第六章 基因打靶
(Gene targeting)
Early mouse development
From Sedivy & Joyner “Gene Targeting” 1992
第六章 基因打靶
Blastocyst
(Gene targeting)
Generating ES Cells
Harvest blastocysts on day 3.5 by flushing from the uterine lumem with M2 medium
After 1-2 days, blastocysts hatch (A) and 1attach to the dish by migration of the trophectoderm (TE), while the inner cell mass (ICM) grows (B)
After about 96-120 hours in culture (C, D), the ICM can be 96dislodged from the TE layer, washed and transplanted to microdrops of medium containing trypsin to disperse the clumps
第六章 基因打靶
(Gene targeting)
打靶载体的设计
选用同源DNA 选用同源DNA 同源臂的长度 正、负选择
第六章 基因打靶
(Gene targeting)
Factors influencing targeting efficiency
isogenic DNA (perfect homology) 1010-25 fold size of region of homology exponential relationship robust screen! Positive controls
~30 cells Inner Cell Mass (128 cell stage)
1-totipotent 2-tissue culture 3-Transfectable 4-Selection 5- Differentiation In vitro
Plate out blastocysts in individual 10 mm dishes on mitotically inactive MEF or STO feeder cells to provide LIF and other factors
Drug or Growth factors
Tissues
Blood Vessels
Blood Cells
Neuronal Development
Bone Cells
Muscle Cells
第六章 基因打靶
(Gene targeting)
Gene targeting in embryonic stem cells Targeting vector with the desired change is electroporated into ES cells Homologous recombinants are identified by drug selection &molecular screening Recombinant ES clones are injected into blastocysts to produce chimeric mice Chimeras are bred to produce heterozygotes Heterozygotes are intercrossed to produce homozygous mutants
(Gene targeting)
X
NEOMYCIN NEOMYCIN
X
Total 4 Kbp (each arm not less than 500bp) Delete coding sequences Change reading frame Transcription of Neo in antisense direction
第六章 基因打靶
(Gene targeting)
SELECTION SELECTION MARKER GENES GENES
Positive selection: - neomycin phosphotransferase (neo) phosphotransferase - hygromycin phosphotransferase (hyg) phosphotransferase (hyg) - puromycine (pur) - hypoxanthine phosphoribosyltransferase (hprt) hypoxanthine phosphoribosyltransferase (hprt) Negative selection： Negative selection： - thymidine kinase (tk) - cytosine deaminase (cd) - hypoxanthine phosphoribosyltransferase (hprt) hypoxanthine phosphoribosyltransferase (hprt)
(Gene targeting)
ES cells spontaneously differentiate when allowed to aggregate in the absence of LIF
- LIF
第六章 基因打靶
(Gene targeting)
ES Cells Differentiation
ES cells: Medium with 15% FCS 1000u/ml LIF CO2 : 7-10% 7LIF (leukaemia inhibitory factor) ： Maintains stem cells in an undifferentiated state
第六章 基因打靶
第六章 基因打靶
(Gene targeting)
转基因动物的基本流程
第六章 基因打靶
(Gene targeting)
Embryonic stem (ES) cells
Pluripotent stem cells derived from the inner cell mass of the blastocyst Can be cultured, manipulated and then reinjected into blastocysts, where they can go on to contribute to all parts of embryo. In principle, ES cells also might be able to generate large quantities of any desired cell .
第六章 基因打靶
(Gene targeting)
NMNM-2细胞的核ቤተ መጻሕፍቲ ባይዱ图
第六章 基因打靶
(Gene targeting)
Stem cell cultures
Mouse Embryonic Fibroblast Cells: Mitotically inactivated by irradiation or mitomycin C
第六章 基因打靶
PolyPoly-A缺失筛选法
(Gene targeting)
Poly- 缺失筛选法的原理与启动子缺失筛选法相似， Poly-A 缺失筛选法的原理与启动子缺失筛选法相似 ， 一 般在打靶基因不能被诱导表达时，采用该方法。 般在打靶基因不能被诱导表达时 ， 采用该方法 。 载体设计特 点是正向选择基因neo 点是正向选择基因neo的3´末端缺乏转录终止信号。由于载体 neo的 末端缺乏转录终止信号。 无转录终止信号，neo基因的表达在转录水平受到抑制 无转录终止信号，neo基因的表达在转录水平受到抑制。但在 基因的表达在转录水平受到抑制。 同源重组的细胞中，neo基因可以利用基因组靶位基因的转录 同源重组的细胞中，neo基因可以利用基因组靶位基因的转录 终止信号得到有效表达，从而使阳性细胞获得Ｇ418抗性 终止信号得到有效表达，从而使阳性细胞获得Ｇ418抗性，实 抗性， 现同源重组转化细胞的富集。 现同源重组转化细胞的富集。
第六章 基因打靶
(Gene targeting)
Homologous recombination
1- Length of homologous sequences 2- Isogenic DNA
Hom. Rec. Efficiency
Base pair 25bp 2000bp
第六章 基因打靶
Gene targeting
Transfer disaggregated contents to a fresh feeder cell tissue culture well and inspect daily for signs of differentiation. Primary cell colonies are clearly visible as distinct clumps
第六章 基因打靶
(Gene targeting)
第六章 基因打靶
(Gene targeting)
历史 Kaufman建立了ES细胞 建立了ES细胞。 1981 Evans and Kaufman建立了ES细胞。 1986－ Robertson，Hooper，Kuehn等对ES细胞进行 等对ES 1986－1987 Robertson，Hooper，Kuehn等对ES细胞进行 遗传操作，并获得ES细胞来源的小鼠个体。 ES细胞来源的小鼠个体 遗传操作，并获得ES细胞来源的小鼠个体。 al在ES细胞中建立基于同源重组的基因 1988 Thomas et al在ES细胞中建立基于同源重组的基因 打靶技术。 打靶技术。 Jaenisch建立 microglobulin-deficient基因剔 1989 Jaenisch建立b2-microglobulin-deficient基因剔 除小鼠。 除小鼠。 Cre-LoxP系统用于ES细胞的打靶 系统用于ES细胞的打靶， 1994 Gu 将Cre-LoxP系统用于ES细胞的打靶，建立条件性 基因剔除技术。 基因剔除技术。