腺病毒介导的高效生产水牛转基因胚胎条件的摸索
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摘要
试验旨在探究腺病毒感染水牛颗粒细胞和胚胎的最佳条件,以提高转基因水牛胚胎的生产效率。以水牛颗粒细胞和体外发育的胚胎为研究对象,分别用0、1×10~0、1×10~(-1)、1×10~(-2)、1×10~(-3)、1×10~(-4)、1×10~(-5) GFU/mL腺病毒感染水牛颗粒细胞,获得最佳感染浓度后,在最佳浓度条件下分别感染24、48、72、96 h,摸索最佳感染时间,用倒置荧光显微镜观察试验结果。利用腺病毒感染水牛颗粒细胞的最佳浓度和时间分别感染2细胞和4细胞期胚胎,对胚胎的最佳感染条件进行摸索,分析胚胎分裂率、囊胚率和转染囊胚率。结果显示,1×10~(-2) GFU/mL浓度和48 h感染时间可获得水牛颗粒细胞的最佳腺病毒感染效率。腺病毒感染2细胞和4细胞期无透明带和非完整透明带胚胎后胚胎发绿光,而感染完整透明带胚胎后不发光,2细胞期胚胎感染后停止发育,4细胞期开始感染的非完整透明带胚胎可继续发育至囊胚。于4细胞期分别感染完整透明带组、非完整透明带组和无透明带组水牛胚胎,结果显示,非完整透明带转染组在囊胚率和囊胚转染效率上均优于其他组。综上,非完整透明带,1×10~(-2) GFU/mL和48 h为感染浓度和时间,4细胞期为感染起始期的腺病毒介导的水牛转基因胚胎生产方法能够实现目标基因在水牛胚胎中的高效表达,从而达到提高转基因水牛胚胎生产效率的目的。
In order to improve the production efficiency of transgenic buffalo embryos,this study was aimed to explore the optimal conditions of adenovirus-infected buffalo granular cells and embryos.Taking buffalo granule cells and embryos developed in vitro as research objects,buffalo granular cells were infected with 0,1×10~0,1×10~(-1),1×10~(-2),1×10~(-3),1×10~(-4),1×10~(-5) GFU/mL adenovirus to obtain the best infection concentration;The buffalo granular cells were infected with the optimum concentration condition for 24,48,72 and 96 h to obtain the optimal infection time,and the results were observed by inverted fluorescence microscope.The optimal concentration and time of adenovirus-infected to buffalo granular cells were infected to the embryos in 2-and 4-cells stages,respectively,the optimal conditions of embryo infection were explored,and the embryo cleavage rate,blastocyst rate and transfection blastocyst rate were analyzed.The results showed that the optimal adenovirus infection efficiency of buffalo granular cells could be obtained by 1×10~(-2) GFU/mL concentration and 48 h infection time of adenovirus.The embryos in 2-and 4-cells stages without zona pellucida and non-complete zona pellucida were infected with adenovirus got green light,while there was no light in the complete zona pellucida embryo.The embryos in 2-cell stage stopped developing after infection,while the incomplete zona pellucida embryos in 4-cell stage could continue developing into blastocyst.The blastocyst rate and blastocyst transfection efficiency of incomplete zona pellucida group was superior to other groups after infected with adenovirus in 4-cell stage.In summary, the incomplete zona pellucida,1×10~(-2) GFU/mL,48 h,and 4-cell stage infection contributed to efficient expression of the target gene in buffalo embryo,and then improved the efficiency of transgenic buffalo embryo production.
In order to improve the production efficiency of transgenic buffalo embryos,this study was aimed to explore the optimal conditions of adenovirus-infected buffalo granular cells and embryos.Taking buffalo granule cells and embryos developed in vitro as research objects,buffalo granular cells were infected with 0,1×10~0,1×10~(-1),1×10~(-2),1×10~(-3),1×10~(-4),1×10~(-5) GFU/mL adenovirus to obtain the best infection concentration;The buffalo granular cells were infected with the optimum concentration condition for 24,48,72 and 96 h to obtain the optimal infection time,and the results were observed by inverted fluorescence microscope.The optimal concentration and time of adenovirus-infected to buffalo granular cells were infected to the embryos in 2-and 4-cells stages,respectively,the optimal conditions of embryo infection were explored,and the embryo cleavage rate,blastocyst rate and transfection blastocyst rate were analyzed.The results showed that the optimal adenovirus infection efficiency of buffalo granular cells could be obtained by 1×10~(-2) GFU/mL concentration and 48 h infection time of adenovirus.The embryos in 2-and 4-cells stages without zona pellucida and non-complete zona pellucida were infected with adenovirus got green light,while there was no light in the complete zona pellucida embryo.The embryos in 2-cell stage stopped developing after infection,while the incomplete zona pellucida embryos in 4-cell stage could continue developing into blastocyst.The blastocyst rate and blastocyst transfection efficiency of incomplete zona pellucida group was superior to other groups after infected with adenovirus in 4-cell stage.In summary, the incomplete zona pellucida,1×10~(-2) GFU/mL,48 h,and 4-cell stage infection contributed to efficient expression of the target gene in buffalo embryo,and then improved the efficiency of transgenic buffalo embryo production.
引文
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[4]鞠丽梅,三好一郎,笠井憲雪,等.显微注射法制备转基因小鼠的技术研究[J].中国实验动物学报,1999,7(1):11-15.JU L M,MIYOSHI L,KEN K S,et al.Study on techniques of making transgenic mice by microinjection method[J].Acta Laboratorium Animalis Scientia Sinica,1999,7(1):11-15.(in Chinese)
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[6]王丹.应用ICSI-Tr和受精卵胞质注射技术生产转基因水牛胚胎的初步研究[D].南宁:广西大学,2011.WANG D.A preliminary study on the production of transgenic buffalo embryos using ICSI-Tr and fertilized egg cytoplasmic injection technology[D].Nanning:Guangxi University,2011.(in Chinese)
[7]韦精卫,孟凡丽,韦英明,等.利用体细胞核移植技术生产黄牛和水牛同种、异种转基因克隆囊胚[J].自然科学进展,2008,18(8):857-862.WEI J W,MENG F L,WEI Y M,et al.The somatic cell nuclear transfer technique was used to produce the homologous and heterogeneous transgenic clones of cattle and buffalo[J].Progress of Natural Sciences,2008,18(8):857-862.(in Chinese)
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[9]邓彦飞,刘真真,李云芳,等.不同逆转录载体系统应用于水牛胎儿成纤维细胞转基因的探索[J].华南农业大学学报,2013,4:558-563.DENG Y F,LIU Z Z,LI Y F,et al.Application of different retroviral carrier systems in the transgenic of buffalo fetal fibroblast cells[J].Journal of South China Agricultural University,2013,4:558-563.(in Chinese)
[10]王仕忠,王勇,关乃富,等.肺癌细胞表面CAR表达水平与腺病毒转基因治疗的可能性研究[J].现代肿瘤医学,2008,16(6):947-950.WANG S Z,WANG Y,GUAN N F,et al.Study on the level of car expression on the surface of lung cancer cells and the possibility of transgenic treatment with adenovirus[J].Modern Oncology Medicine,2008,16(6):947-950.(in Chinese)
[11]卫晋雄.唾液腺腺病毒载体转基因研究进展[J].国际口腔医学杂志,2002,29(2):114-116.WEI J X.Advances in the study of adenovirus vector transgene of salivary gland virus[J].International Journal of Oral Medicine,2002,29(2):114-116.(in Chinese)
[12]段安琴,庞春英,朱鹏,等.腺病毒载体转染水牛不同原代体细胞的效果比较[J].中国畜牧兽医,2016,43(10):2661-2665.DUAN A Q,PANG C Y,ZHU P,et al.Comparison of the effects of adenovirus vectors on different primary somatic cells of buffalo[J].China Animal Husbandry&Veterinary Medicine,2016,43(10):2661-2665.(in Chinese)
[13]PEURA T T,LEWIS I M,TROUNSON A O.The effect of recipient oocyte volume on nuclear transfer in cattle[J].Molecular Reproduction&Development,2015,50(2):185-191.
[14]VAJTA G,LEWIS I M,HYTTEL P,et al.Somatic cell cloning without micromanipulators[J].Cloning,2001,3(2):89-95.
[15]MODLI N'SKI J A.The role of the zona pellucida in the development of mouse eggs in vivo[J].Journal of Embryology&Experimental Morphology,1970,23(3):539-547.
[16]THADANI V M.Mice produced from eggs fertilized in vitro at a very low sperm:Egg ratio[J].Journal of Experimental Zoology Part A Ecological Genetics&Physiology,2010,219(3):277-283.
[17]TSUKUI T,MIYAKE S,AZUMA S,et al.Gene transfer and expression in mouse preimplantation embryos by recombinant adenovirus vector[J].Molecular Reproduction&Development,1995,42(3):291-297.
[18]赵红卫.小鼠非完整透明带胚胎的构建、培养及转染的研究[D].秦皇岛:燕山大学,2012.ZHAO H W.Study on the construction,culture and transfection of the embryo of incomplete transparent zone in mice[D].Qinhuangdao:Yanshan University,2012.(in Chinese)
[19]TSUKUI T,KANEGAE Y I,TOYODA Y.Transgenesis by adenovirus-mediated gene transfer into mouse zona-free eggs[J].Nature Biotechnology,1996,14(8):982-985.
[20]杨丹丹,刘晨,蒋凤兵,等.miR-450a腺病毒对体外培养小鼠早期胚胎发育的影响[J].中国生物制品学杂志,2012,25(7):859-861.YANG D D,LIU C,JIANG F B,et al.Effects of mir-450aadenovirus on early embryonic development of mice cultured in vitro[J].Chinese Journal of Biologics,2012,25(7):859-861.(in Chinese)
[21]武成聪,荣树,任静,等.腺病毒介导增强型绿色荧光蛋白基因转染兔骨髓间充质干细胞的效率及毒性[J].中国组织工程研究,2018,17:2650-2655.WU C C,RONG S,REN J,et al.Transferring adenovirus vector-mediated enhanced green fluorescent protein gene into rabbit bone marrow mesenchymal stem cells:Effectiveness and toxicity[J].Chinese Journal of Tissue Engineering Research,2018,17:2650-2655.(in Chinese)
[22]彭道琥,钟德君,廖烨晖.Oct4、Sox2、C-Myc、Klf4腺病毒载体感染小鼠的胚胎成纤维细胞的实验研究[J].四川医学,2017,38(4):385-388.PENG D H,ZHONG D J,LIAO Y H.Experimental study on embryonic fibroblast of mice infected with Oct4,Sox2,C-Myc and Klf4adenovirus[J].Sichuan Medicine,2017,38(4):385-388.(in Chinese)
[23]姚永曦,吴睿帆,汪以真,等.腺病毒与慢病毒感染猪原代细胞的比较研究[J].中国畜牧杂志,2018,54(4):83-88.YAO Y X,WU R F,WANG Y Z,et al.Comparative study on adenovirus and lentivirus infection of porcine primary cells[J].Chinese Journal of Animal Science,2018,54(4):83-88.(in Chinese)
[24]钱建新,李晓军,董克家.转基因杂交水牛染色体核型分析[J].海南医学院学报,2002,8(1):5-6.QIAN J X,LI X J,DONG K J.The analysis of the chromasomal karyotypes of a transgenic hybrid-buffalo[J].Journal of Hainan Medical College,2002,8(1):5-6.(in Chinese)
[25]杨素芳.水牛卵母细胞孤雌激活与体细胞核移植的研究[D].南宁:广西大学,2002.YANG S F.Studies related to parthenogenetic activation of buffalo oocytes and somatic nuclear transfer[D].Nanning:Guangxi University,2002.(in Chinese)
[26]崔奎青.转基因体细胞克隆水牛与广西水牛的遗传特性研究[D].南宁:广西大学,2007.CUI K Q.Study on the transgenic cloning and genetic analysis of Guangxi buffalos[D].Nanning:Guangxi University,2007.(in Chinese)
[27]陆凤花,罗婵,李楠,等.水牛转基因克隆胚胎的制备[A].中国畜牧兽医学会动物繁殖学分会学术研讨会[C].2010.LU F H,LUO C,LI N,et al.Preparation of transgenic cloned embryos of buffalo[A].Academic Symposium of Animal Reproduction Branch of China Animal Husbandry and Veterinary Society[C].2010.(in Chinese)
[28]刘庆友,王丹,张会娜,等.不同精子处理对应用IC-SI-Tr技术生产转基因水牛胚胎效率的影响[J].基因组学与应用生物学,2011,30(4):294-299.LIU Q Y,WANG D,ZHANG H N,et al.Effects of sperm treatment on efficiency of production transgenic buffalo embryo using ICSI-Tr technique[J].Genomics and Applied Biology,2011,30(4):294-299.(in Chinese)
[29]王建,庞春英,邓廷贤,等.影响Fat-1转基因水牛胚胎发育因素的研究[J].中国畜牧兽医,2012,39(12):121-124.WANG J,PANG C Y,DENG T X,et al.Study on the factors affecting embryo development of Fat-1transgenic buffalo[J].China Animal Husbandry&Veterinary Medicine,2012,39(12):121-124.(in Chinese)
[2]屈红林,陈嘉勤.阿尔茨海默病转基因实验动物模型的构建研究[J].基因组学与应用生物学,2018,37(3):1071-1076.QU H L,CHEN J Q.Study on the construction of transgenic experimental animal model of Alzheimer’s disease[J].Genomics and Applied Biology,2018,37(3):1071-1076.(in Chinese)
[3]CHANEY A,BAUER M,BOCHICCHIO D,et al.Longitudinal investigation of neuroinflammation and metabolite profiles in the APPswe×PS1Δe9transgenic mouse model of Alzheimer’s disease[J].Journal of Neurochemistry,2018,144(3):318-335.
[4]鞠丽梅,三好一郎,笠井憲雪,等.显微注射法制备转基因小鼠的技术研究[J].中国实验动物学报,1999,7(1):11-15.JU L M,MIYOSHI L,KEN K S,et al.Study on techniques of making transgenic mice by microinjection method[J].Acta Laboratorium Animalis Scientia Sinica,1999,7(1):11-15.(in Chinese)
[5]焦钰.原核注射制备转基因小鼠的技术优化[D].呼和浩特:内蒙古大学,2016.JIAO Y.Technology optimization of transgenic mice prepared by prokaryotic injection[D].Hohhot:Inner Mongolia University,2016.(in Chinese)
[6]王丹.应用ICSI-Tr和受精卵胞质注射技术生产转基因水牛胚胎的初步研究[D].南宁:广西大学,2011.WANG D.A preliminary study on the production of transgenic buffalo embryos using ICSI-Tr and fertilized egg cytoplasmic injection technology[D].Nanning:Guangxi University,2011.(in Chinese)
[7]韦精卫,孟凡丽,韦英明,等.利用体细胞核移植技术生产黄牛和水牛同种、异种转基因克隆囊胚[J].自然科学进展,2008,18(8):857-862.WEI J W,MENG F L,WEI Y M,et al.The somatic cell nuclear transfer technique was used to produce the homologous and heterogeneous transgenic clones of cattle and buffalo[J].Progress of Natural Sciences,2008,18(8):857-862.(in Chinese)
[8]KUROME M,UEDA H,TOMII R,et al.Production of transgenic-clone tigs by the combination of ICSI-mediated gene transfer with somatic cell nuclear transfer[J].Transgenic Research,2006,15(2):229-240.
[9]邓彦飞,刘真真,李云芳,等.不同逆转录载体系统应用于水牛胎儿成纤维细胞转基因的探索[J].华南农业大学学报,2013,4:558-563.DENG Y F,LIU Z Z,LI Y F,et al.Application of different retroviral carrier systems in the transgenic of buffalo fetal fibroblast cells[J].Journal of South China Agricultural University,2013,4:558-563.(in Chinese)
[10]王仕忠,王勇,关乃富,等.肺癌细胞表面CAR表达水平与腺病毒转基因治疗的可能性研究[J].现代肿瘤医学,2008,16(6):947-950.WANG S Z,WANG Y,GUAN N F,et al.Study on the level of car expression on the surface of lung cancer cells and the possibility of transgenic treatment with adenovirus[J].Modern Oncology Medicine,2008,16(6):947-950.(in Chinese)
[11]卫晋雄.唾液腺腺病毒载体转基因研究进展[J].国际口腔医学杂志,2002,29(2):114-116.WEI J X.Advances in the study of adenovirus vector transgene of salivary gland virus[J].International Journal of Oral Medicine,2002,29(2):114-116.(in Chinese)
[12]段安琴,庞春英,朱鹏,等.腺病毒载体转染水牛不同原代体细胞的效果比较[J].中国畜牧兽医,2016,43(10):2661-2665.DUAN A Q,PANG C Y,ZHU P,et al.Comparison of the effects of adenovirus vectors on different primary somatic cells of buffalo[J].China Animal Husbandry&Veterinary Medicine,2016,43(10):2661-2665.(in Chinese)
[13]PEURA T T,LEWIS I M,TROUNSON A O.The effect of recipient oocyte volume on nuclear transfer in cattle[J].Molecular Reproduction&Development,2015,50(2):185-191.
[14]VAJTA G,LEWIS I M,HYTTEL P,et al.Somatic cell cloning without micromanipulators[J].Cloning,2001,3(2):89-95.
[15]MODLI N'SKI J A.The role of the zona pellucida in the development of mouse eggs in vivo[J].Journal of Embryology&Experimental Morphology,1970,23(3):539-547.
[16]THADANI V M.Mice produced from eggs fertilized in vitro at a very low sperm:Egg ratio[J].Journal of Experimental Zoology Part A Ecological Genetics&Physiology,2010,219(3):277-283.
[17]TSUKUI T,MIYAKE S,AZUMA S,et al.Gene transfer and expression in mouse preimplantation embryos by recombinant adenovirus vector[J].Molecular Reproduction&Development,1995,42(3):291-297.
[18]赵红卫.小鼠非完整透明带胚胎的构建、培养及转染的研究[D].秦皇岛:燕山大学,2012.ZHAO H W.Study on the construction,culture and transfection of the embryo of incomplete transparent zone in mice[D].Qinhuangdao:Yanshan University,2012.(in Chinese)
[19]TSUKUI T,KANEGAE Y I,TOYODA Y.Transgenesis by adenovirus-mediated gene transfer into mouse zona-free eggs[J].Nature Biotechnology,1996,14(8):982-985.
[20]杨丹丹,刘晨,蒋凤兵,等.miR-450a腺病毒对体外培养小鼠早期胚胎发育的影响[J].中国生物制品学杂志,2012,25(7):859-861.YANG D D,LIU C,JIANG F B,et al.Effects of mir-450aadenovirus on early embryonic development of mice cultured in vitro[J].Chinese Journal of Biologics,2012,25(7):859-861.(in Chinese)
[21]武成聪,荣树,任静,等.腺病毒介导增强型绿色荧光蛋白基因转染兔骨髓间充质干细胞的效率及毒性[J].中国组织工程研究,2018,17:2650-2655.WU C C,RONG S,REN J,et al.Transferring adenovirus vector-mediated enhanced green fluorescent protein gene into rabbit bone marrow mesenchymal stem cells:Effectiveness and toxicity[J].Chinese Journal of Tissue Engineering Research,2018,17:2650-2655.(in Chinese)
[22]彭道琥,钟德君,廖烨晖.Oct4、Sox2、C-Myc、Klf4腺病毒载体感染小鼠的胚胎成纤维细胞的实验研究[J].四川医学,2017,38(4):385-388.PENG D H,ZHONG D J,LIAO Y H.Experimental study on embryonic fibroblast of mice infected with Oct4,Sox2,C-Myc and Klf4adenovirus[J].Sichuan Medicine,2017,38(4):385-388.(in Chinese)
[23]姚永曦,吴睿帆,汪以真,等.腺病毒与慢病毒感染猪原代细胞的比较研究[J].中国畜牧杂志,2018,54(4):83-88.YAO Y X,WU R F,WANG Y Z,et al.Comparative study on adenovirus and lentivirus infection of porcine primary cells[J].Chinese Journal of Animal Science,2018,54(4):83-88.(in Chinese)
[24]钱建新,李晓军,董克家.转基因杂交水牛染色体核型分析[J].海南医学院学报,2002,8(1):5-6.QIAN J X,LI X J,DONG K J.The analysis of the chromasomal karyotypes of a transgenic hybrid-buffalo[J].Journal of Hainan Medical College,2002,8(1):5-6.(in Chinese)
[25]杨素芳.水牛卵母细胞孤雌激活与体细胞核移植的研究[D].南宁:广西大学,2002.YANG S F.Studies related to parthenogenetic activation of buffalo oocytes and somatic nuclear transfer[D].Nanning:Guangxi University,2002.(in Chinese)
[26]崔奎青.转基因体细胞克隆水牛与广西水牛的遗传特性研究[D].南宁:广西大学,2007.CUI K Q.Study on the transgenic cloning and genetic analysis of Guangxi buffalos[D].Nanning:Guangxi University,2007.(in Chinese)
[27]陆凤花,罗婵,李楠,等.水牛转基因克隆胚胎的制备[A].中国畜牧兽医学会动物繁殖学分会学术研讨会[C].2010.LU F H,LUO C,LI N,et al.Preparation of transgenic cloned embryos of buffalo[A].Academic Symposium of Animal Reproduction Branch of China Animal Husbandry and Veterinary Society[C].2010.(in Chinese)
[28]刘庆友,王丹,张会娜,等.不同精子处理对应用IC-SI-Tr技术生产转基因水牛胚胎效率的影响[J].基因组学与应用生物学,2011,30(4):294-299.LIU Q Y,WANG D,ZHANG H N,et al.Effects of sperm treatment on efficiency of production transgenic buffalo embryo using ICSI-Tr technique[J].Genomics and Applied Biology,2011,30(4):294-299.(in Chinese)
[29]王建,庞春英,邓廷贤,等.影响Fat-1转基因水牛胚胎发育因素的研究[J].中国畜牧兽医,2012,39(12):121-124.WANG J,PANG C Y,DENG T X,et al.Study on the factors affecting embryo development of Fat-1transgenic buffalo[J].China Animal Husbandry&Veterinary Medicine,2012,39(12):121-124.(in Chinese)