锌指核酸酶介导的陆川猪Fat1基因打靶载体构建及体外转基因研究
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摘要
秀丽隐杆线虫(Caenorhabditis elegans) Fat1基因翻译产物是ω-3多不饱和脂肪酸脱氢酶,哺乳动物因为缺少这个基因,不能将ω-6多不饱和脂肪酸转化为ω-3多不饱和脂肪酸,因此只能通过饮食获得ω-3多不饱和脂肪酸,以保证ω-6/ω-3的平衡,满足机体的健康所需。为获得整合了Fat1基因且能稳定表达的转基因猪(Sus scrofa),本实验采用锌指核酸酶(zinc finger nuclease, ZFN)介导的多位点打靶技术从细胞水平上开展了转基因技术的研究。首先构建了陆川猪Fat1基因打靶载体pCAGGS-DS2-eGFP-Fat1-DS1,并设计和构建了两个靶向陆川猪r DNA基因中内转录间隔区1 (internal transcribed spacer-1, ITS-1)序列的锌指核酸酶真核表达载体pcDNA3.1-NLS-ZFN-R和pcDNA3.1-NLS-ZFN-L。然后从猪的肾脏中成功分离出原代成纤维细胞(primary kidney fibroblast, PKF),通过脂质体共转染将Fat1基因打靶载体和锌指核酸酶载体导入细胞,转染细胞7 d后,提取细胞基因组DNA,PCR检测外源基因整合情况,结果表明Fat1基因成功整合到陆川猪PKF细胞基因组中,本研究为进一步获得转基因猪提供了一定的理论依据。
3 polyunsaturated fatty acid(PUFA) dehydrogenase, the expression product of Fat1 in Caenorhabditis elegans, can convert ω-6 PUFAs to ω-3 PUFAs. Mammals cannot synthesize ω-3 PUFAs due to lack of Fat1 gene. ω-3 PUFAs can only be obtained through the diet to ensure the balance of ω-6/ω-3 and body's health. In order to obtain the transgenic pig(Sus scrofa) with stable integration and expression of Fat1,experiments on transgenic technology at the cellular level were performed by zinc finger nuclease(ZFN)-mediated multi-site targeting technology. Expression vectors pcDNA3.1-NLS-ZFN-R and pcDNA3.1-NLSZFN-L, which were used to target to internal transcribed spacer-1(ITS1) sequence in Luchuan pig rDNA gene, were constructed. Secondly, primary kidney fibroblast(PKF) cells were successfully isolated from kidney. After 7 d of co-transfection of targeting vector and ZFN vectors into PKF cells, the genomic DNA was extracted and the integration of exogenous genes were detected by PCR. The results showed that Fat1 gene was successfully integrated into the genome of Luchuan pig PKF cells, but not in a site-specific way. This study provides a theoretical basis for further obtaining transgenic pigs.
3 polyunsaturated fatty acid(PUFA) dehydrogenase, the expression product of Fat1 in Caenorhabditis elegans, can convert ω-6 PUFAs to ω-3 PUFAs. Mammals cannot synthesize ω-3 PUFAs due to lack of Fat1 gene. ω-3 PUFAs can only be obtained through the diet to ensure the balance of ω-6/ω-3 and body's health. In order to obtain the transgenic pig(Sus scrofa) with stable integration and expression of Fat1,experiments on transgenic technology at the cellular level were performed by zinc finger nuclease(ZFN)-mediated multi-site targeting technology. Expression vectors pcDNA3.1-NLS-ZFN-R and pcDNA3.1-NLSZFN-L, which were used to target to internal transcribed spacer-1(ITS1) sequence in Luchuan pig rDNA gene, were constructed. Secondly, primary kidney fibroblast(PKF) cells were successfully isolated from kidney. After 7 d of co-transfection of targeting vector and ZFN vectors into PKF cells, the genomic DNA was extracted and the integration of exogenous genes were detected by PCR. The results showed that Fat1 gene was successfully integrated into the genome of Luchuan pig PKF cells, but not in a site-specific way. This study provides a theoretical basis for further obtaining transgenic pigs.
引文
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Beumer K J,Trautman J K,Liu J L,et al.2008.Efficient gene targeting in Drosophila by direct embryo injection with zinc-finger nucleases[J].Procedings of the National Academy of Sciences,105(50):19821-19826.
Bibikova M,Carroll D,Segal D J,et al.2001.Stimulation of homologous recombination through targeted cleavage by chimericnucleases[J].Molecular and Cell Biology,21:289-297.
Brown R S,San der C,Argos P.1985.The primary structure of transcription factor TFIIIA has 12 consecutive repeats[J].FEBS Letters,186(2):271-274.
Doyon,Yannick,McCammon,et al.2008.Heritable targeted gene disruption in zebrafish using designed zinc-fingernucleases[J].Nature Biotechnol,26:702-708.
Flisikowska T,ThoreyI S,Ofner S,et al.2011.Efficient immunoglobulin gene disruption and targeted replacement in rabbit usingzinc finger nucleases[J].PLoS ONE,6(6):e21045.
Frew L,Sugiarto N U,Rajagopal S P,et al.2013.The effect of omega-3 polyunsaturated fatty acid on the inflammatory response of the amnion[J].Prostaglandins LeukotEssent Fatty Acids,89(4):221-225.
Gavin W,Blash S,Buzzell N,et al.2018.Generation of transgenic goats by pronuclear microinjection:A retrospective analysis of a commercial operation(1995-2012)[J].Technical Report,27(1):115-122.
Handel E M,Alwin S and Cathomen T.2008.Expanding or restricting the target site repertoire of zinc-finger nucleases:The inter-domain linker as a major determinant of target site selectivity[J].Molecular Therapy,17(1):104-111.
Hong Jo Lee,Lee H C,Kim Y M,et al.2016.Site-specific recombination in the chicken genome using Flipase recombinase-mediated cassette exchange[J].The FASEBJournal,30(2):555-63.
Kim Y G,Cha J,Chan drasegarans.1996.Hybrid restriction enzymes:Zinc finger fusions to Fok I cleavage domain[J].PNAS,93:1156-1160.
Lai L,Kang J X,Li R,et al.2006.Generation of cloned transgenic pigs rich in omega-3 fatty acids[J].Nature Biotechnol,24(4):435-436.
Lee H J,Lee H C,Kim Y M,et al.2016.Site-specific recombination in the chicken genome using flipase recombinase-mediated cassette exchange[J].FASEB Journal,30(2):555-563.
Lorgeril M,Salen P,Defaye P,et al.2013.Recent findings on the health effects of omega-3 fatty acids and statins,and their interactions:Do statins inhibit omega-3[J].BMCMeddical,11:5.
Ma L,Wang Y,Wang H,et al.2018.Screen and verification for transgene integration sites in pigs[J].Scientific Reports,8(1):7433.
Mashimo T,Takizawa A,Volgt B,et al.2010.Generation of knockout rats with X-linked severe combined immunodeficiency(X-SCID)using zinc-finger nucleases[J].PLOS ONE,5(1):e8870.
Maskrey B H,Megson I L,Rossi A G,et al.2013.Emerging importance of omega-3 fatty acids in the innate immune response:Molecular mechanisms and lipidomic strategies for their analysis[J].Molecular Nutrition&Food Research,57(8):1390-1400.
Menchaca A,Anegon I,Whitelaw C B,et al.2016.New insights and current tools for genetically engineered(GE)sheep and goats[J].Theriogenology,86(1):160-169.
Meyer M,de Angelis M H,Wurst W,et al.2010.Genetargeting by homologous recombination in mouse zygotes mediated byzinc-finger nucleases[J].Procedings of the National Academyof Sciences,107(34):15022-15026.
Miller J C,McLachlan A D,Klug A.1985.Repetitive zincbinding domains in the protein transcription factor IIIAfrom Xenopus oocytes[J].EMBO Journal,4(6):1609-1614.
Miller J C,Holmes M C,Wang J,et al.2007.An improved zinc-finger nuclease architecture for highly specific genome editing[J].Nature Biotechnology,25(7):778-785.
Pang D K,Zhang L,Zhou Y R,et al.2009.Production ofω-3fattyacid desaturase gene(sfat-1)transgenic pigs bysomatic cell nuclear transfer[J].Science in China(Series C:Life Sciences),39(3):295-302.
Riediger N D,Othman R A,Suh M.2009.Moghadasian MHA systemic review of the roles of n-3 fatty acids in health and disease[J].Journal of The American Dietetic Association109:668-679.
Saeki K,Matsumoto K,Kinoshita M,et al.2004.Functional expression of a Delta12 fattyacid desaturase gene from spinach in transgenic pigs[J].Proceedings of the National Academy of Sciences of the USA,101(17):6361-6366.
Santiago Y,Chan E,Orlando S,et al.2008.Targeted gene knockout in mammalian cells byusing engineered zincfinger nucleases[J].Proceedings of the National Academy of Sciences of the USA,105:5809-5814.
Sheng L Y,Luo J J,Song Z Y,et al.2011,Highlyeficient modification of beta-lactoglobulin(BLG)gene via zinc-finger nucleases in catle[J].Cell Research,21:1638-1640.
Spychalla J P,K inney A J,Browse J,et al.1997.Identification of an animal omega-3 fatty acid desaturase by heterologous expression in Arabidopsis[J].Proceedings of the National Academy of Sciences of America,94(4):1142-1147.
Suzuki K,Mitsui K,Aizawa,et al.2008.Highly efficient transient gene expression and gene targeting in primateembryonic stem cells with helper-dependent adenoviral vectors[J].Proceedings of the National Academy of Sciences of the USA,105:13781-13786.
Urnov F D,Miller J C,Lee Y L,et al.2005.Highly efficient endogenous human gene correction usingde-signed zincfinger nucleases[J].Nature,435(7042):646-651.
Watts J L,Browse J.2002.Genetic dissection of polyunsaturated fatty acidsynthesisin Caenorhabditis elegans[J].Proceedings of the National Academy of Sciences of America,99(9):5854-5859.
Yang D,Yang H,Li W,et al.2011.Generation of PPARγmono-alelic knockout pigs via zinc-finger nucleases and muclear transfer cloning[J].Cell Research,21:979-982.
Yashodhara B M,Umakanth S,Pappachan J M,et al.2009.Omega-3 fatty acids:A comprehensive review of their role in health and disease[J].Postgraduate Medical Journal,85:84-90.
Yum S Y,Lee S J,Kim H M,et al.2016.Efficient generation of transgenic cattle using the DNA transposon and their analysis by next-generation sequencing[J].Scientific Reports,6:27185.
Zeng F,Li Z,Zhu Q,et al.2017.Production of functional human nerve growth factor from the saliva of transgenic mice by using salivary glands as bioreactors[J].Scientific Reports,7:41,270.
Zhang Q,Chen J Q,Lin J,et al.2014.Production GH transgenic goat improving mammogenesis by somatic cell nuclear transfer[J].Molecular Biology Reports,41(7):4759-4768.
罗婷婷,严爱芬,唐冬生,等.2018.类转录激活因子效应物核酸酶介导的E-cad和Bmi-1基因联合干预鼻咽癌的体外研究[J].中南大学学报(医学版),43(3):229-239.(Luo T T,Yan A F,Tang D S,et al.In vitro study of joint intervention of E-cad and Bmi-1 mediated by transcription activator-like effector nuclease in nasopharyngeal carcinoma[J].Journal of Central South University(Medical Science),43(3):229-239.)
唐冬生,蒋泓,张细权,等.2012.锌指核酸酶介导的高效多位点基因打靶[J].科学通报,57(9):711-719.(Tang DS,Jiang H,Zhang X Q,et al.2012.Multi-locus,high efficiency gene targeting mediated by zinc finger nucleases[J].Science China Press,57(9):711-719.)
赵宇航,云亭,李荣凤,等.2014.锌指核酸酶(ZFN)介导的牛肌肉抑制素基因(MSTN)突变的研究[J].农业生物技术学报,22(10):1204-1212.(Zhao Y H,Yun T,Li R F,et al.2014.Study on the mutation of bovine(Bos taurus)myostatin gene(MSTN)using zinc finger nuclease(ZFN)[J].Journal of Agricultural Biotechnology,22(10):1204-1212.)
曾芳,李紫聪,董锐,等.2016.转基因猪技术及其在农业上的应用[J].畜牧兽医学报,47(2):218-224.(Zeng F,Li Z C,Dong R,et al.Transgenic pig technology and its application in agriculture[J].ActaVeterinaria et Zootechnica Sinica,47(2):218-224.)
Arterbum L M,Hall E B,Oken H,et al.2006.Distribution,interconversion,and dose response of n-3 fatty acids in humans[J].The American Journal of Clinical Nutrition,83(6 Suppl):1467S-1476S.
Beumer K J,Trautman J K,Liu J L,et al.2008.Efficient gene targeting in Drosophila by direct embryo injection with zinc-finger nucleases[J].Procedings of the National Academy of Sciences,105(50):19821-19826.
Bibikova M,Carroll D,Segal D J,et al.2001.Stimulation of homologous recombination through targeted cleavage by chimericnucleases[J].Molecular and Cell Biology,21:289-297.
Brown R S,San der C,Argos P.1985.The primary structure of transcription factor TFIIIA has 12 consecutive repeats[J].FEBS Letters,186(2):271-274.
Doyon,Yannick,McCammon,et al.2008.Heritable targeted gene disruption in zebrafish using designed zinc-fingernucleases[J].Nature Biotechnol,26:702-708.
Flisikowska T,ThoreyI S,Ofner S,et al.2011.Efficient immunoglobulin gene disruption and targeted replacement in rabbit usingzinc finger nucleases[J].PLoS ONE,6(6):e21045.
Frew L,Sugiarto N U,Rajagopal S P,et al.2013.The effect of omega-3 polyunsaturated fatty acid on the inflammatory response of the amnion[J].Prostaglandins LeukotEssent Fatty Acids,89(4):221-225.
Gavin W,Blash S,Buzzell N,et al.2018.Generation of transgenic goats by pronuclear microinjection:A retrospective analysis of a commercial operation(1995-2012)[J].Technical Report,27(1):115-122.
Handel E M,Alwin S and Cathomen T.2008.Expanding or restricting the target site repertoire of zinc-finger nucleases:The inter-domain linker as a major determinant of target site selectivity[J].Molecular Therapy,17(1):104-111.
Hong Jo Lee,Lee H C,Kim Y M,et al.2016.Site-specific recombination in the chicken genome using Flipase recombinase-mediated cassette exchange[J].The FASEBJournal,30(2):555-63.
Kim Y G,Cha J,Chan drasegarans.1996.Hybrid restriction enzymes:Zinc finger fusions to Fok I cleavage domain[J].PNAS,93:1156-1160.
Lai L,Kang J X,Li R,et al.2006.Generation of cloned transgenic pigs rich in omega-3 fatty acids[J].Nature Biotechnol,24(4):435-436.
Lee H J,Lee H C,Kim Y M,et al.2016.Site-specific recombination in the chicken genome using flipase recombinase-mediated cassette exchange[J].FASEB Journal,30(2):555-563.
Lorgeril M,Salen P,Defaye P,et al.2013.Recent findings on the health effects of omega-3 fatty acids and statins,and their interactions:Do statins inhibit omega-3[J].BMCMeddical,11:5.
Ma L,Wang Y,Wang H,et al.2018.Screen and verification for transgene integration sites in pigs[J].Scientific Reports,8(1):7433.
Mashimo T,Takizawa A,Volgt B,et al.2010.Generation of knockout rats with X-linked severe combined immunodeficiency(X-SCID)using zinc-finger nucleases[J].PLOS ONE,5(1):e8870.
Maskrey B H,Megson I L,Rossi A G,et al.2013.Emerging importance of omega-3 fatty acids in the innate immune response:Molecular mechanisms and lipidomic strategies for their analysis[J].Molecular Nutrition&Food Research,57(8):1390-1400.
Menchaca A,Anegon I,Whitelaw C B,et al.2016.New insights and current tools for genetically engineered(GE)sheep and goats[J].Theriogenology,86(1):160-169.
Meyer M,de Angelis M H,Wurst W,et al.2010.Genetargeting by homologous recombination in mouse zygotes mediated byzinc-finger nucleases[J].Procedings of the National Academyof Sciences,107(34):15022-15026.
Miller J C,McLachlan A D,Klug A.1985.Repetitive zincbinding domains in the protein transcription factor IIIAfrom Xenopus oocytes[J].EMBO Journal,4(6):1609-1614.
Miller J C,Holmes M C,Wang J,et al.2007.An improved zinc-finger nuclease architecture for highly specific genome editing[J].Nature Biotechnology,25(7):778-785.
Pang D K,Zhang L,Zhou Y R,et al.2009.Production ofω-3fattyacid desaturase gene(sfat-1)transgenic pigs bysomatic cell nuclear transfer[J].Science in China(Series C:Life Sciences),39(3):295-302.
Riediger N D,Othman R A,Suh M.2009.Moghadasian MHA systemic review of the roles of n-3 fatty acids in health and disease[J].Journal of The American Dietetic Association109:668-679.
Saeki K,Matsumoto K,Kinoshita M,et al.2004.Functional expression of a Delta12 fattyacid desaturase gene from spinach in transgenic pigs[J].Proceedings of the National Academy of Sciences of the USA,101(17):6361-6366.
Santiago Y,Chan E,Orlando S,et al.2008.Targeted gene knockout in mammalian cells byusing engineered zincfinger nucleases[J].Proceedings of the National Academy of Sciences of the USA,105:5809-5814.
Sheng L Y,Luo J J,Song Z Y,et al.2011,Highlyeficient modification of beta-lactoglobulin(BLG)gene via zinc-finger nucleases in catle[J].Cell Research,21:1638-1640.
Spychalla J P,K inney A J,Browse J,et al.1997.Identification of an animal omega-3 fatty acid desaturase by heterologous expression in Arabidopsis[J].Proceedings of the National Academy of Sciences of America,94(4):1142-1147.
Suzuki K,Mitsui K,Aizawa,et al.2008.Highly efficient transient gene expression and gene targeting in primateembryonic stem cells with helper-dependent adenoviral vectors[J].Proceedings of the National Academy of Sciences of the USA,105:13781-13786.
Urnov F D,Miller J C,Lee Y L,et al.2005.Highly efficient endogenous human gene correction usingde-signed zincfinger nucleases[J].Nature,435(7042):646-651.
Watts J L,Browse J.2002.Genetic dissection of polyunsaturated fatty acidsynthesisin Caenorhabditis elegans[J].Proceedings of the National Academy of Sciences of America,99(9):5854-5859.
Yang D,Yang H,Li W,et al.2011.Generation of PPARγmono-alelic knockout pigs via zinc-finger nucleases and muclear transfer cloning[J].Cell Research,21:979-982.
Yashodhara B M,Umakanth S,Pappachan J M,et al.2009.Omega-3 fatty acids:A comprehensive review of their role in health and disease[J].Postgraduate Medical Journal,85:84-90.
Yum S Y,Lee S J,Kim H M,et al.2016.Efficient generation of transgenic cattle using the DNA transposon and their analysis by next-generation sequencing[J].Scientific Reports,6:27185.
Zeng F,Li Z,Zhu Q,et al.2017.Production of functional human nerve growth factor from the saliva of transgenic mice by using salivary glands as bioreactors[J].Scientific Reports,7:41,270.
Zhang Q,Chen J Q,Lin J,et al.2014.Production GH transgenic goat improving mammogenesis by somatic cell nuclear transfer[J].Molecular Biology Reports,41(7):4759-4768.