大载体转染猪胎儿成纤维细胞的电转条件优化
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摘要
【背景】随着生物技术发展,研究的生理机制和生物功能日益复杂,提高大载体的转染效率对多基因共表达系统、基因编辑技术、转基因育种等具有重要的意义。在转基因育种中,使用的转基因载体相对较大,而且转基因动物的制备效率也与供体细胞猪胎儿成纤维(porcine Fetal Fibroblasts,PFFs)细胞的转染效率有关。【目的】研究主要从转染参数、质粒用量和拓扑结构三方面,比较3种电转仪ECM~?830/NEPA21/Nucleofector~(TM)2b的大载体转染效率,以探索大载体转染PFFs的最佳条件。【方法】使用3种不同电转仪将长达26 kb的携带增强型绿荧光蛋白基因的pPXAT-EGFP质粒转染1×10~6个PFFs,48 h后使用流式细胞仪测定荧光细胞比例,从电转参数、质粒用量和拓扑结构三方面分别比较瞬时转染效率。【结果】首先比较电转仪不同参数的转染效率,结果显示当电转参数为脉冲电压300 V,脉冲长度1 ms,脉冲间隔50 ms,脉冲次数3次,NEPA 21转染PFFs的效率最高,为13.24%±1.63%,而Nucleofector~(TM) 2b的最佳电转参数为U-023,其转染效率高达46.36%±3.95%。然后在最佳电转参数下分别比较6、8、10和12μg的26 kb超螺旋质粒的转染效率,ECM~?830和Nucleofector~(TM) 2b转染PFFs的最佳质粒用量为12μg,其转染效率分别为8.44%±0.90%(电转参数:脉冲电压300 V,脉冲长度1 ms,脉冲次数3次)和14.63%±3.21%(电转参数:U-023),而NEPA 21使用10μg质粒转染PFFs时效率达到最高(6.09%±0.72%)。最后比较不同质粒拓扑结构的转染效率,结果显示线性化质粒的转染效率较低,仅为超螺旋质粒转染效率的34.96%—48.39%。【结论】因此Nucleofector~(TM) 2b转染PFFs的最佳条件为:U-023程序、12μg超螺旋质粒;NEPA 21为:脉冲电压200 V,脉冲长度3 ms,脉冲间隔50 ms,脉冲次数3次、10μg超螺旋质粒;ECM~?830则在脉冲电压300 V,脉冲长度1 ms,脉冲次数3次条件下转染12μg超螺旋质粒可获得最佳转染效率。综合比较上述3种电转仪,26 kb大载体转染PFFs的最佳电转仪是Nucleofector~(TM) 2b。
【Background】With the development of biotechnology,the physiological mechanisms and biological functions of research are becoming more and more complex,and improving the transfection efficiency of large vectors is of great significance for multi-gene co-expression systems,genome editing,and transgenic breeding.In transgenic breeding,the transgenic vectors is relatively large,and the efficiency of generating transgenic animals is also attributed to the transfection efficiency of porcine fetal fibroblasts(PFFs).【Objective】Therefore,this study mainly compared the electroporation efficiency of three electroporation apparatus ECM~?830/NEPA 21/Nucleofector~(TM) 2b on various parameters,plasmid dosage and topology,to explore the optimal electroporation condition of large vector in PFFs.【Method】We transfected a 26 kb plasmid,namely pPXAT-EGFP,into the PFFs,and then the electroporation efficiencies of various parameters of three electroporation apparatus ECM?830/NEPA21/Nucleofector~(TM)2b,and the dosage and topological structure of the plasmid by the flow cytometry were compared.【Result】By comparing the transfection efficiency of different transfection parameters,the results showed that the highest transfection efficiency of NEPA 21 was 13.24%±1.63%at pulse voltage 300 V,pulse length 1 ms,pulse interval 50 ms,pulse number 3 times,and the optimal electrical rotation parameter of Nucleofector~(TM) 2b was U-023,whose transfection efficiency was 46.36%±3.95%.In addition,the transfection efficiencies of 6,8,10 and 12μg 26 kb supercoiled plasmids were compared under the optimal electroporation parameters,and the results showed that the optimal plasmid dosage for ECM?830 and Nucleofector ~(TM) 2b transfected PFFs was 12μg with 8.44%±0.90%efficiency(transfection parameters:pulse voltage 300 V,pulse length 1 ms,pulse number 3 times)and14.63%±3.21%(U-023),while the NEPA 21 achieved the highest efficiency of 6.09%±0.72%with 10μg plasmid.Finally,we compared the transfection efficiency of different quality topologies and found that the transfection efficiency of linearized plasmids was low,only 34.96%-48.39%of the supercoiled plasmids.【Conclusion】Therefore,the optimal electroporation condition of PFFs by Nucleofector~(TM)2b was U-023 procedure,12μg supercoiled plasmid;NEPA 21 was pulse voltage 200 V,pulse length 3 ms,pulse interval 50 ms,pulse number 3 times,and 10μg supercoiled plasmid;ECM?830 was transfected with 12μg supercoiled plasmid at pulse voltage 300 V,pulse length 1 ms,and pulse number 3 times to obtain the highest transfection efficiency.Comprehensive comparison of the above three kinds of electroporation apparatus,the optimum for transfecting PFFs with 26 kb large carrier was Nucleofector~(TM)2b.
【Background】With the development of biotechnology,the physiological mechanisms and biological functions of research are becoming more and more complex,and improving the transfection efficiency of large vectors is of great significance for multi-gene co-expression systems,genome editing,and transgenic breeding.In transgenic breeding,the transgenic vectors is relatively large,and the efficiency of generating transgenic animals is also attributed to the transfection efficiency of porcine fetal fibroblasts(PFFs).【Objective】Therefore,this study mainly compared the electroporation efficiency of three electroporation apparatus ECM~?830/NEPA 21/Nucleofector~(TM) 2b on various parameters,plasmid dosage and topology,to explore the optimal electroporation condition of large vector in PFFs.【Method】We transfected a 26 kb plasmid,namely pPXAT-EGFP,into the PFFs,and then the electroporation efficiencies of various parameters of three electroporation apparatus ECM?830/NEPA21/Nucleofector~(TM)2b,and the dosage and topological structure of the plasmid by the flow cytometry were compared.【Result】By comparing the transfection efficiency of different transfection parameters,the results showed that the highest transfection efficiency of NEPA 21 was 13.24%±1.63%at pulse voltage 300 V,pulse length 1 ms,pulse interval 50 ms,pulse number 3 times,and the optimal electrical rotation parameter of Nucleofector~(TM) 2b was U-023,whose transfection efficiency was 46.36%±3.95%.In addition,the transfection efficiencies of 6,8,10 and 12μg 26 kb supercoiled plasmids were compared under the optimal electroporation parameters,and the results showed that the optimal plasmid dosage for ECM?830 and Nucleofector ~(TM) 2b transfected PFFs was 12μg with 8.44%±0.90%efficiency(transfection parameters:pulse voltage 300 V,pulse length 1 ms,pulse number 3 times)and14.63%±3.21%(U-023),while the NEPA 21 achieved the highest efficiency of 6.09%±0.72%with 10μg plasmid.Finally,we compared the transfection efficiency of different quality topologies and found that the transfection efficiency of linearized plasmids was low,only 34.96%-48.39%of the supercoiled plasmids.【Conclusion】Therefore,the optimal electroporation condition of PFFs by Nucleofector~(TM)2b was U-023 procedure,12μg supercoiled plasmid;NEPA 21 was pulse voltage 200 V,pulse length 3 ms,pulse interval 50 ms,pulse number 3 times,and 10μg supercoiled plasmid;ECM?830 was transfected with 12μg supercoiled plasmid at pulse voltage 300 V,pulse length 1 ms,and pulse number 3 times to obtain the highest transfection efficiency.Comprehensive comparison of the above three kinds of electroporation apparatus,the optimum for transfecting PFFs with 26 kb large carrier was Nucleofector~(TM)2b.
引文
[1]RUAN J,LI H,XU K,WU T,WEI J,ZHOU R,LIU Z,MU Y,YANGS,OUYANG H,CHEN-TSAI R Y,LI K.Highly efficient CRISPR/Cas9-mediated transgene knock in at the H11 locus in pigs.Scientific Reports,2015,5:14253.
[2]王金霞,徐影琪,魏政立,杨葳,张梅英,郑志红.一种能实现蛋白可逆表达的敲入载体构建方法.辽宁农业科学,2016(4):27-32.WANG J X,XU Y Q,WEI Z L,YANG W,ZHANG M Y,ZHENG ZH.A method of constructing a knock-in vector which can achieve reversible protein expression.Liaoning Agricultural Sciences,2016(4):27-32.(in Chinese)
[3]张驹.CRISPR/Cas9系统介导羊MSTN基因敲除和定点整合fat-1基因的研究[D].呼和浩特:内蒙古大学,2016.ZHANG J.Generation of MSTN gene knock-out and fat-1 gene Knock-in goat via CRISPER/CAS9[D].Huhhot:Inner Mongolia University,2016.(in Chinese)
[4]GUAN L Z,SUN Y P,XI Q Y,WANG J L,ZHOU J Y,SHU G,JIANG Q Y,ZHANG Y L.β-Glucanase specific expression in the parotid gland of transgenic mice.Transgenic Research,2013,22(4):805-812.
[5]CLARK K J,CARLSON D F,FAHRENKRUG S C.Pigs taking wing with transposons and recombinases.Genome Biology,2007,8(Suppl1):S13.
[6]DING S,WU X,LI G,HAN M,ZHUANG Y,XU T.Efficient transposition of the piggyBac(PB)transposon in mammalian cells and mice.Cell,2005,122(3):473-483.
[7]LI M A,TURNER D J,NING Z,YUSA K,LIANG Q,ECKERT S,RAD L,FITZGERALD T W,CRAIG N L,BRADLEY A.Mobilization of giant piggyBac transposons in the mouse genome.Nucleic Acids Research,2011,39(22):e148.
[8]WU S C,MEIR Y J,COATES C J,HANDLER A M,PELCZAR P,MOISYADI S,KAMINSKI J M.piggyBac is a flexible and highly active transposon as compared to sleeping beauty,Tol2,and Mos1 in mammalian cells.Proceedings of the National Academy of Sciences of the United States of America,2006,103(41):15008-15013.
[9]WILSON M H,COATES C J,GEORGE AL JR.PiggyBac transposon-mediated gene transfer in human cells.Molecular Therapy,2007,15(1):139-145.
[10]杜新华,高雪,张路培,高会江,李俊雅,许尚忠.Piggybac转座子在牛基因组的整合位点及特征分析.遗传,2013,35(6):771-777.DU X H,GAO X,ZHANG L P,GAO H J,LI J Y,XU S Z.Integration sites and their characteristic analysis of piggyBac transposon in cattle genome.Hereditas,2013,35(6):771-777.(in Chinese)
[11]XIE Z,PANG D,WANG K,LI M,GUO N,YUAN H,LI J,ZOU X,JIAO H,OUYANG H,LI Z,TANG X.Optimization of a CRISPR/Cas9-mediated knock-in strategy at the porcine Rosa26 locus in porcine foetal fibroblasts.Scientific Reports,2017,7(1):3036.
[12]CHU V T,WEBER T,GRAF R,SOMMERMANN T,PETSCH K,SACK U,VOLCHKOV P,RAJEWSKY K,KüHN R.Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6zygotes.BMC Biotechnology,2016,16:4.
[13]WU M,WEI C,LIAN Z,LIU R,ZHU C,WANG H,CAO J,SHEN Y,ZHAO F,ZHANG L,MU Z,WANG Y,WANG X,DU L,WANG C.Rosa26-targeted sheep gene knock-in via CRISPR-Cas9 system.Scientific Reports,2016,6:24360.
[14]LAI S,WEI S,ZHAO B,OUYANG Z,ZHANG Q,FAN N,LIU Z,ZHAO Y,YAN Q,ZHOU X,LI L,XIN J,ZENG Y,LAI L,ZOU Q.Generation of knock-in pigs carrying oct4-tdtomato reporter through CRISPR/Cas9-mediated genome engineering.PLoS ONE,2016,11(1).doi:10.1371/journal.pone.0146562.
[15]覃兆鲜,潘天彪,谢炳坤.猪成纤维细胞转染方法的比较.江苏农业科学,2011(3):250-251.QIN Z X,PAN T B,XIE B K.Comparison of the transfection methods for porcine fibroblasts cell.Jiangsu Agricultural Sciences,2011(3):250-251.(in Chinese)
[16]RICHTER A,KUROME M,KESSLER B,ZAKHARTCHENKO V,KLYMIUK N,NAGASHIMA H,WOLF E,WUENSCH A.Potential of primary kidney cells for somatic cell nuclear transfer mediated transgenesis in pig.BMC Biotechnology,2012,12(1):84.
[17]NAKAYAMA A,SATO M,SHINOHARA M,MATSUBARA S,YOKOMINE T,AKASAKA E,YOSHIDA M,TAKAO S.Efficient transfection of primarily cultured porcine embryonic fibroblasts using the Amaxa Nucleofection System?.Cloning and Stem Cells,2007,9(4):523-534.
[18]ROSS J W,WHYTE J J,ZHAO J,SAMUEL M,WELLS K D,PRATHER R S.Optimization of square-wave electroporation for transfection of porcine fetal fibroblasts.Transgenic Research,2009,19(4):611-620.
[19]BARNABé-HEIDER F,MELETIS K,ERIKSSON M,BERGMANNO,SABELSTR?M H,HARVEY MA,MIKKERS H,FRISéN J.Genetic manipulation of adult mouse neurogenic niches by in vivo electroporation.Nature Methods,2008,5(2):189-196.
[20]ZOU M,KONINCK P D,NEVE R L,FRIEDRICH R W.Fast gene transfer into the adult zebrafish brain by herpes simplex virus 1(hsv-1)and electroporation:methods and optogenetic applications.Frontiers in Neural Circuits,2014,8(7):41.
[21]ISHINO T,HASHIMOTO M,AMAGASA M,SAITO N,DOCHI O,KIRISAWA R.Establishment of protocol for preparation of gene-edited bovine ear-derived fibroblasts for somatic cell nuclear transplantation.Biomedical Research,2018,39(2):95-104.
[22]钟翠丽,李国玲,莫健新,全绒,王豪强,李紫聪,吴珍芳,张献伟.不同电转仪的电转参数、质粒用量和拓扑结构对猪胎儿成纤维细胞转染效率的影响.遗传,2017(10):930-938.ZHONG C L,LI G L,MO J X,QUAN R,WANG H Q,LI Z C,WU ZF,ZHANG X W.Effects of parameters,plasmid dosages and topological structures on transfection efficiency of porcine fetal fibroblasts using different electroporators.Hereditas,2017(10):930-938.(in Chinese)
[23]VON GROLL A,LEVIN Y,BARBOSA M C,RAVAZZOLO A P.Linear DNA low efficiency transfection by liposome can be improved by the use of cationic lipid as charge neutralizer.Biotechnology Progress,2006,22(4):1220-1224.
[24]AUER T O,DUROURE K,DE CIAN A,CONCORDET J P,DELBENE F.Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair.Genome Research,2014,24(1):142-153.
[25]楚素霞,姚伦广,邢延豪,周延清.多基因表达系统研究进展.中国生物工程杂志,2011,31(6):116-123.CHU S X,YAO L G,XING Y H,ZHOU Y Q.Progress in research on multiple gene expression system.China Biotechnology,2011,31(6):116-123.(in Chinese)
[26]李瑞国,苗朝华,侯健,关宏,安晓荣,陈永福.牛β-酪蛋白座位无启动子基因打靶载体的构建.华北农学报,2010,25(4):17-24.LI R G,MIAO Z H,HOU J,GUAN H,AN X R,CHEN Y F.Non-promotor gene targeting vector construction for bovineβ-casein site.Acta Agriculturae Boreali-Sinica,2010,25(4):17-24.(in Chinese)
[27]沈俊杰,单娟娟,骆菁菁,刘立,钱程.构建多顺反子表达载体系统的新策略[J].浙江理工大学学报,2009,26(4):561-566.SHEN J J,SHAN J J,LUO J J,LIU L,QIAN C.New strategy for construction of polycistron expression vector.Journal of Zhejiang University of Science and Technology,2009,26(4):561-566.(in Chinese)
[28]DENG W,YANG D,ZHAO B,OUYANG Z,SONG J,FAN N,LIU Z,ZHAO Y,WU Q,NASHUN B,TANG J,WU Z,GU W,LAI L.Use of the 2A Peptide for Generation of multi-transgenic pigs through a single round of nuclear transfer.PLoS ONE,2011,6(5):e19986.
[29]SATO M,KAGOSHIMA A,SAITOH I,INADA E,MIYOSHI K,OHTSUKA M,NAKAMURA S,SAKURAI T,WATANABE S.Generation ofα-1,3-galactosyltransferase-deficient porcine embryonic fibroblasts by CRISPR/Cas9-mediated knock-in of a small mutated sequence and a targeted toxin-based selection system.Reproduction in Domestic Animals,2015,50(5):872-880.
[30]NAKADE S,TSUBOTA T,SAKANE Y,KUME S,SAKAMOTO N,OBARA M,DAIMON T,SEZUTSU H,YAMAMOTO T,SAKUMAT,SUZUKI K T.Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9.Nature Communications,2014,5:5560.
[2]王金霞,徐影琪,魏政立,杨葳,张梅英,郑志红.一种能实现蛋白可逆表达的敲入载体构建方法.辽宁农业科学,2016(4):27-32.WANG J X,XU Y Q,WEI Z L,YANG W,ZHANG M Y,ZHENG ZH.A method of constructing a knock-in vector which can achieve reversible protein expression.Liaoning Agricultural Sciences,2016(4):27-32.(in Chinese)
[3]张驹.CRISPR/Cas9系统介导羊MSTN基因敲除和定点整合fat-1基因的研究[D].呼和浩特:内蒙古大学,2016.ZHANG J.Generation of MSTN gene knock-out and fat-1 gene Knock-in goat via CRISPER/CAS9[D].Huhhot:Inner Mongolia University,2016.(in Chinese)
[4]GUAN L Z,SUN Y P,XI Q Y,WANG J L,ZHOU J Y,SHU G,JIANG Q Y,ZHANG Y L.β-Glucanase specific expression in the parotid gland of transgenic mice.Transgenic Research,2013,22(4):805-812.
[5]CLARK K J,CARLSON D F,FAHRENKRUG S C.Pigs taking wing with transposons and recombinases.Genome Biology,2007,8(Suppl1):S13.
[6]DING S,WU X,LI G,HAN M,ZHUANG Y,XU T.Efficient transposition of the piggyBac(PB)transposon in mammalian cells and mice.Cell,2005,122(3):473-483.
[7]LI M A,TURNER D J,NING Z,YUSA K,LIANG Q,ECKERT S,RAD L,FITZGERALD T W,CRAIG N L,BRADLEY A.Mobilization of giant piggyBac transposons in the mouse genome.Nucleic Acids Research,2011,39(22):e148.
[8]WU S C,MEIR Y J,COATES C J,HANDLER A M,PELCZAR P,MOISYADI S,KAMINSKI J M.piggyBac is a flexible and highly active transposon as compared to sleeping beauty,Tol2,and Mos1 in mammalian cells.Proceedings of the National Academy of Sciences of the United States of America,2006,103(41):15008-15013.
[9]WILSON M H,COATES C J,GEORGE AL JR.PiggyBac transposon-mediated gene transfer in human cells.Molecular Therapy,2007,15(1):139-145.
[10]杜新华,高雪,张路培,高会江,李俊雅,许尚忠.Piggybac转座子在牛基因组的整合位点及特征分析.遗传,2013,35(6):771-777.DU X H,GAO X,ZHANG L P,GAO H J,LI J Y,XU S Z.Integration sites and their characteristic analysis of piggyBac transposon in cattle genome.Hereditas,2013,35(6):771-777.(in Chinese)
[11]XIE Z,PANG D,WANG K,LI M,GUO N,YUAN H,LI J,ZOU X,JIAO H,OUYANG H,LI Z,TANG X.Optimization of a CRISPR/Cas9-mediated knock-in strategy at the porcine Rosa26 locus in porcine foetal fibroblasts.Scientific Reports,2017,7(1):3036.
[12]CHU V T,WEBER T,GRAF R,SOMMERMANN T,PETSCH K,SACK U,VOLCHKOV P,RAJEWSKY K,KüHN R.Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6zygotes.BMC Biotechnology,2016,16:4.
[13]WU M,WEI C,LIAN Z,LIU R,ZHU C,WANG H,CAO J,SHEN Y,ZHAO F,ZHANG L,MU Z,WANG Y,WANG X,DU L,WANG C.Rosa26-targeted sheep gene knock-in via CRISPR-Cas9 system.Scientific Reports,2016,6:24360.
[14]LAI S,WEI S,ZHAO B,OUYANG Z,ZHANG Q,FAN N,LIU Z,ZHAO Y,YAN Q,ZHOU X,LI L,XIN J,ZENG Y,LAI L,ZOU Q.Generation of knock-in pigs carrying oct4-tdtomato reporter through CRISPR/Cas9-mediated genome engineering.PLoS ONE,2016,11(1).doi:10.1371/journal.pone.0146562.
[15]覃兆鲜,潘天彪,谢炳坤.猪成纤维细胞转染方法的比较.江苏农业科学,2011(3):250-251.QIN Z X,PAN T B,XIE B K.Comparison of the transfection methods for porcine fibroblasts cell.Jiangsu Agricultural Sciences,2011(3):250-251.(in Chinese)
[16]RICHTER A,KUROME M,KESSLER B,ZAKHARTCHENKO V,KLYMIUK N,NAGASHIMA H,WOLF E,WUENSCH A.Potential of primary kidney cells for somatic cell nuclear transfer mediated transgenesis in pig.BMC Biotechnology,2012,12(1):84.
[17]NAKAYAMA A,SATO M,SHINOHARA M,MATSUBARA S,YOKOMINE T,AKASAKA E,YOSHIDA M,TAKAO S.Efficient transfection of primarily cultured porcine embryonic fibroblasts using the Amaxa Nucleofection System?.Cloning and Stem Cells,2007,9(4):523-534.
[18]ROSS J W,WHYTE J J,ZHAO J,SAMUEL M,WELLS K D,PRATHER R S.Optimization of square-wave electroporation for transfection of porcine fetal fibroblasts.Transgenic Research,2009,19(4):611-620.
[19]BARNABé-HEIDER F,MELETIS K,ERIKSSON M,BERGMANNO,SABELSTR?M H,HARVEY MA,MIKKERS H,FRISéN J.Genetic manipulation of adult mouse neurogenic niches by in vivo electroporation.Nature Methods,2008,5(2):189-196.
[20]ZOU M,KONINCK P D,NEVE R L,FRIEDRICH R W.Fast gene transfer into the adult zebrafish brain by herpes simplex virus 1(hsv-1)and electroporation:methods and optogenetic applications.Frontiers in Neural Circuits,2014,8(7):41.
[21]ISHINO T,HASHIMOTO M,AMAGASA M,SAITO N,DOCHI O,KIRISAWA R.Establishment of protocol for preparation of gene-edited bovine ear-derived fibroblasts for somatic cell nuclear transplantation.Biomedical Research,2018,39(2):95-104.
[22]钟翠丽,李国玲,莫健新,全绒,王豪强,李紫聪,吴珍芳,张献伟.不同电转仪的电转参数、质粒用量和拓扑结构对猪胎儿成纤维细胞转染效率的影响.遗传,2017(10):930-938.ZHONG C L,LI G L,MO J X,QUAN R,WANG H Q,LI Z C,WU ZF,ZHANG X W.Effects of parameters,plasmid dosages and topological structures on transfection efficiency of porcine fetal fibroblasts using different electroporators.Hereditas,2017(10):930-938.(in Chinese)
[23]VON GROLL A,LEVIN Y,BARBOSA M C,RAVAZZOLO A P.Linear DNA low efficiency transfection by liposome can be improved by the use of cationic lipid as charge neutralizer.Biotechnology Progress,2006,22(4):1220-1224.
[24]AUER T O,DUROURE K,DE CIAN A,CONCORDET J P,DELBENE F.Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair.Genome Research,2014,24(1):142-153.
[25]楚素霞,姚伦广,邢延豪,周延清.多基因表达系统研究进展.中国生物工程杂志,2011,31(6):116-123.CHU S X,YAO L G,XING Y H,ZHOU Y Q.Progress in research on multiple gene expression system.China Biotechnology,2011,31(6):116-123.(in Chinese)
[26]李瑞国,苗朝华,侯健,关宏,安晓荣,陈永福.牛β-酪蛋白座位无启动子基因打靶载体的构建.华北农学报,2010,25(4):17-24.LI R G,MIAO Z H,HOU J,GUAN H,AN X R,CHEN Y F.Non-promotor gene targeting vector construction for bovineβ-casein site.Acta Agriculturae Boreali-Sinica,2010,25(4):17-24.(in Chinese)
[27]沈俊杰,单娟娟,骆菁菁,刘立,钱程.构建多顺反子表达载体系统的新策略[J].浙江理工大学学报,2009,26(4):561-566.SHEN J J,SHAN J J,LUO J J,LIU L,QIAN C.New strategy for construction of polycistron expression vector.Journal of Zhejiang University of Science and Technology,2009,26(4):561-566.(in Chinese)
[28]DENG W,YANG D,ZHAO B,OUYANG Z,SONG J,FAN N,LIU Z,ZHAO Y,WU Q,NASHUN B,TANG J,WU Z,GU W,LAI L.Use of the 2A Peptide for Generation of multi-transgenic pigs through a single round of nuclear transfer.PLoS ONE,2011,6(5):e19986.
[29]SATO M,KAGOSHIMA A,SAITOH I,INADA E,MIYOSHI K,OHTSUKA M,NAKAMURA S,SAKURAI T,WATANABE S.Generation ofα-1,3-galactosyltransferase-deficient porcine embryonic fibroblasts by CRISPR/Cas9-mediated knock-in of a small mutated sequence and a targeted toxin-based selection system.Reproduction in Domestic Animals,2015,50(5):872-880.
[30]NAKADE S,TSUBOTA T,SAKANE Y,KUME S,SAKAMOTO N,OBARA M,DAIMON T,SEZUTSU H,YAMAMOTO T,SAKUMAT,SUZUKI K T.Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9.Nature Communications,2014,5:5560.