sfat-1真核表达载体构建及其转基因细胞系建立
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摘要
n-3多不饱和脂肪酸作为细胞构成的重要成分,一直以来受到人们的广泛关注。研究表明,哺乳动物体内一定量的n-3多不饱和脂肪酸含量,可以起到预防心血管疾病、神经退行性疾病甚至癌症的作用。但n-3多不饱和脂肪酸在大多数动物体内不能合成,只能从食物中补充。fat-1基因的发现改变了这一现状,它可以将多不饱和脂肪酸从n-6形式转化为n-3形式,并发挥功能。研究人员已经利用转基因的方法制备了能够表达fat-1基因的转基因动物,如:小鼠、猪等,为研究n-3多不饱和脂肪酸的功能提供了高效、准确的医学、营养学动物模型。随着人们对n-3多不饱和脂肪酸认识的增加,对富含这一成分的食品需求也大大增加,fat-1基因在生产特殊农产品方面也具有十分大的潜力,亟待我们开发利用。本实验采用经密码子优化了的线虫基因sfat-1,使用脂质体转染的方法获得转sfat-1基因荷斯坦奶牛细胞,并将其应用到克隆研究中。实验中通过PCR-酶切连接方法人工合成了sfat-1基因,并构建了基于真核表达载体pCDNA3.1(+)的转基因载体pCDNA3.1(+)-sfat-1-EGFP及阴性对照载体pCDNA3.1(+)-EGFP;利用阳离子脂质体转染法,对自建8079TE细胞系进行转基因操作,经过G418筛选获得阳性转基因细胞,经PCR、RT-PCR检测证明外源基因已整合进入细胞基因组内。
作为阶段性成果,本实验构建的pCDNA3.1(+)-EGFP对照载体为实验室后来转基因研究提供了良好的对照;建立的8079TE细胞系可以为多种基因的转基因克隆提供良好供体;筛选获得的转sfat-1基因细胞为后期n-3多不饱和脂肪酸细胞水平生物学研究及转sfat-1基因克隆牛研究奠定了基础。
n-3 polyunsaturated fatty acids (PUFAs) that is an important component of the fatty acid, are concerned by scienists for a long time. It is necessary to maintain the appropriate proportion of n-3 PUFAs physiological and it can serve to prevent cardiovascular disease, neurodegenerative diseases and even cancer role. However most animals have to obtain n-3 PUFAs from diet, because they can not synthesize them by themselves. Fat-1 gene, translating into n-3 PUFA desaturase, can convert PUFAs from n-6 to n3 form. Researchers have used the method of gene transfer to the fat-1 gene expression in transgenic animals, such as: mice, pigs for the study of n-3 polyunsaturated fatty acids to provide the functions of an efficient and accurate medical, nutritional animal model. As the awareness of n-3 polyunsaturated fatty acids increasing,the rich ingredients of food demand also increased significantly in people. In the production of special agricultural products,fat-1 gene also has very great potential and we need development and utilization. The nematode gene sfat-1 which optimized the codon is used in this study, we use lipid-transfer method to obtain sfat-1 gene transfered Holstein cow cells, and apply it to cloning research.We synthetic sfat-1 gene using PCR-restriction method. Then reconstruct the gene vector pCDNA3.1(+)-sfat-1-EGFP and negative control vector pCDNA3.1(+)-EGFP based on the Construction of eukaryotic expression vector pCDNA3.1(+).The 8079 TE cell line is transferred using cationic lipid-transfer method, and the positive transgenic cells are obtained after G418 selection. After testing, the PCR, RT-PCR test proved that foreign genes has been integrated within the genome.
As initial results, construction of the pCDNA3.1 (+)-EGFP vector is a good negative control in transgenic rearch in my lab.. The establishment of the 8079 TE cell lines can be good donors in variety of transgenic cloned research.Obtaining of sfat-1 gene transfected positive cells lays the foundation for research of n-3 polyunsaturated fatty acids biology at cell level and sfat-1 transgenic cattle.
作为阶段性成果,本实验构建的pCDNA3.1(+)-EGFP对照载体为实验室后来转基因研究提供了良好的对照;建立的8079TE细胞系可以为多种基因的转基因克隆提供良好供体;筛选获得的转sfat-1基因细胞为后期n-3多不饱和脂肪酸细胞水平生物学研究及转sfat-1基因克隆牛研究奠定了基础。
n-3 polyunsaturated fatty acids (PUFAs) that is an important component of the fatty acid, are concerned by scienists for a long time. It is necessary to maintain the appropriate proportion of n-3 PUFAs physiological and it can serve to prevent cardiovascular disease, neurodegenerative diseases and even cancer role. However most animals have to obtain n-3 PUFAs from diet, because they can not synthesize them by themselves. Fat-1 gene, translating into n-3 PUFA desaturase, can convert PUFAs from n-6 to n3 form. Researchers have used the method of gene transfer to the fat-1 gene expression in transgenic animals, such as: mice, pigs for the study of n-3 polyunsaturated fatty acids to provide the functions of an efficient and accurate medical, nutritional animal model. As the awareness of n-3 polyunsaturated fatty acids increasing,the rich ingredients of food demand also increased significantly in people. In the production of special agricultural products,fat-1 gene also has very great potential and we need development and utilization. The nematode gene sfat-1 which optimized the codon is used in this study, we use lipid-transfer method to obtain sfat-1 gene transfered Holstein cow cells, and apply it to cloning research.We synthetic sfat-1 gene using PCR-restriction method. Then reconstruct the gene vector pCDNA3.1(+)-sfat-1-EGFP and negative control vector pCDNA3.1(+)-EGFP based on the Construction of eukaryotic expression vector pCDNA3.1(+).The 8079 TE cell line is transferred using cationic lipid-transfer method, and the positive transgenic cells are obtained after G418 selection. After testing, the PCR, RT-PCR test proved that foreign genes has been integrated within the genome.
As initial results, construction of the pCDNA3.1 (+)-EGFP vector is a good negative control in transgenic rearch in my lab.. The establishment of the 8079 TE cell lines can be good donors in variety of transgenic cloned research.Obtaining of sfat-1 gene transfected positive cells lays the foundation for research of n-3 polyunsaturated fatty acids biology at cell level and sfat-1 transgenic cattle.
引文
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2.陈秀,昀赵,彭卫平张峰,冯晓黎,黄君霆,陆长德.新霉素抗性基因在家蚕中的插入和表达.生物化学与生物物理学报1999:31(1):90-92.
3.龚国春,戴蕴平,樊宝良,朱化彬,王海平,王莉莉,方昌阁,万荣,刘颖,李荣andothers.以不同类型的转基因细胞为核供体生产牛的转基因克隆胚胎.中国科学(C辑)2003:33(6):532-538.
4.关伟军,马月辉,丁鸿,于太永,张海艳,梁海青.小尾寒羊耳组织成纤维细胞系的建立与生物学特性研究.畜牧兽医学报2005:36:511-516.
5.郭瑜.牛成纤维细胞库的建立及生物学特性研究[硕士学位论文].雅安:四川农业大学.2005
6.李扬,吴凯峰,郭旭东.脂质体介导外源基因体外转染牛胎儿成纤维细胞条件的优化.遗传2002:24(6):653-655.
7.陆会宁,刘丑生,王志刚,张利平,赵俊金,飞孟.贵德黑裘皮羊耳成纤维细胞系的建立和生物学特性的研究.中国草食动物2007:2:3-6.
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9.唐孝青,李斌,伍小兵,张亮.绿色荧光蛋白及其应用的研究进展.陕西农业科学2007:1:123-124,145.
10.薛庆善.体外培养的原理与技术(第一版).北京:科学出版社2001:157.
11.徐佳怡.浅谈EDTA在水泥化学分析中的应用.水泥工程2007:4:65-68.
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2.陈秀,昀赵,彭卫平张峰,冯晓黎,黄君霆,陆长德.新霉素抗性基因在家蚕中的插入和表达.生物化学与生物物理学报1999:31(1):90-92.
3.龚国春,戴蕴平,樊宝良,朱化彬,王海平,王莉莉,方昌阁,万荣,刘颖,李荣andothers.以不同类型的转基因细胞为核供体生产牛的转基因克隆胚胎.中国科学(C辑)2003:33(6):532-538.
4.关伟军,马月辉,丁鸿,于太永,张海艳,梁海青.小尾寒羊耳组织成纤维细胞系的建立与生物学特性研究.畜牧兽医学报2005:36:511-516.
5.郭瑜.牛成纤维细胞库的建立及生物学特性研究[硕士学位论文].雅安:四川农业大学.2005
6.李扬,吴凯峰,郭旭东.脂质体介导外源基因体外转染牛胎儿成纤维细胞条件的优化.遗传2002:24(6):653-655.
7.陆会宁,刘丑生,王志刚,张利平,赵俊金,飞孟.贵德黑裘皮羊耳成纤维细胞系的建立和生物学特性的研究.中国草食动物2007:2:3-6.
8.陶云霞(译).用于转化实验的最佳DNA连接反应.生物工程杂志1987:06.
9.唐孝青,李斌,伍小兵,张亮.绿色荧光蛋白及其应用的研究进展.陕西农业科学2007:1:123-124,145.
10.薛庆善.体外培养的原理与技术(第一版).北京:科学出版社2001:157.
11.徐佳怡.浅谈EDTA在水泥化学分析中的应用.水泥工程2007:4:65-68.
12.杨凤.动物营养学(第二版).北京:中国农业出版社1993
13.周雪雁,关伟军,马月辉,刘桂林.脂质体介导转染法原理及其研究近展.上海畜牧兽医通讯2004:4:5-6.
14.朱贵明.ω-3脂肪酸去饱和酶基因的克隆、鉴定及转基因小鼠的制备[博士学位论文].杨凌:西北农林科技大学2006
15.朱贵明,宏陈,卢建申,周艳荣,吴晓洁,陈红星,邓继先.应用PCR-酶切连接法合成全长sFat-1基因.遗传2006:28(9):1123-1128.
16. Abe Y, El-Masri B, Kimball KT, Pownall H, Reilly CF, Osmundsen K, et al. Soluble cell adhesion molecules in hypertriglyceridemia and potential significance on monocyte adhesion. Arterioscler Thromb Vasc Biol 1998; 18: 723-31.
17. Agren JJ, Vaisanen S, Hanninen O, Muller AD, Hornstra G. Hemostatic factors and platelet aggregation after a fish-enriched diet or fish oil or docosahexaenoic acid supplementation. Prostaglandins Leukot Essent Fatty Acids 1997; 57: 419-21.
18. Akbar M, Calderon F, Wen Z, Kim HY. Docosahexaenoic acid: a positive modulator of Akt signaling in neuronal survival. Proc Natl Acad Sci U S A 2005; 102: 10858-63.
19. Bang HO, Dyerberg J, Hjoorne N. The composition of food consumed by Greenland Eskimos. Acta Med Scand 1976; 200: 69-73.
20. Bhattacharya A, Chandrasekar B, Rahman MM, Banu J, Kang JX, Fernandes G. Inhibition of inflammatory response in transgenic fat-1 mice on a calorie-restricted diet. Biochem Biophys Res Commun 2006; 349: 925-30.
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