3.08%(2/65).【Conclusion】 Recombinant eukaryotic expression vector pEGFP-N1-p53/MAR can contribute to the apoptosis of human liver cancer cells Hep3 B.Liposome mediated pEGFP-N1-p53/MAR plasmid with MAR sequence control element could improve the exogenous gene p53 stability of consolidation in transgenic chicken genome and transfer to future generations.
引文
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[18] Park S H.CpG methylation modulates tissue-specific expression of a transgene in chickens [J].Theriogenology,2010,74:805-816.
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[20] Ley D,Harraghy N,Le Fourn V,et al.MAR elements and transposons for improved transgene integration and expression [J].PLoS One,2013,31(4):62784.
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[23] 沈强,俞彰,法京,等.电镜下几种凋亡细胞的形态特征 [J].复旦学报(医学版),2010,37(3):322-325.Shen Q,Yu Z,Fa J,et al.Morphological features of different apoptotic cells under the electron microscope [J].Fudan Univ J(Med Sci),2010,37(3):322-325.
[24] Majno G,Joris I.Apoptosis,oncosis and necrosis:an overview of cell death [J].Am J Pathol,1995,146(1):3-15.
[25] Hacker G.The morphology of apoptosis [J].Cell Tissue Res,2000,301(1):5-17.
[26] Tseng C L,Peng C L,Huang J Y,et al.Gelatin nanoparticles as gene carriers for transgenic chicken applications [J].Journal of Biomaterials Applications,2013,27(8):1055-1065.
[27] Kwon M S,Koo B C,Choi B R,et al.Development of transgenic chickens expressing enhanced green fluorescent protein [J].Biochemical and Biophysical Research Communications,2004,320(2):442-448.
[28] Mozdziak P E,Borwornpinyo S,McCoy D W,et al.Development of transgenic chickens expressing bacterial beta-galactosidase [J].Developmental Dynamics,2003,226(3):439-445.
[29] Harvey A J,Speksnijder G,Baugh L R,et al.Expression of exogenous protein in the egg white of transgenic chickens [J].Nature Biotechnology,2002,20(4):396-399.
[30] Xu S,Sun Y,Ding H,et al.Efficient production of transgenic chickens using self-inactive HIV-based lentiviral vectors [J].Current Zoology,2009,55(5):383-387.
[31] Kwon M S,Koo B C,Roh J Y,et al.Production of transgenic chickens expressing a tetracycline-inducible GFP gene [J].Biochemical and Biophysical Research Communications,2011,410(4):890-894.
[32] Koo B C,Kwon M S,Lee H,et al.Tetracycline-dependent expression of the human erythropoietin gene in transgenic chickens [J].Transgenic Research,2010,19(3):437-447.
[33] 孙国锋,张智英,白仪春,等.慢病毒转基因鸡的研究 [J].西北农林科技大学学报(自然科学版),2015,43(12):1-6.Sun G F,Zhang Z Y,Bai Y C,et al.Transgenic chicken generated by testis injection of recombinant lentivirus [J].Journal of Northwest A & F University (Nat Sci Ed),2015,43(12):1-6.
[34] Furlan-Magaril M,Rebollar E,Guerrero G,et al.An insulator embedded in the chicken alpha-globin locus regulates chromatin domain configuration and differential gene expression [J].Nucleic Acids Research,2011,39(1):89-103.
[35] Cool S K,Breyne K,Meyer E,et al.Comparison of in vivo optical systems for bioluminescence and fluorescence imaging [J].Journal of Fluorescence,2013,23(5):909-920.
[36] Chapman S C,Lawson A,Macarthur W C,et al.Ubiquitous GFP expression in transgenic chickens using a lentiviral vector [J].Development,2005,132(5):935-940.
[37] Koo B C,Kwon M S,Choi B R,et al.Production of germline transgenic chickens expressing enhanced green fluorescent protein using a MoMLV-based retrovirus vector [J].The Faseb Journal,2006,20(13):2251-2260.
[38] 李振红.转人抗癌基因P53鸡的研究 [D].河南洛阳:河南科技大学,2011.Li Z H.The study on transferring of human tumor suppressor gene P53 in chicken [D].Luoyang,Henan:Henan University of Science and Technology,2011.
[39] Song Z,Li Z H,Lei X Q,et al.Construction of the mammalian expressing vector pEGFP-N1-P53 and its expression successful in chicken fibroblast cells and blastoderm [J].Genet Mol Res,2015,14(1):931-939.
[2] Harrison R L,Jarvis D L.Protein N-glycosylation in the baculovirus-insect cell expression system and engineering of insect cells to produce “mammalianized” recombinant glycoproteins [J].Adv Virus Res,2006,68:159-191.
[3] O’Callaghan P M,James D C.Systems biotechnology of mammalian cell factories [J].Brief Funct Genomic Proteomic,2008,7(2):95-110.
[4] Houdebine L M.Production of pharmaceutical proteins by transgenic animals [J].Comp Immunol Microbiol Infect Dis,2009,32(2):107-121.
[5] Rudolph N S.Biopharmaceutical production in transgenic livestock [J].Trends Biotechnol,1999,17(9):367-374.
[6] Houdebine L M.Transgenic animal bioreactors [J].Transgenic Res,2000,9(4/5):305-320.
[7] Kues W A,Niemann H.The contribution of farm animals to human health [J].Trends Biotechnol,2004,22(6):286-294.
[8] Ivarie R.Avian transgenesis:progress towards the promise [J].Trends Biotechnol,2003,21(1):14-19.
[9] Motono M,Yamada Y,Hattori Y,et al.Production of transgenic chickens from purified primordial germ cells infected with a lentiviral vector [J].J Biosci Bioeng,2010,109(4):315-321.
[10] Macdonald J,Taylor L,Sherman A,et al.Efficient genetic modification and germ-line transmission of primordial germ cells using piggyBac and Tol2 transposons [J].Proc Natl Acad Sci USA,2012,109(23):1466-1472.
[11] Park S H,Kim J N,Park T S,et al.CpG methylation modulates tissuespecific expression of a transgene in chickens [J].Theriogenology,2010,74(5):805-816.
[12] Naldini L,Blomer U,Gallay P,et al.In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector [J].Science,1996,272(5259):263-267.
[13] McGrew M J,Sherman A,Ellard F M,et al.Efficient production of germline transgenic chickens using lentiviral vectors [J].Embo Reports,2004,5(7):728-733.
[14] Park T S,Lee H G,Moon J K,et al.Deposition of bioactive human epidermal growth factor in the egg white of transgenic hens using an oviduct-specific minisynthetic promoter [J].The Faseb Journal,2015,29(6):2386-2396.
[15] Sang H.Transgenic chickens:methods and potential applications [J].Trends Biotechnol,1994,12(10):415-420.
[16] Park T S,Han J Y.PiggyBac transposition into primordial germ cells is an efficient tool for transgenesis in chickens [J].Proc Natl Acad Sci USA,2012,109(24):9337-9341.
[17] Leighton P A,van de Lavoir M C,Diamond J H,et al.Genetic modification of primordial germ cells by gene trapping,gene targeting,and phiC31 integrase [J].Mol Reprod Dev,2008,75:1163-1175.
[18] Park S H.CpG methylation modulates tissue-specific expression of a transgene in chickens [J].Theriogenology,2010,74:805-816.
[19] van de Lavoir M C,Diamond J H,Leighton P A,et al.Germline transmission of genetically modified primordial germ cells [J].Nature,2006,441(7094):766-769.
[20] Ley D,Harraghy N,Le Fourn V,et al.MAR elements and transposons for improved transgene integration and expression [J].PLoS One,2013,31(4):62784.
[21] Nishijima K,Iijima S.Transgenic chickens [J].Development Growth and Differentiation,2013,55(1):207-216.
[22] 郭军,房殿春,范宗江,等.HCCR-2基因沉默后HepG2肝癌细胞形态变化 [J].昆明医学院学报,2010,31(10):27-30.Guo J,Fang D C,Fan Z J,et al.The morphology of HepG2 liver cancer cells after silencing HCCR-2 gene [J].Journal of Kunming Medical University,2010,31(10):27-30.
[23] 沈强,俞彰,法京,等.电镜下几种凋亡细胞的形态特征 [J].复旦学报(医学版),2010,37(3):322-325.Shen Q,Yu Z,Fa J,et al.Morphological features of different apoptotic cells under the electron microscope [J].Fudan Univ J(Med Sci),2010,37(3):322-325.
[24] Majno G,Joris I.Apoptosis,oncosis and necrosis:an overview of cell death [J].Am J Pathol,1995,146(1):3-15.
[25] Hacker G.The morphology of apoptosis [J].Cell Tissue Res,2000,301(1):5-17.
[26] Tseng C L,Peng C L,Huang J Y,et al.Gelatin nanoparticles as gene carriers for transgenic chicken applications [J].Journal of Biomaterials Applications,2013,27(8):1055-1065.
[27] Kwon M S,Koo B C,Choi B R,et al.Development of transgenic chickens expressing enhanced green fluorescent protein [J].Biochemical and Biophysical Research Communications,2004,320(2):442-448.
[28] Mozdziak P E,Borwornpinyo S,McCoy D W,et al.Development of transgenic chickens expressing bacterial beta-galactosidase [J].Developmental Dynamics,2003,226(3):439-445.
[29] Harvey A J,Speksnijder G,Baugh L R,et al.Expression of exogenous protein in the egg white of transgenic chickens [J].Nature Biotechnology,2002,20(4):396-399.
[30] Xu S,Sun Y,Ding H,et al.Efficient production of transgenic chickens using self-inactive HIV-based lentiviral vectors [J].Current Zoology,2009,55(5):383-387.
[31] Kwon M S,Koo B C,Roh J Y,et al.Production of transgenic chickens expressing a tetracycline-inducible GFP gene [J].Biochemical and Biophysical Research Communications,2011,410(4):890-894.
[32] Koo B C,Kwon M S,Lee H,et al.Tetracycline-dependent expression of the human erythropoietin gene in transgenic chickens [J].Transgenic Research,2010,19(3):437-447.
[33] 孙国锋,张智英,白仪春,等.慢病毒转基因鸡的研究 [J].西北农林科技大学学报(自然科学版),2015,43(12):1-6.Sun G F,Zhang Z Y,Bai Y C,et al.Transgenic chicken generated by testis injection of recombinant lentivirus [J].Journal of Northwest A & F University (Nat Sci Ed),2015,43(12):1-6.
[34] Furlan-Magaril M,Rebollar E,Guerrero G,et al.An insulator embedded in the chicken alpha-globin locus regulates chromatin domain configuration and differential gene expression [J].Nucleic Acids Research,2011,39(1):89-103.
[35] Cool S K,Breyne K,Meyer E,et al.Comparison of in vivo optical systems for bioluminescence and fluorescence imaging [J].Journal of Fluorescence,2013,23(5):909-920.
[36] Chapman S C,Lawson A,Macarthur W C,et al.Ubiquitous GFP expression in transgenic chickens using a lentiviral vector [J].Development,2005,132(5):935-940.
[37] Koo B C,Kwon M S,Choi B R,et al.Production of germline transgenic chickens expressing enhanced green fluorescent protein using a MoMLV-based retrovirus vector [J].The Faseb Journal,2006,20(13):2251-2260.
[38] 李振红.转人抗癌基因P53鸡的研究 [D].河南洛阳:河南科技大学,2011.Li Z H.The study on transferring of human tumor suppressor gene P53 in chicken [D].Luoyang,Henan:Henan University of Science and Technology,2011.
[39] Song Z,Li Z H,Lei X Q,et al.Construction of the mammalian expressing vector pEGFP-N1-P53 and its expression successful in chicken fibroblast cells and blastoderm [J].Genet Mol Res,2015,14(1):931-939.
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