动物转基因克隆的分子生物学基础.ppt

立项依据及科学意义科学意义,Click to edit Master text styles,Second Level,Third Level,Fourth Level,Fifth Level,*,热烈祝贺“生命科学博士后研讨会”召开！,中国农业大学,动物转基因与克隆的研究进展,李宁,超级,鼠,转GH基因的鼠,异种器官移植,乳腺生物反应器,转基因育种,血液,器官,药用蛋白,生长,羊毛,营养保健,Hemoglobin,DNX Biotherapeutics Inc,Heart,Imutran Limited,Kidney,Nextran,a,1,-Antitrypin,PPL Therapeuticals plc,Antithrombin III,Genzyme Transgenic Corp,Lactoferrin,Pharming B.V.,Glycoconjugates,Ross Products Corp,GH,IGF-I,Pig Breeding,Bresgen Ltd,MP,IGF-I,Sheep Breeding,AgRes.,TRANSGENIC ANIMALS,Where Is the Potential for Application,4,转基因动物研究的里程碑,1966,1977,1980-1981,1981,1982,1983,1985,1987,1989,1994,1997,1998,1999,配子的微注射技术建立,mRNA和DNA转移爪蟾卵细胞,转基因小鼠获得成功,小鼠胚胎干细胞转移,转基因“超级鼠”,转基因小鼠组织特异性表达,转基因家畜,嵌合体“敲除”鼠成功,定位整合的种系嵌合体小鼠,精子细胞的移植,绵羊的体细胞克隆,利用核移植技术产生转基因绵羊,“敲除”家畜获得,成功,和,“Doogie”,鼠,5,Robert(Bob)Wall,Key Player in USA,Keith Campbell,Key Player in UK,6,利用转基因动物制药的途径,血液、,尿腺,、,乳腺,、禽卵、昆虫,120,亿美元,年的全球市场、,20,亿美元,年药用蛋白专利过期,9,Strategy for Production of Human Medicine by Transgenic Animals,10,转基因奶牛和细胞培养技术生产抗体蛋白的成本比较,美国商业交流公司、,Genzyme,公司和,D&MD,医药投资发展公司报告,11,美国商业交流公司、,Genzyme,公司和,D&MD,医药投资发展公司报告,动物乳腺生物反应器生产人用药物蛋白的现状,12,InterNutria,报告、,Nature Biotechnology 16（8）：728731，1998,动物乳腺反应器生产人医用营养保健蛋白的现状,13,“人乳化”牛奶是最好的医用营养保健品,人乳与牛乳主要成分的比较,Human milk,Bovine milk,Carbohydrares,Lactose,Oligosaccharides,Proteins,Caseins,a,-Lactalbumin,Lactoferrin,Secretory IgA,Lysozyme,b,-lactoglobulin,Lipids,Triglycerides,Phospholipids,Minerals,Sodium,Potassium,Chloride,Calcium,Magnesium,73 g/l,12 g/l,40 g/l,1.0 g/l,3.0 g/l,1.8 g/l,1.5 g/l,1.4 g/l,1.2 g/l,none,28 g/l,1.2 g/l,Trace,0.03 g/l,0.03 g/l,3.6 g/l,4.0%,0.04%,4.0%,0.04%,5.0 mM15 mM,15 mM,8.0 mM1.4 mM,15 mM45 mM,35 mM,30 mM,4.0 mM,15,人奶与牛奶的功能差异及其优缺点,Casein:whey protein,30:70,80:20,Problems:hard curd in the stomach and dietary protein allergy,Cysteine:methionine,high,low,Phenylalanine&tyrosine,low,high,Problems:retardation of brain development and PKU babies,HUMAN,COW,Calcium:phosphate,2.3:1,1.26:1,Problems:hypocalcemia,tetany and hyperphosphatemia,16,Personal Ballistic Protection:,“superior energy-to-break”mechanical properties with weight 25%lighter than aramid-based fibers,Medical Materials:,high strength sutures and prosthetic devices,Personal Healthcare:,cosmetic formulation to provide support,and elasticity to skin,Industrial Component:,improve the strength profile for automotive,and military and aerospace components,“生物钢”材料的应用领域,17,“生物钢”的应用领域与发展需求,国防用“生物钢”-新型生物材料的生产,医用人工韧带、关节等生物材料的生产,高附加值,特殊用途-粘性蛋白等的生产,欧美市场估计为,150亿美元/年,中国市场估计为,100以人民币/年,18,转基因奶羊和细胞培养技术生产抗体蛋白的成本比较,19,The Comparison between Different Fermentation Technology,Cell Culture,Methods,Complex Protein,Production,Large Scale,Production,Low Cost,Production,Bacteria,Yeast,Mammalian,Transgenic,No,No,Yes,Yes,Yes,Yes,No,Yes,Yes/No,Yes/No,No,Yes,20,美国商业交流公司、,Genzyme,公司和,D&MD,医药投资发展公司报告,21,Time&Financial Required for Development,of Therapeuticals by Transgenic cattle,0 1 100 300 550 700,Feasibility,Founder,Transgenicals,Transgenic breeding,Process development,Commercial,manufacture,Clinical trials,Marketing,0 1 2 3 4 5 6 7 8 9 10 11,22,Genzyme Transgenic Corp,Found in 1992,and the farm was,Constructed in 1995.The total,Employee is 672 people but only,112 of them closely working in,The transgenic platform.The all,Others are in a pharmacology,Division.The NASDAQ price per,Share is around 18-68 US$.,23,Goat Barn in Genzyme Transgenic Corp,24,The Situation in side the Goat Barn,25,The demo GAP barn for transgenic milking goat,26,Transgenic Goats with a Sensor Label,27,Milking Facilities for Transgenic Goats,28,PURIFICATON FACILITIES,29,PPL Therapeutics plc,China“863”High-Tech Program,Z21-04 Production of therapeutical proteins,Development,1.CAU,2.SMI,3.MMI,4.NGC,5.XJI,Research,1.CAU,2.MAU,3.HIU,4.GXU,5.SBI,6.VRI,31,Transgenic Dairy Goat with,Human,mATT Gene,32,Transgenic Dairy Goat with,Human,mATT Gene,33,生产人营养保健（医疗）品！,“一种药物需要,10,年以上的开发时间和至少,2.5,亿美元的资金才有可能走向市场，而营,养医用品只需要几百万美元和几年的时间。,更重要的是，营养医用品的利润也非常大”,Dr.Lewis Lepene，,InterNutria,公司执行总裁,34,InterNutria,报告、,Nature Biotechnology 16（8）：728731，1998,35,Production the Human-Like Milk,by State-of-the-Art Technology,Program for the Production of Nutraceuticals,by Transgenic Cow,Gene cassette,Microinjection,Transfection,Selection,NT,Transfer,Newborns,Market,人奶基因,基因转染,药物筛选,怀孕产犊,核卵融合,胚胎移植,克隆胚胎,繁育扩群,转基因克隆牛,代孕母亲,工厂化生产牛群,市场销售,全球市场营销,体细胞,核移植,细胞培养,38,Expansion of the Founder Transgenics,A.I.&Selection,Time&Financial Required for Development,of Nutraceuticals by Transgenic cattle,Feasibility,Founder&Breeding,Transgenicals,Trials,Marketing,0 1 2 3 4,0 1 30 40 50,40,Comparison of Milk Componentbetween Human and Cow,Human milk,Bovine milk,Carbohydrares,Lactose,Oligosaccharides,Proteins,Caseins,a,-Lactalbumin,Lactoferrin,Secretory IgA,b,-lactoglobulin,Lipids,Triglycerides,Phospholipids,Minerals,Sodium,Potassium,Chloride,Calcium,Magnesium,73 g/l,12 g/l,40 g/l,1.0 g/l,3.0 g/l,1.8 g/l,1.5 g/l,1.4 g/l,none,28 g/l,1.2 g/l,Trace,0.03 g/l,3.6 g/l,4.0%,0.04%,4.0%,0.04%,5.0 mM15 mM,15 mM,8.0 mM1.4 mM,15 mM45 mM,35 mM,30 mM,4.0 mM,41,Problems Presented by Cow-Milk Feeding Infants or Human,Casein:whey protein,30:70,80:20,Problems:hard curd in the stomach and dietary protein allergy,Cysteine:methionine,high,low,Phenylalanine&tyrosine,low,high,Problems:retardation of brain development and PKU babies,HUMAN,COW,Calcium:phosphate,2.3:1,1.26:1,Problems:hypocalcemia,tetany and hyperphosphatemia,42,Medical Function of Human Lactoferrin,Bacteriostatic effect on micro-organisms,Bacteriolytic effect on broad spectrum of bacteria,Growth stimulating effect on bifidus strains,Iron transport protein,Zinc transport protein,Trophic effect on mucosal cells,43,Possible Application of Human Lactoferrin as Therapeutical,hLF/hLZ,as anti-infectious factors for people at,risk of gastric&intestinal infection,e.g.young infants,elderly people,patients with,compromised immunological resistance,hLF,as iron carrier for,anaemic,people,e.g.pregnant women,elderly people,anaemic,patients,44,More Lactoproteins in Development,Lactoperoxidase,Anti or cytotoxic effect to pathogenic cells,Prolactin,Improvement of immuno-response,Zinc-transporter,Protection of stunted growth,alopecia，,Dermatitis and zinc transportation,Growth factors,Close of the leaky mucosal lining of newborn,Inhibition factors,Direct lysis of infected cells,45,More Lactoproteins in Development,Aldolase,treatment of hereditary fructose intolerance,Catalase,endothelial recovery and aging process,Superoxide dismutase,skin and organ in adversity,Lipoprotein lipase,different hyperlipidemic disorders,Peroxidase,against peroxynitrite-mediated oxidation,Xanthine oxidoreductase,cardiocirculatory disease,Galactosyl transferase,resistant to parasite&bacteria,46,影响转,基因动物产业化的根本原因,落后的转基因技术-微注射,1、转移效率极低(1%),2、表达效率极低(1g/l),3、不能定点整合,定点进行基因组修饰必将带,来转基因动物产业化的革命！,47,基因,怀孕产犊,胚胎移植,转基因牛,代孕母亲,表达检测,分子筛选,微注射,胚胎培养,随机整合,传统微注射技术生产转基因动物示意图,总效率0.5%,48,核供体动物,体细胞,细胞培养,卵供体动物,未受精卵,除去卵核,细胞融合,克隆胚胎,胚胎移植,无核卵子,代孕母亲,代孕母亲,与核供体遗传相同的克隆牛,动物体细胞克隆提供解决方案？,49,核供体动物,体细胞,细胞培养,卵供体动物,未受精卵,除去卵核,细胞融合,克隆胚胎,胚胎移植,无核卵子,代孕母亲,代孕母亲,基因组经过随机整合修饰的转基因克隆牛,细胞筛选,基因整合,阳性细胞扩群,目标基因,基因转移,随机整合,转基因克隆提高了转基因效率,但未解决表达效率和时空表达,50,依赖于基因同源重组的转基因克隆,可以同时提高基因整合和表达效率,核供体动物,体细胞,细胞培养,卵供体动物,未受精卵,除去卵核,细胞融合,克隆胚胎,胚胎移植,无核卵子,代孕母亲,代孕母亲,基因组经过同源重组修饰的转基因克隆牛,细胞筛选,基因整合,目标基因,基因转移,同源重组,51,同源重组进行基因整合的机理,A,B,Neo,R,P,Killing,B,A,A,B,Neo,R,P,双交换,内源性酪蛋白基因,突变的酪蛋白基因,外源基因结构,抗新霉素的细胞系（群）,52,目标基因,基因转染,药物筛选,怀孕产犊,核卵融合,胚胎移植,克隆胚胎,繁育扩群,转基因克隆牛,基因高效表达,代孕母亲,工厂化生产牛群,市场销售,全球市场营销,体细胞,核移植,细胞培养,同源重组,依赖于基因组修饰的转基因克隆技术图示,53,Production of Transgenic Animal via Cloning,54,Knock-out Technology,55,HOMOLOGOUS RECOMBINATION,A,B,Neo,R,P,Killing,B,A,A,B,Neo,R,P,Double crossover,Endogenous locus,Mutated locus,Foreign DNA,Neomycin resistant cloning cell line,56,HOMOLOGOUS RECOMBINATION,A,B,Neo,R,P,Killing,B,A,A,B,Neo,R,P,Single crossover,Endogenous locus,Mutated locus,Foreign DNA,Cells will be killed by toxin genes product,Killing,B,A,Endogenous locus,57,Inducible expression system,Tet-on or Tet-off,X,58,Cre-LoxP system,Lox P,Lox P,Cre,Protein,Lox P,59,Integration of multiple copies,RNA-mediated DNA methylation,Insertion into hypermethylated,Genomic regions,Transgene recognition,DNA pairing,DNA-RNA,pairing,“Spreading”of,Methylation,pattern,Age,Environmental,stress,60,How to overcome“positional effect”,Chromosome DNA loop,Nuclear matrix,channel,Active genes attached to,matrix channels through,the regulatory domains,and DNA pol II,Matrix,Attachment,Region,61,A,B,C,D,E,LCR,A,B,C,D,E,Locus A,Locus B,Locus C,Locus D,Insulator,Insulator,Locus Control Region,Insulator,62,Animal Transgenics,Mammary bioreactor,Pharmaceuticals,Neutraceuticals,Bio-materials,Bio-cosmetics,63,国内外研究现状与趋势,体细胞克隆动物,64,国内外研究现状与趋势,PPL公司Knock-out绵羊,65,PPL公司Knock-out猪,66,核供体动物,体细胞,细胞培养,卵供体动物,未受精卵,除去卵核,细胞融合,克隆胚胎,胚胎移植,无核卵子,代孕母亲,代孕母亲,基因组经过同源重组修饰的转基因克隆牛,细胞筛选,基因整合,目标基因,基因转移,同源重组,利用同源重组进行基因组修饰的难点,长期培养,67,研究技术路线,体细胞克隆,细胞长期培养,细胞克隆筛选,基因的同源重组,转基因克隆,基因组修饰技术平台,68,研究方案和内容,化学去核法以使体细胞克隆操作简便、提高工作规模和克隆效率,卵供体动物,未受精卵,69,研究方案和内容,双标记筛选已确定同源重组的细胞克隆（群）,细胞筛选,同源重组,新霉素,荧光GFP,克隆胚胎,荧光细胞,70,转有人mAAT基因的奶山羊,71,核供体动物,体细胞,细胞培养,遗传修饰,卵供体动物,未受精卵,除去卵核,细胞融合,克隆胚胎,胚胎移植,无核卵子,代孕母亲,代孕母亲,与核供体遗传相同的克隆牛,72,Animal Transgenics,Xenotransplantation,Organs,GM-cells,Tissues,Frog development without head?,73,Immunological Barriers,Hyperacute rejection,:Rejection mediated by antibody-comple,-ment complexes which damages the endo,-thelial cells of vascular interity and killing,the organ within minutes,Acute rejection,:Rejection mediated by T cells which damage,the blood vessels and destroying the organ,within days,Chronic rejection,:Rejection mediated by a complex immune,Process(humoral immunity and cell-mediated,immunity)and damaging the organ in the range,of many years,74,用于人体器官移植的动物器官,PPL Interim,报告和,Nature Biotechnology 16（3）：224，1998,75,美国,D&MD,医药投资发展公司,美国等待器官移植的病人数量（,1998,年）,76,Porcine endogenous,retrovirus may hurt the,human when pig organs,was transplanted into,the human.,Whether pig pathogens,Will infect human who,harboring a pig organs,remain unknown?,77,治疗型克隆技术将更具有挑战性和广泛应用前景？,固体器官：60,亿美元,年的美国市场,Xenotransplatation pigs,The skin was grafted from other species,79,Production of Biosteel by Tg Goat,5 mm,80,The strongest materials in the World,Spider dragline silk is widely recognized,as the strongest material known,81,Personal Ballistic Protection:,“superior energy-to-break”mechanical properties with weight 25%lighter than aramid-based fibers,Medical Materials:,high strength sutures and prosthetic devices,Personal Healthcare:,cosmetic formulation to provide support,and elasticity to skin,Industrial Component:,improve the strength profile for automotive,and military and aerospace components,Potential Applications of Biosteel in Future,82,SPIDER MAN-USA ARMY?,83,USA Biosteel Production Basement,Plattsbaerg,NY,84,诊断、治疗人类疾病及新药筛选的转基因动物模型,基因过量的表达,基因敲除,(Knock-out),或敲入,(Knock-in),病毒和其他病原的,DNA,插入诱变,318 knock-out-in mice,47 over-expression mice,15 virus-induced mice,1825,亿美元,/,年市场？,85,The R&D of Global Biotech Company,Based on Transgenic Platform,Total Numbers in 1999:56(21 in USA&the North America,12 in EU,13 in Asia&Pacific Island),The numbers in stock market:10(5 in USA and Canada,4 in,European countries,1 in Australia.,The price ranged from 7 to 158 US$,The current situation in China:3+3+2 pending?,86,Xeno-medicine Care,Oxygen,therapeuticals,Biopure,Enzoy,corp.,In situ,ES cells therapy,Chiron,Baxter Inc.,16.7 billion in USA but 5.8 billion in blood transfusion,87,BIOPURE:a cinderala technology,Hemopure:,an immediate oxygen deliver medicine for,many diseases(clinical trial III finished),Oxyglobin:,an oxygen therapeuticals for pet animals,88,Biopure Headquarter in Boston,NASDAQ:22.5 US$/per share.But 300 millions spent,100 million in hand for heading to market,89,ANIMAL CLONING&ES CELL,TECHNOLOGY,1997,1997,90,91,Cloning technology of animal proposed,1938,Spemann,Adult frogs from the nuclear of somatic cells,1962,Gurdon,Nuclear transplantation in sheep embryo,1986,Willadsen,Production of identical bovine offspring,1990,Bondioli,Sheep cloned from a cultured cell line,1995,Campbell,Viable offspring from adult mammalian cells,1997,Wilmut,Cloned mice produced by injected cell nuclei,1998,Wakayama,Milestone of Animal Cloning,92,93,Mice were cloned in a large scale by“Honolulu”technology,egg,nucleus,mother,cloned,cloned,94,95,Collecting tissue and cell culture,Pet or farm animal to be cloned,96,Cells Storage for Cloning to relive,97,Manipulation of recipient oocytes,98,NUCLEAR TRANSFER,99,Embryo Transfer,100,101,Cloned transgenic calves produced from nonquiescent fetal fibroblasts,CMV,Lac Z,Neo,R,Transfection,Neo-resistant,Nuclear transfer,Embryo transfer,Cloned calves,102,Cloning Panda and Mammoth,Rare in the world,Dead over 12,000 yrs,103,Cross-species animal cloning,104,Cloning Human or Organ?,105,Therapeutical cloning procedure,106,The cells can be induced and differentiated to many types,107,Somatic cell,Nuclear transfer,In vitro fertilization,Fetal tissue,Patient or,Great-man,Therapeutic,Cells&Tissues,Human Embryonic Pluripotent Stem Cells,108,Why People Fear,:,Cloning Technology,The Global Animal Cloning Industry,The total number:32(12 in USA,10 in European,6 in,Pacific country and 12 in the other countries,The company in stock market:7(3 in USA,3 in Euro-,countries and 1 in Australia)and the price,per share ranged from 12 to 187.5 US$,The number in China:3+6 pending in the year 2000?,110,Estimated global development in 21,st,Century in Animal Biotech,2005 2010 2020,Breeding,Medicine,Xenograft,Pets animal,Farm animal,Human,Transgenics,Cloning,3,30,2,10,175,25,35,480,80,4,8,2,15,35,30,35,100,180,100 million US$/year,111,动物生产质量管理规范,无“骚痒”或“疯牛”病原的动物,隔离其他物种的安全装置,动物管理和兽检的标准操作程序（,SOPs,）,计算机辅助鉴别和示踪,严格的健康检查与监控,饲料质量和消化吸收监控,利用精子和胚胎库保存种群,不同生产群的隔离,奶的收集、储藏、运输的标准程序,单个产品的分区管理,大群,(bulk),资源样品的追踪,Good Agricultural Practice,，,GAP,112,安全性评估要点,转基因结构的产生和定性,转基因始祖动物的创建和定性、遗传和表达,建立可靠持续的转基因动物、精子和胚胎库,生产群的产生和选择,转基因动物饲养管理,转基因产品的纯化和定性,临床前的安全评估,113,转基因动物的专利现状,1988,年,4,月,12,日美国专利局批准了第一个转基因动物“原癌小鼠”（,Oncomouse,）专利，打破“动物生命不允许专利”的铁律,1998,年，美国和欧洲专利局共批准了,42,种转基因动物专利，登记注册了,88,种专利申请,114,转基因动物文献引用：,1984,年,32,篇有关，,1999,年,4162,篇,1997,年转基因,(transgenic),小鼠,804,种，基因“敲除”（,knock-out,）或“定点整合”鼠,(knock-in)782,种，涉及到,647,种不同的基因结构,以转基因动物技术为核心技术的公司目前全球大约为56家,发展迅猛的转基因动物研究与开发,115,转基因动物技术的新革命？,体细胞表达评价系统,基因组修饰,(knock-out-in),多基因协同表达结构,非预测性表达结构,全天然性表达结构,克隆！克隆！,116,谢谢!,117,