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 :Gene Targeting
Part 3 :Applications
Part 1
Transgenic Technology
What’s transgenic ?
• Narrow Definition : Artifacial insertion of DNA fragment into genome
• Broad Definition : Artifacial modification of genome, including insertion, mutation and deletion
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
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
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
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
…
Transgenic Construct
• Selection for promoters• cDNA• Poly A• Introns and insulators• Construct design
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
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
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
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
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:
( 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)
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
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
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
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
•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
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
Transgenic Technology
Part 1 :Basis of classic transgenics
Part 2 :Gene Targeting
Part 3 :Applications
Part 2 : Gene Targeting
•Theory
•Technique
•Application
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
小鼠早期发育示意图
3.5 天
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
Targeting strategy
• Vector design
• Conditional construction
• Reporter knocking– Tissue-specific for study gene regul
ation– Universal expression: functional stu
dies
NEO1 3 TK
X X
1 NEO 3
1 32Wild type alelle
Vector
After recombination
Basic Targeting Vector
NEO TK
FRTFRT
loxPloxP
Wild type
Vector
Flipase to remove NEO
FRT
Tissue-specific Cre to inactivate gene
loxP
FRT
Conditional knockout
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
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
Time Table
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
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
(Gene Trapping)
LacZ-Neo(GEO)
Gene B-likeGene B-likeExpressionExpressionpossiblepossible
No No ExpressionExpression
Gene BGene BGene AGene A
Gene CGene C
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
Gene trap transgenic embryos
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
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
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
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