OSP-1启动子及与CMV结合的双启动子真核表达载体在奶山羊卵巢壁颗粒细胞中的表达与鉴定
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摘要
中国是世界上奶山羊生产大国,近几年国内外的山羊奶消费热激发了业内专家对高产奶羊品种选育的高度关注。我国是养殖大国也是人口大国,随着畜牧业的快速发展,人畜争粮问题也日益严峻,因此,大力发展节粮环保和优质高效的奶山羊产业,对于转变我国奶业发展方式,促进奶业的可持续发展具有重要意义,而有效增加母羊胎产羔数和产后成活数不仅可以显著提高生产效益,而且可以促使我国的养羊业可持续发展。但由于多年以来,对奶山羊品种的繁殖性状缺乏有效的系统选育,致使产羔率下降到150%左右,因此,急需进一步选育提高奶山羊的产羔性能。为了快速提高奶山羊的产羔性能,必须尽快的把现代生物技术应用到奶山羊的产羔性状育种上来,最大程度上解决制约了奶羊业的快速发展的生产瓶颈问题。
卵巢是动物繁殖中最重要的器官,且颗粒细胞在维持奶山羊繁殖功能上发挥着非常重要的作用,筛选和研究调控卵泡颗粒细胞内与产羔性能密切相关的功能基因表达的特异性启动子是当前动物生殖科学研究的热点之一。目前发现的卵巢组织中特异性的启动子有OSP-1、OSP-2 (卵巢组织特异性启动子),而这些启动子能否有效的调控奶山羊卵巢组织中与产羔数相关的功能基因的特异性表达还缺乏试验证明。
本试验旨在筛选和研究适合于调控奶山羊卵巢组织的特异性启动子,以便通过基因工程等分子生物学技术探索调控奶山羊卵泡形成、成熟,以及维持卵巢功能等的功能基因对于奶山羊排卵数、受精率和产羔数的影响,进而为奶山羊的分子育种繁殖工作提供一个可行的方法。本研究采用卵巢特异性启动子表达载体的构建、卵泡颗粒细胞分离培育、脂质体转染、实时定量PCR检测等实验方法,分析OSP-1的卵巢组织特异性及双启动子调控报告基因在卵巢组织中的表达效率,试验结果如下:
1、设计并克隆了卵巢特异性启动子(OSP-1)。构建了三种含有不同启动子的EGFP (报告基因)载体:pCMV-EGFP (巨细胞病毒启动子报告基因载体)、pOSP1-EGFP (卵巢特异性启动子的报告基因载体)、pCMV-OSP1-EGFP(巨细胞病毒启动子和卵巢特异性启动子报告基因载体)。
2、使用机械分离法,成功分离培养得到奶山羊卵泡颗粒细胞。原代贴壁的奶山羊卵泡颗粒细胞大部分为梭形或者不规则多边形。刚开始接种后,细胞呈悬浮的球型状;随着培养时间的增加,细胞开始接触、贴壁,同时有彼此接近运动并出现细胞团的现象;继续培养发现,细胞团底部贴壁的伪足相互接触;大约培养5-6天后,可铺满培养皿底部,形成颗粒细胞单层。
3、将pOSP1-EGFP转染到奶山羊卵泡颗粒细胞、乳腺上皮细胞、胎儿成纤维细胞及人卵巢癌细胞系HO-8910。通过倒置荧光显微镜观察和实时定量PCR鉴定,只在奶山羊卵泡颗粒细胞和人卵巢癌细胞系HO-8910中检测到EGFP的表达,表明pOSP1-EGFP在卵巢组织中有表达转录活性。
4、分别将含有OSP-1、CMV和CMV-OSP1双启动子的EGFP表达载体转染不同细胞,采用实时定量PCR检测不同启动子调控下EGFP在不同细胞中的表达效率,结果显示:在卵泡颗粒细胞中,pCMV-OSP1-EGFP组EGFP的相对表达量分别比单启动子组pOSP1-EGFP、pCMV-EGFP提高了29%( p<0.05)、51.22%( p <0.05);在人卵巢癌细胞系HO-8910中,pCMV-OSP1-EGFP组EGFP的相对表达量分别比单启动子组pOSP1-EGFP、pCMV-EGFP提高了21%( p <0.05)、44.07%( p <0.05)。表明增加启动子数目可以有效地提高外源基因的表达效率。
Reproductive rate is an important economic trait in goat reproduction. To improve the fecundity, also known as litter size, is of special meaning in the selection of animals with high reproductivity. Ovary is the main functional organ in reproduction, and Granulosa cells playe an important role in maintaining the function of dairy goat breeding.It is a hot topic in animal reproductive science to screen and research ovarian tissue specific promoter, which could regulate the genes related with lambing performance in the ovarian granulosa cells.Currently, it was found that the ovarian tissue-specific promoter were only OSP-1, OSP-2. But there lack experimental proof whether these promoter can regulate functional genes associated with the litter size-specific in the dairy goat ovarian tissue.
This study was designed to analyse the ovarian tissue-specific of OSP-1 and the expression efficiency of EGFP in the ovarian tissue with the regulation of two promoters, with the use of the constrction of ovarian specific promoter expression vector, the isolation and cultivation of granulosa cells, lipofection, real-time quantitative PCR and so on. In this research, we design and clone ovarian-specific promoter1 (OSP-1). Three vectors carried different promoters were constructed, which were carrying EGFP driven by CMV promoter, OSP-1 promoter and CMV-SP conjoint promoters, respectively. Those were named: pCMV-EGFP, pOSP1-EGFP and p CMV-OSP1-EGFP.
In this study, the use of mechanical separation have successfully isolated and cultured ovarian mural granulose cells of dairy goat, and most of primary adherent granulose cells were spindle or irregular polygon. After the inoculation, cells were suspended spherical shape; with the increase of culture time, cells begin to contact and adherence, while there are close to each movement and the emergence of the phenomenon of cell clusters. Continuing to been cultured, the pseudopods in the bottom of cell clusters contact with each other. After 5-6 days, the bottom of petri dish can be covered, and form a granular cell layer.
The vector pOSP1-EGFP have been transfected into four different cell: ovarian mural granulose cells of dairy goat, breast epithelial cells, fetal fibroblast cells and human ovarian cancer cell line HO-8910. The expression of OSP-1 were been detected only in the ovarian mural granulose cells of dairy goat and HO-8910, after observed by inverted fluorescence microscope and real-time quantitative PCR. This shows pOSP1-EGFP has the specific expressional activity in ovarian tissues.
The EGFP expression vectors containing OSP-1, CMV and CMV-OSP1 promoter have been transfected into different cell. We detected the level of EGFP expression of different promoters in different cells by real-time quantitative PCR. The results showed: in the ovarian mural granulose cells of dairy goat, the expression efficacy of EGFP by two promoters was higher than the groups of pOSP1-EGFP and pCMV-EGFP (29%, P<0.01 and 51.22%, P<0.01); in the human ovarian cancer cell line HO-8910, the expression efficacy of EGFP by two promoters was higher than the groups of pOSP1-EGFP and pCMV-EGFP (21%, P<0.01 and 44.07%, P<0.01). These data indicated that it can improve the efficiency of exogenous gene expression effectively.
卵巢是动物繁殖中最重要的器官,且颗粒细胞在维持奶山羊繁殖功能上发挥着非常重要的作用,筛选和研究调控卵泡颗粒细胞内与产羔性能密切相关的功能基因表达的特异性启动子是当前动物生殖科学研究的热点之一。目前发现的卵巢组织中特异性的启动子有OSP-1、OSP-2 (卵巢组织特异性启动子),而这些启动子能否有效的调控奶山羊卵巢组织中与产羔数相关的功能基因的特异性表达还缺乏试验证明。
本试验旨在筛选和研究适合于调控奶山羊卵巢组织的特异性启动子,以便通过基因工程等分子生物学技术探索调控奶山羊卵泡形成、成熟,以及维持卵巢功能等的功能基因对于奶山羊排卵数、受精率和产羔数的影响,进而为奶山羊的分子育种繁殖工作提供一个可行的方法。本研究采用卵巢特异性启动子表达载体的构建、卵泡颗粒细胞分离培育、脂质体转染、实时定量PCR检测等实验方法,分析OSP-1的卵巢组织特异性及双启动子调控报告基因在卵巢组织中的表达效率,试验结果如下:
1、设计并克隆了卵巢特异性启动子(OSP-1)。构建了三种含有不同启动子的EGFP (报告基因)载体:pCMV-EGFP (巨细胞病毒启动子报告基因载体)、pOSP1-EGFP (卵巢特异性启动子的报告基因载体)、pCMV-OSP1-EGFP(巨细胞病毒启动子和卵巢特异性启动子报告基因载体)。
2、使用机械分离法,成功分离培养得到奶山羊卵泡颗粒细胞。原代贴壁的奶山羊卵泡颗粒细胞大部分为梭形或者不规则多边形。刚开始接种后,细胞呈悬浮的球型状;随着培养时间的增加,细胞开始接触、贴壁,同时有彼此接近运动并出现细胞团的现象;继续培养发现,细胞团底部贴壁的伪足相互接触;大约培养5-6天后,可铺满培养皿底部,形成颗粒细胞单层。
3、将pOSP1-EGFP转染到奶山羊卵泡颗粒细胞、乳腺上皮细胞、胎儿成纤维细胞及人卵巢癌细胞系HO-8910。通过倒置荧光显微镜观察和实时定量PCR鉴定,只在奶山羊卵泡颗粒细胞和人卵巢癌细胞系HO-8910中检测到EGFP的表达,表明pOSP1-EGFP在卵巢组织中有表达转录活性。
4、分别将含有OSP-1、CMV和CMV-OSP1双启动子的EGFP表达载体转染不同细胞,采用实时定量PCR检测不同启动子调控下EGFP在不同细胞中的表达效率,结果显示:在卵泡颗粒细胞中,pCMV-OSP1-EGFP组EGFP的相对表达量分别比单启动子组pOSP1-EGFP、pCMV-EGFP提高了29%( p<0.05)、51.22%( p <0.05);在人卵巢癌细胞系HO-8910中,pCMV-OSP1-EGFP组EGFP的相对表达量分别比单启动子组pOSP1-EGFP、pCMV-EGFP提高了21%( p <0.05)、44.07%( p <0.05)。表明增加启动子数目可以有效地提高外源基因的表达效率。
Reproductive rate is an important economic trait in goat reproduction. To improve the fecundity, also known as litter size, is of special meaning in the selection of animals with high reproductivity. Ovary is the main functional organ in reproduction, and Granulosa cells playe an important role in maintaining the function of dairy goat breeding.It is a hot topic in animal reproductive science to screen and research ovarian tissue specific promoter, which could regulate the genes related with lambing performance in the ovarian granulosa cells.Currently, it was found that the ovarian tissue-specific promoter were only OSP-1, OSP-2. But there lack experimental proof whether these promoter can regulate functional genes associated with the litter size-specific in the dairy goat ovarian tissue.
This study was designed to analyse the ovarian tissue-specific of OSP-1 and the expression efficiency of EGFP in the ovarian tissue with the regulation of two promoters, with the use of the constrction of ovarian specific promoter expression vector, the isolation and cultivation of granulosa cells, lipofection, real-time quantitative PCR and so on. In this research, we design and clone ovarian-specific promoter1 (OSP-1). Three vectors carried different promoters were constructed, which were carrying EGFP driven by CMV promoter, OSP-1 promoter and CMV-SP conjoint promoters, respectively. Those were named: pCMV-EGFP, pOSP1-EGFP and p CMV-OSP1-EGFP.
In this study, the use of mechanical separation have successfully isolated and cultured ovarian mural granulose cells of dairy goat, and most of primary adherent granulose cells were spindle or irregular polygon. After the inoculation, cells were suspended spherical shape; with the increase of culture time, cells begin to contact and adherence, while there are close to each movement and the emergence of the phenomenon of cell clusters. Continuing to been cultured, the pseudopods in the bottom of cell clusters contact with each other. After 5-6 days, the bottom of petri dish can be covered, and form a granular cell layer.
The vector pOSP1-EGFP have been transfected into four different cell: ovarian mural granulose cells of dairy goat, breast epithelial cells, fetal fibroblast cells and human ovarian cancer cell line HO-8910. The expression of OSP-1 were been detected only in the ovarian mural granulose cells of dairy goat and HO-8910, after observed by inverted fluorescence microscope and real-time quantitative PCR. This shows pOSP1-EGFP has the specific expressional activity in ovarian tissues.
The EGFP expression vectors containing OSP-1, CMV and CMV-OSP1 promoter have been transfected into different cell. We detected the level of EGFP expression of different promoters in different cells by real-time quantitative PCR. The results showed: in the ovarian mural granulose cells of dairy goat, the expression efficacy of EGFP by two promoters was higher than the groups of pOSP1-EGFP and pCMV-EGFP (29%, P<0.01 and 51.22%, P<0.01); in the human ovarian cancer cell line HO-8910, the expression efficacy of EGFP by two promoters was higher than the groups of pOSP1-EGFP and pCMV-EGFP (21%, P<0.01 and 44.07%, P<0.01). These data indicated that it can improve the efficiency of exogenous gene expression effectively.
引文
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Lieto LD, Borrego F, You CH.2003.Human CD94 gene expression: dual promoters differing in responsiveness to IL-2 or IL-15. Journal of Immunology, 171: 5277-5286
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Heid CA, Stevens J, Livak KJ, Williams PM. 1996. Real time quantitative PCR. Genome Res, 6: 986-994 Higuchi R, Dollinger G, Walsh PS. 1992. Simultaneous amplification and detection of specific DNA sequences. Biotechnology(N Y), 10: 413-417
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Kemp PR, Osbourn JK, Grainger DJ, Metcalf JC. 1995. Cloning and analysis of the promoter region of the rat SM22αgene. J Biochem, 310 :1037-1043
Knipe D M, Howley P M, Griffin D E. 2001. Fields Virology. 4th. Philadelphia: Lippincott William & Wilkins: 2399-2420
Kubista M, Andrade J, Bengtsson M. 2006. The real-time polymerase chain reaction. Mol Aspects Med, 27:95-125.
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Kyung-Jin Kim,Han-Eol Kim,Kwang-Hoon Lee.2004.Two-promoter vector is highly efficient for overproduction of protein complexes. Protein Sci, 13: 1698-1703
Lee LG, Connell CR, Bloch W.1993. Allelic discrimination by nick-translation PCR with fluorogenic probes. Nucleic Acids Res, 21:3761-3766
Li X Y, Eastman EM, Schwartz RJ, Schwartz, Ruxandra Draghia-Akli. 1999. Synthetic muscle promoters : activities exceeding naturally occurring regulatory sequences. Nat Biotechnol, 17 (3): 241-245
Lieto LD, Borrego F, You CH.2003.Human CD94 gene expression: dual promoters differing in responsiveness to IL-2 or IL-15. Journal of Immunology, 171: 5277-5286
Livak KJ, Flood SJ, Marmaro J, Giusti W, Deetz K.1995. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleicacid hybridization. PCR Methods Appl, 4:357-362
Mertens N, Remaut E, Fiers W. 1995. Versatile multi-featured plasmids for high-level expression of heterologous genes in Escherichia coli:overproduction of human and murine cytokines.Gene, 164: 9-15
Min BH, Foster DN, Strauch AR. 1990. The 5′2 flanking region of the mouse vascular smooth muscleα2actin gene contains ev olutionarily conserved sequence motifs within a functional promoter . J Biol .Chem , 265 (27): 16667-16675
Ming-liang He, Leng Wen,Christine E, Campbell.1999. Transcription repression by Xenopus ET and its human ortholog TBX3,a gene involved in ulnar-mammary syndrome. Developmental Biology, 96 (18): 10212-10217
Muller CW.2001. Transcription factors:global and detailed views.Curr. Opin. Struct. Biol, 11:26-32 Mullis KB. 1990. Target amplification for DNA analysis by the polymerase chain reaction. Ann Biol Clin(Paris), 48:579-582.
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Nandi S, Girish KV, Manjunatha BM. 2008. Follicular fluid concentrations of glucose, lactate and pyruvate in buffalo and sheep, and their effects on cultured oocytes, granulosa and cumulus cells. Theriogenology, 69: 186-196
Paul S, Zhang X, Hulett FM.2001. Two ResD-Controlled Promoters Regulate ctaA Expression in Bacillus subtilis, Journal of Bacteriology, 183: 3237-3246
Saha BK, Tian B, Bucy RP. 2001. Quantitation of HIV-1 by real-time PCR with a unique fluorogenic probe. J Virol Methods, 93:33-42
Schweitzer B, Kingsmore S. 2001. Combining nucleic acid amplification and detection. Curr Opin Biotechnol, 12:21-27
Stewart V,Bledsoe PJ,wiUi SB.2003. Dual overlapping promoters control napF (pefiplasmic nitrate reductase)operon expresion in Escherichia eoli K-12. Journal of Bacteriology, l85:5862-5870
Tyagi S, Bratu DP, Kramer FR. 1998. Multicolor molecular beacons for allele discrimination. Nat Biotechnol, 16:49-53
Tyagi S, Kramer FR. 1996. Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol, 14: 303-308
U Ranga, C Woffendin, ZY Yang. 1997.Cell and viral regulatory elements enhance the expression and function of a human immunodeficiency virus inhibitory gene.J Virol, 71 (9): 7020-7029
W. Bruening, B. Giasson, W. Mushynski, H. D. Durham. 1998. Activation of stress-activated MAP protein kinases up-regulates expression of transgenes driven by the cytomegalovirus immediate/early promoter. Nucleic Acids Research, 26 (2): 486-489
Wadsworth S, Jacob R J, Roizman B. 1975. Anatomy of herpes simplex virus DNA.Ⅱ.Size, composition,and arrangement of inverted terminal repetitions. J Virol, 15(06):1487-1497
Walburger DK, Afonina IA, Wydro R. 2001. An improved real time PCR method for simultaneous detection of C282Y and H63D mutations in the HFE gene associated with hereditary hemochromatosis. Mutat Res, 432:69-78
Warren AJ.2002.Eukaryotic transcription factors.Curr. Opin. Struct. Biol, 12: 107-l l4 Wittwer CT, Herrmann MG, Moss AA. 1997. Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques, 22:130-131; 134-138
Xiaobing Yu, Xiangcan Zhan, Jenice D'Costa, Vivek M. Tanavde, Zhaohui Ye, Tien Peng, Matthew T. Malehorn, Xiaoming Yang, Curt I. Civin, Linzhao Cheng.2003. Lentiviral vectors with two independent internal promoters transfer high-level expression of multiple transgenes to human hematopoietic stem-progenitor cells. Molecular Therapy, 7: 827-838
Yang P, Wang J, Gong G, Sun X, Zhang R, et al. 2008. Cattle Mammary Bioreactor Generated by a Novel Procedure of Transgenic Cloning for Large-Scale Production of Functional Human Lactoferrin. PLoS ONE 3(10): e3453.
Yoon SY, Chung GT, Kang DH. 2005. Application of real-time PCR for quantitative detection of Clostridium botulinum type A toxin gene in food. Microbiol Immunol, 49:505-511.
Zhi Quan Xiang, Steve L. Spitalnik, Jun Cheng, Jan Erikson, Boguslaw Wojczyk and Hildegund C. J. Ertl.1995. Immune responses to nucleic acid vaccines to rabies virus.Virology, 209 (2):569-579