Fat-1转基因小鼠模型的构建
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摘要
多不饱和脂肪酸(Polyunsaturated Fatty acids, PUFAs)参与构成生物膜,对生物膜的形成和物理性质、膜脂中脂肪酸的组成和不饱和度等方面起着重要的调节作用。人体中ω-3多不饱和脂肪酸含量低,而ω-6多不饱和脂肪酸含量相对偏高,且二者的平衡对于有机体自身的稳定和正常发育非常重要。除此之外,ω-3多不饱和脂肪酸对人类及其他哺乳动物的正常发育和维持健康极其重要,且在人类的多种疾病的预防与治疗中也发挥着突出的作用。但是ω-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。研究发现Fat-1基因编码的蛋白是多不饱和脂肪酸合成过程中一个重要的脂肪酸脱氢酶,即ω-3脂肪酸脱氢酶,它可以催化ω-6脂肪酸转化为ω-3脂肪酸。本实验首先根据Genebank上公布的秀丽隐杆线虫Fat-1基因序列,按照牛乳蛋白密码子的使用原则,对Fat-1基因进行了改造,同时在改造基因序列的5’端和3’端引入XhoI酶切位点,设计好的整个序列由上海生物工程技术服务有限公司合成。之后构建了基于乳腺特异性表达载体pBC1的转基因载体pBC1-Fat-1,采用原核显微注射方法获得了转Fat-1基因的转基因小鼠,经PCR、Southern blot检测证明外源基因已整合进入细胞基因组内,并对阳性母鼠奶样中ω-3多不饱和脂肪酸含量进行检测,用以分析Fat-1基因的表达情况。检测结果显示阳性母鼠奶样中ω-3/ω-6多不饱和脂肪酸的比值有所提高,说明此Fat-1基因可以在小鼠体内行使其功能,它的表达可以使小鼠乳中的ω-6多不饱和脂肪酸转化为ω-3多不饱和脂肪酸。
Polyunsaturated fatty acids(PUFAs)are present in all groups of organisms and play a key role in the maintenance of the proper structure and functioning of biological membanes. The amount ofω-3 PUFAs is insufficient in most people, whereas the level ofω-6 PUFAs is relatively too high, the balance of these two classes of PUFAs is important for homeostasis and normal development. Besidesω-3 PUFAs have shown to exert many preventive and therapeutic actions besides their important role in maintenances human health and normal development. However most animals have to obtainω-3 PUFAs from diet, because they can not synthesize them by themselves. The protein encoded by Fat-1 gene isω-3 fatty acid desaturase, which is an important fatty acid desaturase in the biosynthesis of PUFAs.This enzyme can catalyze the conversion ofω-6 PUFAs toω-3 PUFAs. In this study , based on the published seqence of the C.elegans Fat-1 gene in the Genebank and the regulation of the codons usage of the bovine milk protein with the bias, we optimized the codons of Fat-1 gene and synthesized the optimized gene, and inserted the restriction enzyme digestion sites XhoI to the 5’and 3’ends of sequende. After the whole modified sequence was synthesized by the Shanghai Sangon Biological Engineering Technology and Service Co,Ltd. Then we reconstruct the gene vector pBC1-Fat-1 based on the pBC1. After that we transferred this Fat-1 gene into mice by the method of microinjection. Then we proved that foreign genes has been integated within the mice genome by the PCR and Southern blot.Meanwhile we analysis theω-3/ω-6 PUFAs of the transgenic mice milk, the result demonstrated clearly thatω-6 PUFAs in milk of the transgenic mice are reduced,indicating thatω-6 PUFAs have been converted toω-3 PUFAs,causing the ratio ofω-3/ω-6 PUFAs to increased.
Polyunsaturated fatty acids(PUFAs)are present in all groups of organisms and play a key role in the maintenance of the proper structure and functioning of biological membanes. The amount ofω-3 PUFAs is insufficient in most people, whereas the level ofω-6 PUFAs is relatively too high, the balance of these two classes of PUFAs is important for homeostasis and normal development. Besidesω-3 PUFAs have shown to exert many preventive and therapeutic actions besides their important role in maintenances human health and normal development. However most animals have to obtainω-3 PUFAs from diet, because they can not synthesize them by themselves. The protein encoded by Fat-1 gene isω-3 fatty acid desaturase, which is an important fatty acid desaturase in the biosynthesis of PUFAs.This enzyme can catalyze the conversion ofω-6 PUFAs toω-3 PUFAs. In this study , based on the published seqence of the C.elegans Fat-1 gene in the Genebank and the regulation of the codons usage of the bovine milk protein with the bias, we optimized the codons of Fat-1 gene and synthesized the optimized gene, and inserted the restriction enzyme digestion sites XhoI to the 5’and 3’ends of sequende. After the whole modified sequence was synthesized by the Shanghai Sangon Biological Engineering Technology and Service Co,Ltd. Then we reconstruct the gene vector pBC1-Fat-1 based on the pBC1. After that we transferred this Fat-1 gene into mice by the method of microinjection. Then we proved that foreign genes has been integated within the mice genome by the PCR and Southern blot.Meanwhile we analysis theω-3/ω-6 PUFAs of the transgenic mice milk, the result demonstrated clearly thatω-6 PUFAs in milk of the transgenic mice are reduced,indicating thatω-6 PUFAs have been converted toω-3 PUFAs,causing the ratio ofω-3/ω-6 PUFAs to increased.
引文
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[2] Simopoulos AP.Essential fatty acids in health and chronic desease[J].The American Journal of Clinical Nutrition,1999,70(3 Suppl):560S-569S.
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[10] Cleland LG, James MJ, Proudman SM. The role of fish oils in the treatment of rheumatoid arthritis[J].Drugs,2003,63(9):845.
[11] Healy DA,Wallace FA,Miles EA,et a1.Effect of low-to-moderate amounts of dietary fish oil on neutrophil lipid composition and function[J].Lipids,2000,35(7):763.
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[14] McMurray DN,Jolly CA,Chapkin RS.Effects of dietary n一3 fatty acids on T cell activation and T cell receptor mediated signaling in a murine model[J].J Infect Dis,2000,182(1):103.
[15] Sanderson P,Macpherson GG,Jenkins CH,et a1.Dietary fish oil diminishes the antigen presentation activity of rat dendritic cells[J].J Leuk Biol,1997,62(11):771.
[16] Mayer K,Meyer S,Reinholz·Mubly M,et a1.Short-time infusion of fish oil-based lipid emulsions,approved for parenteral nutrition,reduces monocyte proinflammatory cytokine generation and adhesive interaction with endothelium in humans[J].J Immunol,2003,171(9):4837.
[17] Hughes DA.Pinder AC.n-3 polyunsaturated fatty acids inhibit the antigen presenting function of human monocytes[J].Am J Clin Nutr,2000,71(1):357.
[18] Hu FB, Bronner L, Willett WC, et al.Fish and omega-3 fatty acid intake and risk of coronary heart disease in women.JAMA,2002,287(14):1815-1821.
[19] Iso H, Rexrode KM,Stampfer MJ, et al.Intake of fish and omega-3 fatty acids and risk of stroke in women.JAMA,2001,285(3):304-312.
[20] Yam D,Peled A, Shinitzky M.Suppression of tumor growth and metastasis by dietary fish oil combined with vitamins E and C and cisplatin[J].Cancer Chemother Pharmacol,2001,47(1):34.
[21] Bhattacharya A,Lawrence RA, Krishnan A, et al.Effect of dietary n-3 and n-6 oils with and without food restriction on activity of antioxidant enzymes and lipid peroxidation in livers of cyclophosphamide treated autoimmune-prone NZB/W female mice[J].Am J Coll Nutr,2003,22(5):388.
[22] Pratt VC, Watanabe S,Buera E, et al. Plasma and neutrophil fatty acid composition in advanced cancer patients and response to fish oil supplementation[J].Br J Cancer,2002,87(12):1370.
[23] Larsson SC, Kumlin M, Ingelman-Sundberg M, et al. Dietary long-chain n-3 fatty acids for the prevention of cancer:a review of potential mechanisms[J]. Am J Clin Nutr,2004,79(6):935.
[24] Schwartz SA, Hernandez A, Mark E B.The role of NFkappaB/IkappaB proteins in cancer:implications for novel treatment strategies[J].Surg Oncol,1999;8:143.
[25] Minami A, Ishimura N, Sakamoto S, et al. Effect of eicosapentaenoic acid ethyl ester v.oleic acid-rich safflower oil on insulin resistance in type 2 diabetic model rats with hypertriacylglycerolaemia[J].Br J Nutr,2002,87:157.
[26] Nemets B, Stahl Z. Addition of omega-3 fatty acid to maintenance medication treatment for recurrent unipolar depressive disorder[J].Am J Psychiatry,2002,159(3):477.
[27] Per M, Brind J, Ramchand CN, et al. Two double-blind placebocontrolled pilot studies of eicosape-ntaenoic acid in the treatment of schizophenia [J].SchizophrRes,2001,49(3):243.
[28] James G. Wallis, Jennifer L. Watts, John Browse. Polyunsaturated fatty acid synthesis: what will they think of next? TREND in Biochemical Sciences, 2002,27(9):467-473.
[29] Tocher DR, leaver MJ, Hodgson PA. Recent advances in the biochemistry and molecular biology of fatty acid desaturase.Prog Lipid Res,1998,37:73-117.
[30] Takahiro Oura, Susumu Kajiwara, Saccharomyces Kluyveri. FAD3 encodes anω-3 fatty acid desaturase. Microbiology,2004,150:1983-1990.
[31] Ge Y,Chen Z,Kang ZB et al. Effects of adenoviral gene transfer of C.elegans n-3 fatty acid desaturase on the lipid profile and growth of human breast cancer cells. Anticancer Res,2002,22(2A):537-543.
[32] Spychalla J P, Kinney A J, Browse J. Identification of an animalω-3 fatty acid desaturase by heterologous expression in Arabidopsis.Proc Natl Acad Sci USA,1997,94:1142-1147.
[33] Inagaki K, Aki T,Fukuda Y, Kawamoto S et al. Identification and expression of a rat fatty acid elongase involved the biosynthesis of C18 fatty acid.Biosci Biochem,2002,66:613-620.
[34] Kang J X, Wang J, Wu L, et al. Transgenic mice: Fat-1 mice convert n-6 to n-3 fatty acids. Nature,2004,427:504.
[35]陈永福主编:《转基因动物》“863”生物高技术丛书[M],科学出版社2002年第1版.
[36]陈祥.转基因动物研究进展[C].会议论文2007,2:40-42.
[37] Gordon JW,Scangos GA, Plotkin DJ,Barbosa JA, Ruddle FA.Genetic transformation mouse embryos by microinjection of purified DNA[J].Proc Nat Acad Sci USA 1980,77:7380-7384.
[38] FELGNER P L.Lipofection:A highly efficient lipimediated DNA-transfection procedure[J].Proc.Natl.Acad.Sci,1987(84):7413-7417.
[39]程焉平,炳昌.转基因动物的研究与应用[J].松辽学刊(自然科学版),2002(1):5-7.
[40] Wheeler MB, Walters EM, Clark SG. Transgenic animals in biomedicine and agriculture:outlook for the future[J].Anim Reprod.Sci ,2003,79:265-289.
[41]卢一凡,邓继先,肖成祖,等.转基因动物理论与技术的研究进展[J].生物技术通报,1997(4):17.
[42]杨章平,常洪.转基因动物的研究进展[J].世界农业,2001(3):39-42.
[43]刘薇,卢光.转基因动物技术的研究进展[J].遗传,2001(3):289-291.
[44] WILMUT I,SCHNIEKE A E,MC WHIR J,et al.Viable offspring derives from fetal and adult mammalian cells[J].Nature,1997,385:810-813.
[45] LAI L,PARK K W,CHEONG H T,et al.Transgenic pig expressing the enhanced green fluorescent protein produced by nuclear transfer using colchicines treated fibroblasts as donor cells[J].Mol Reprod Dev,2002,62:300-306.
[46] Cibelli J B,Stice S L,Golueke P J,et al.Cloned transgenic calves produced from nonquiescent fetal fiboblasts[J].Science,1998,280(5367):1256-1258.
[47] Lai L, Kang JX,Li R,Wang J,Witt WT,Yong HY,et al.Generation of cloned transgenic pigs rich in omega-3 fatty acids[J].Nat Biotechnol 2006,24:435-436.
[48] Lai L,Kolber-Simonds D,Park KW,Cheong HT,Greenstein JL,Im GS,et al.Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear tansfer cloning[J].Science2002,295:1089-1092.
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