mouse model for gene regulation studies
DESCRIPTION
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 : - PowerPoint PPT PresentationTRANSCRIPT
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Mouse Model for Gene Regulation Studies
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Course Materials
• Introduction to Gene Regulations
• Introduction to mouse models
• Introduction to transgenic techniques
• Examples: VEGF gene regulation and pathologic development
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Transgenic Technology
Part 1 :Basis of classic transgenics
Part 2 :Gene Targeting
Part 3 :Applications
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Part 1
Transgenic Technology
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What’s transgenic ?
• Narrow Definition : Artifacial insertion of DNA fragment into genome
• Broad Definition : Artifacial modification of genome, including insertion, mutation and deletion
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Importance of Transgenic Technology
Basic Research • Gene regulation , promoter function• Gene expression tracing ( Knockout , Gene Tra
p )• Cell tracing , tumor cell labeling• Functional study , embryonic, developmental and pathological
Commercial • Protein product• Low cost• Disease model• Organ donor• Strain improvement• Gene Therapy
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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 ran
dom , limited DNA size , safety consern• Embryonic stem cell / Blastocyst microinject
ion : Gene targeting, only for mice• Nuclear transpalntation/animal cloning / onl
y way to generate targeted modification. High cost and low efficiency
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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
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Gene Structure
mRNA structure
IV
3’-UT
AATAAA
Exon IGG
5’-UTR
AAAAAAAAA
3’-UTR
II III IV
Intron I
Translation
Exon IICCAAT boxTATA boxEnhancer
Transcription
Exon I
5’UT
…
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Transgenic Construct
• Selection for promoters• cDNA• Poly A• Introns and insulators• Construct design
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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
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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
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绿色荧光蛋白 (GFP) -活体细胞追踪
半乳糖干酶显示基因 (lacZ) -基因表达组织定位
LacZ and GFP
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Parts of transgenic construct
• poly A : stable mRNA• poly A: SV40 poly A, -Globin p
oly A• First Intron : -Globin • Insulator : Chicken beta-globin
gene• Loxp, FRT, tetO
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基因 A 启动子
Typical transgene
CCAAT boxTATA boxEnhancer
转录起始点
基因 B cDNA
翻译起始点
基因 C polyA
5’-UT
3’-UT
拼接区域:
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( 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)
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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
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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
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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
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转基因鼠建立时间表转基因鼠建立时间表
注射注射 出生出生 分窝分窝 传代传代 F1F1 出生出生 分窝分窝 分析分析
孕期孕期 转基因鼠转基因鼠鉴定鉴定
传代传代鉴定鉴定
性成熟性成熟 孕期孕期
时间时间(月)(月)
00 11 22 33 44 55
出生率出生率3030 -- 5050%%
转基因比率转基因比率1515 -- 5050%%
传代效率 传代效率 ~90~90%%
性成熟性成熟
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•PMSF and HCG superovulationPMSF and HCG superovulation•Donor eggDonor egg ,, 100100 -- 200 each time200 each time•DNA concentration for injection: 2ng/ulDNA concentration for injection: 2ng/ul•Embryo transfer back to recipient: 20-30Embryo transfer back to recipient: 20-30•PCR or Southern to detect founders, 15-50%, PCR or Southern to detect founders, 15-50%, random insertionrandom insertion ,, copy 1 to over 100copy 1 to over 100•First generationFirst generation (( F1) 0-100%F1) 0-100% ,, some may insome may integrate at 2 cell stage, some may have more ttegrate at 2 cell stage, some may have more than one insertion locushan one insertion locus•Second generationSecond generation ,, Mendel inheritance, 50Mendel inheritance, 50%%•Characterization, E10 to 5 monthsCharacterization, E10 to 5 months
Related Data
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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 (founder
s)• 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 E
DTA 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
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Transgenic Technology
Part 1 :Basis of classic transgenics
Part 2 :Gene Targeting
Part 3 :Applications
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Part 2 : Gene Targeting
•Theory
•Technique
•Application
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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
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小鼠早期发育示意图
3.5 天
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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
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Targeting strategy
• Vector design
• Conditional construction
• Reporter knocking– Tissue-specific for study gene regul
ation– Universal expression: functional stu
dies
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NEO1 3 TK
X X
1 NEO 3
1 32Wild type alelle
Vector
After recombination
Basic Targeting Vector
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NEO TK
FRTFRT
loxPloxP
Wild type
Vector
Flipase to remove NEO
FRT
Tissue-specific Cre to inactivate gene
loxP
FRT
Conditional knockout
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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
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Chimera Mating
• Chimera ( Founders ) – ES come from 129 strain , brown mice
(agouti) – C57 / BL6 blastocyst (black) – ES , coat color can tell how much ES
get integrated– 40-100% male chimera mate with C57/B
L6 female • 6 week old chimera male with two C57 /
BL6 female, female changed every week.• Look for brown mice
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Time Table
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输卵管移植
子宫移植
代孕鼠
电击筛选
囊胚注射
Comparison of Trangenic and targeting proceduresComparison of Trangenic and targeting procedures
Founder 的产生
传代,分析
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Comparison of Trangenic and targetingComparison of Trangenic and targetingTransgenic Targeting
Genetic property
Dominant, Unstable
Dominant or recessive ,Stable
Insertion position
Random , uncontrolled
Fixed , Controllable
Copy number Vary,
1 to >100
Het1, homo 2
Expression Up to construct and insertion site
Mimic original gene
Time >5 month >9 month
Cost >$3000 USD >$10,000USD
Influence to nearby gene
Likely, unpredictable
Usually no
predictable
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(Gene Trapping)
LacZ-Neo(GEO)
Gene B-likeGene B-likeExpressionExpressionpossiblepossible
No No ExpressionExpression
Gene BGene BGene AGene A
Gene CGene C
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基因捕捉可以做为基因敲除的替代
电转干细胞,筛选 neoR
PGK neoR pA
剪接受体
用 Inverse PCR 、序列分析确认被捕捉基因
被随机捕捉的基因
启动子 外显子
转录产物
从基因捕捉库筛选出基因进行囊胚注射
lacZ pA
基因功能分析
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Gene trap transgenic embryos
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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
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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
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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
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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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