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Mouse Model for Gene Regulation Studies

Mouse Model for Gene Regulation Studies. Course Materials. Introduction to Gene Regulations Introduction to mouse models Introduction to transgenic techniques Examples: VEGF gene regulation and pathologic development. Transgenic Technology. Part 1 : Basis of classic transgenics Part 2 :

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Mouse Model for Gene Regulation Studies

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  1. Mouse Model for Gene Regulation Studies

  2. Course Materials • Introduction to Gene Regulations • Introduction to mouse models • Introduction to transgenic techniques • Examples: VEGF gene regulation and pathologic development

  3. Transgenic Technology Part 1: Basis of classic transgenics Part 2: Gene Targeting Part 3: Applications

  4. Part 1 Transgenic Technology

  5. What’s transgenic? • Narrow Definition:Artifacial insertion of DNA fragment into genome • Broad Definition:Artifacial modification of genome, including insertion, mutation and deletion

  6. Importance of Transgenic Technology Basic Research • Gene regulation,promoter function • Gene expression tracing(Knockout,Gene Trap) • Cell tracing,tumor cell labeling • Functional study,embryonic, developmental and pathological Commercial • Protein product • Low cost • Disease model • Organ donor • Strain improvement • Gene Therapy

  7. Common Techniques Used for Making Transgenic animals • Most Comkon :Pronuclei DNA injection,random insertion,low predictability,variable • Sperm associated DNA transduction,low repetibility • Transposons, not common • Viral infection:High efficiency,less random,limited DNA size,safety consern • Embryonic stem cell/Blastocyst microinjection:Gene targeting, only for mice • Nuclear transpalntation/animal cloning/only way to generate targeted modification. High cost and low efficiency

  8. 真核基因结构与调节 • 真核基因结构:启动子(promoter),外显子(exon),内含子(intron),polyA信号, 活化信号(enhancers), 沉默信号(silencers),封闭区(Insulators) • 转录因子(transcription factors),活化因子(activators),抑制因子(inhibitors) • 真核mRNA结构:Cap, 5’非编码区(5’-UT), 编码区(coding region),3’非编码区(3’-UT), polyA • 组织专一性调节(tissue-specific) • 发育阶段调节(developmental) • 诱导性调节(inducible) • 转录后调节: mRNA剪接和修饰, mRNA的稳定性,蛋白质合成效率,蛋白质半衰期

  9. Transcription Translation CCAAT box TATA box Enhancer Exon I Intron I Exon II Exon I 5’UT 5’-UTR 3’-UTR AAAAAAAAA GG 3’-UT II IV III IV AATAAA Gene Structure … mRNAstructure

  10. Transgenic Construct • Selection for promoters • cDNA • Poly A • Introns and insulators • Construct design

  11. Promoter • Tissue-specific:研究基因调节,启动子的功能,或调节其它基因的表达。如果用于调节其它基因,一定要查询启动子在转基因动物应用方面的文献,了解启动子的完整性 • High expression:用于高表达某个基因,调节其它基因,或高表达后蛋白的生产 • Universal expression:显示基因细胞标记,基因调节研究 • Inducible:用于基因调节,有毒蛋白表达,致命基因的可逆调控等 • Conbination:可诱导,高表达,组织专一,构成基因调节系统,用于调节基因或高表达蛋白

  12. Types of cDNAs • Reporters: lacZ, GFP,CAT, AP等 • Regulators: Cre, ER-Cre, tTA, PTX等 • Protein function:蛋白突变体的表达 • Gene splicing:研究外显子、内含子功能 • Commercial gene:药用蛋白,改良基因等 • Functional gene: 基因治疗,干细胞移植等 • Non-coding:基因治疗,如siRNA

  13. LacZ and GFP 半乳糖干酶显示基因(lacZ)-基因表达组织定位 绿色荧光蛋白(GFP)-活体细胞追踪

  14. Parts of transgenic construct • poly A:stable mRNA • poly A: SV40 poly A, -Globin poly A • First Intron:-Globin • Insulator:Chicken beta-globin gene • Loxp, FRT, tetO

  15. Typical transgene 翻译起始点 转录起始点 基因A启动子 CCAAT box TATA box Enhancer 基因B cDNA 基因C polyA 拼接区域: 5’-UT 3’-UT

  16. (Founders)detection • Check for insertion: • PCR • Southern • Copy:Real time PCR • mRNA expression: • RT-PCR • Northern • Protein Expression: • Reporter:lacZ,GFP,AP • Functional analysis • Immunohistochemistry (IHC)

  17. Mouse Characteristics Great immune system,high efficiency of propagation,small size,the most economic animal model Variety of phenotypes,genome sequenced,classical mammal model Long genetic study history, hundreds of inbred strains Gestation19-21days Sex maturity:4-6 week Estrus: as short as 5 days Body weight of adults: 20-50g Pregnancy average 5-6 times Litter size: 6-14 pups

  18. Mouse Requirement for Transgenic Production • SPF facility,High fat, high protein feed(breeder chows) • Egg donor:4-6week old F1 femals (C57B6xCBA or DBA),10-12 each time,ywice a week. • Stuck males: 20-24 2-12 month old F1 males (C57B6xCBA or DBA). • Recipient:50-100 2-6 month old (CD1, Kunming) • Stuck male:Vecectomysed (C57B6xCBA or DBA) or (CD1)males,2-18months old

  19. Steps of making Transgenic mice • Construct, remove vector • Superovulation,set mating,collect E0.5 egg • DNA microinjection • Overnight culture • Embryo transfer • Tail and numbering • Detection, mating • Expression analysis

  20. 转基因鼠建立时间表 F1出生 注射 出生 分窝 传代 分窝 分析 0 1 2 3 4 5 时间 (月) 孕期 孕期 转基因鼠 鉴定 性成熟 传代 鉴定 出生率 30-50% 转基因比率 15-50% 传代效率 ~90% 性成熟

  21. Related Data • PMSF and HCG superovulation • Donor egg,100-200 each time • DNA concentration for injection: 2ng/ul • Embryo transfer back to recipient: 20-30 • PCR or Southern to detect founders, 15-50%, random insertion,copy 1 to over 100 • First generation(F1) 0-100%,some may integrate at 2 cell stage, some may have more than one insertion locus • Second generation,Mendel inheritance, 50% • Characterization, E10 to 5 months

  22. Discussion • 有限的启动子信息,不完全的启动子,造成基因不表达或表达于错误的组织 • DNA插入的随机性,多拷贝性,不表达,低表达,鼠系间差异 • 转基因受插入位点的影响及封闭区(insulaters)序列的发现 • 基因高表达可能造成的毒性,得不到转基因鼠或只有不表达的转基因鼠系(founders) • 第一个内含子的重要性 • 转基因片段的大小: • 2kb到mb • 大部分2-10kb,容易注射 • P1质粒(PAC),70kb左右,黏度大 • 细菌人工染色体BAC,120kb左右, 黏度更大 • 酵母人工染色体YAC,500kb-1mb大小,非常难 • 同时注射两个以上的转基因: 效率高,一般插入同一位点 • 多拷贝插入方式:头尾相接 • 拷贝数与表达水平之间的关系:表达水平与拷贝数无紧密关联,多数拷贝被甲基化及有限的转录因子浓度 • 影响转基因成功与否的其它因素:DNA的浓度(2-3ng/ul), 纯度,如嗅化乙锭和EDTA含量等 • 载体DNA对转基因表达的影响:不稳定 • C57/BL6纯系转基因鼠受精卵注射

  23. Transgenic Technology Part 1: Basis of classic transgenics Part 2: Gene Targeting Part 3: Applications

  24. Part 2:Gene Targeting Theory Technique Application

  25. Importance of Gene Targeting • Gene function study:Gene knockout is first choice to identify function for genes predicted from whole genome sequencing • Gene Regulation:More accurate control, more confirmative results, better disease models • Due to the stability and predictability, better for protein production or human gene replacement • Major progress in Gene Therapy and Regeneration Medicine

  26. 小鼠早期发育示意图 3.5天

  27. 干细胞的特性 • Projenitor cells • Can duplicate, few division, uneven division • Stem cells • 可以分化成一种或多种组织器官,可以不断增殖的少数细胞 • Embryonic stem cell (ES) Can develop into any tissues, whole body • Source of ES cells: Inner cell mass (ICM) Strain:129SV/jae, fewer from C57/BL6 Coat color:Brown (129), Black (C57/BL6) Sex:Male, more stable

  28. Targeting strategy • Vector design • Conditional construction • Reporter knocking • Tissue-specific for study gene regulation • Universal expression: functional studies

  29. Wild type alelle 1 2 3 Basic Targeting Vector X X Vector 1 NEO 3 TK After recombination 1 NEO 3

  30. Conditional knockout Wild type FRT FRT Vector NEO TK loxP loxP Flipase to remove NEO FRT loxP Tissue-specific Cre to inactivate gene FRT

  31. Steps for targeting • ES cell isolation and culture • Targeting vector construction • ES electroporation and selection for recombination • ES cell injection • Chimera production and mating • Heterologous mice generation • Homozygous generation • Function analysis

  32. Chimera Mating • Chimera(Founders) • ES come from 129 strain,brown mice (agouti) • C57/BL6blastocyst (black) • ES,coat color can tell how much ES get integrated • 40-100% male chimera mate with C57/BL6 female • 6 week old chimera male with two C57/BL6female, female changed every week. • Look for brown mice

  33. Time Table

  34. Comparison of Trangenic and targeting procedures 电击筛选 囊胚注射 子宫移植 输卵管移植 代孕鼠 Founder的产生 传代,分析

  35. Comparison of Trangenic and targeting

  36. LacZ-Neo (GEO) Gene B-like Expression possible No Expression Gene A Gene B (Gene Trapping) Gene C

  37. 启动子 外显子 lacZ pA 剪接受体 被随机捕捉的基因 转录产物 用Inverse PCR、序列分析确认被捕捉基因 从基因捕捉库筛选出基因进行囊胚注射 基因捕捉可以做为基因敲除的替代 PGK neoR pA 电转干细胞,筛选neoR 基因功能分析

  38. Gene trap transgenic embryos

  39. 核移植技术与大动物克隆 • 供体细胞核 • 越胚性越容易成功,如胚胎成纤维细胞 • 细胞分裂状态:分裂静止期 • 受体细胞 • 超排卵 • 去细胞核 • 核移植 • 核显微注射 • 诱导发育 • 目前还存在问题

  40. 转基因动物发展趋势 • 以研究为导向,以技术突破为核心 • 以应用为目标,以加速技术转化为宗旨 • DNA序列解析加速基因功能的解析 • 小动物为研究材料,大动物为生物反应器 • 干细胞、克隆技术进一步成熟 • 在疾病治疗、品种改良方面扩展、深入 • 由间接的转基因药物到直接的基因治疗、器官移植 • 从人们对转基因食品的不接受到转基因制品管理的逐步完善 • 转基因技术与基因定位技术的有机结合,对启动子特性的不断了解,在组织专一性,可诱导性方面将更加精确 • 大动物转基因技术的不断完善,商业应用将更为普遍,如品种改良,蛋白药抗体药生产等 • RNAi在转基因动物和基因治疗方面的应用 • 转基因技术对人类生存将产生更为深远的影响,人们对转基因产品的认识将不断发生改变,相应的政策法规将得到进一步完善

  41. 干细胞研究的进展与前景 • 第一个人干细胞来源于人胚胎 • 定向分化后的干细胞可以用于治疗 • Induced pluripotent stem (iPS) • 病人特异性干细胞可以通过基因诱导获得:Oct3/4, Sox2, Klf4, and c-Myc • 病人特异性干细胞可以通过核移植获得 • 需要解决问题: • 诱导干细胞与正常干细胞的差异 • 表观遗传学问题 • 定向诱导分化问题 • 安全问题

  42. 核移植和大动物克隆的应用前景 成本高,目前主要用于品种改良,器官移植等商业价值较高的方面 最近已经成功用于干细胞生产,有望将来用于临床再生治疗 理论上可行,实际上还有很多问题,但它是唯一的大动物基因修饰途径

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