小鼠基因剔除技术( knockout) 鼠胚胎干细胞 体外传代 发育全能性(特别是能发育为生殖细胞) DNA同源重组技术 基因捕获技术(随机方法) 化学诱变技术(ENU) ES细胞 J鼠个体
小鼠基因剔除技术（knockout） • 小鼠胚胎干细胞 – 体外传代 – 发育全能性（特别是能发育为生殖细胞） • DNA同源重组技术 • 基因捕获技术（随机方法） • 化学诱变技术（ENU） – ES细胞 – 小鼠个体
小鼠基因别除技术的里程碑工作 1981Evans and kaufman:( Nature. 1981 Jul 9: 292(5819): 154-6, Proc Natl Acad UsA.1981Dec;78(12:7634-8 )建立了ES细胞 1986-1987 Robertson et al, Hooper et al, Kuehn etω,:对S细胞进行遗传操作,并获得 ES细胞来源的小鼠个体( re.1986oct2-8;323(6087):445-8. Nature1987Mar 19-25;326(6110:292-5, Nature.1987Mar19-25;326(6110:295-8. 1988 Thomas et al.,;在S细胞中建立基于同源重 组的基因打靶技术( ce.1987Nov651(3):503-12. 1989 Jaenisch et al:建立b2- microglobulin deficien基因剔除小鼠( Nature.1989NoV23;342(6248)435-8 1994 Gu et al,将Cre一LoxP系统用于ES细胞的打靶, 建立条件性基因别除技术(sm,m,2m)
小鼠基因剔除技术的里程碑工作 • 1981Evans and Kaufman:（Nature. 1981 Jul 9;292(5819):154-6，Proc Natl Acad Sci U S A. 1981 Dec;78(12):7634-8）建立了ES细胞 • 1986－1987 Robertson et al ，Hooper et al， Kuehn et al，：对ES细胞进行遗传操作，并获得 ES细胞来源的小鼠个体（Nature. 1986 Oct 2-8;323(6087):445-8. Nature 1987 Mar 19-25;326(6110):292-5， Nature. 1987 Mar 19-25;326(6110):295-8.） • 1988 Thomas et al.,：在ES细胞中建立基于同源重 组的基因打靶技术（Cell. 1987 Nov 6;51(3):503-12. ） • 1989 Jaenisch et al: 建立b2-microglobulindeficient基因剔除小鼠(Nature. 1989 Nov 23;342(6248):435-8.) • 1994 Gu et al, 将Cre-LoxP系统用于ES细胞的打靶， 建立条件性基因剔除技术（Science. 1994 . 265:103. ）
基因打靶( Gene targeting) ·基因剔除(常规、条件性) Gene Knock-out, conventional and conditional) 基因敲入( Gene Knock-in) 基因捕荻( Gene Trap)
基因打靶 (Gene targeting) • 基因剔除（常规、条件性） (Gene Knock-out, conventional and conditional) • 基因敲入(Gene Knock-in) • 基因捕获(Gene Trap)
Early mouse development ♀ Pronucleus d Pronucleus Cleavage Zona A. Zygote B. 2 cell 1.5d C. 4 cell 2d nner cell mass Trophectoderm Blastocoel (ICM) vity Compaction Formation of blastocoel D. Morula 2.5d E. 3d F. Blastocyst 3.5d Primitive ectoderm Trophectoderm From endoderm Primitive Sedivy Uterus ectoderm Joyner Gene Targeting Uterus 1992 G. Implantation 4.5d H. Egg cylinder 6d
Early mouse development From Sedivy & Joyner “Gene Targeting” 1992
Blastocyst 3.5 days Generating ES Cells 30 cells Harvest blastocysts on day 3.5 by flushing from the uterine lumen with Inner Cell mass Plate out blastocysts in individual 10 mm dishes (128 cell stage) on mitotically inactive MEF or STO feeder cells to provide liF and other factors 1-totipotent After 1-2 days, blastocysts hatch(A) and attach to the dish by migration of 2-tissue culture the trophectoderm TE), while the nner cell mass(ICM)grows(B) 3-Transfectable After about 96-120 hours in culture (C, D), the ICM can be dislodged from the TE layer, washed and transplanted to microdrops of medium containing trypsin to disperse the 4-Selection clumps 5- Differentiation Transfer disaggregated contents to a fresh feeder cell tissue culture well and inspect daily for signs of In vitro differentiation. Primary cell colonies are clearly visible as
Blastocyst 3.5 days Inner Cell Mass (128 cell stage) 1-totipotent 2-tissue culture 3-Transfectable 4-Selection 5- Differentiation In vitro Harvest blastocysts on day 3.5 by flushing from the uterine lumem with M2 medium Plate out blastocysts in individual 10 mm dishes on mitotically inactive MEF or STO feeder cells to provide LIF and other factors After about 96-120 hours in culture (C, D), the ICM can be dislodged from the TE layer, washed and transplanted to microdrops of medium containing trypsin to disperse the clumps After 1-2 days, blastocysts hatch (A) and attach to the dish by migration of the trophectoderm (TE), while the inner cell mass (ICM) grows (B) 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 Generating ES Cells ~30 cells
NM-2细胞的族型囝 AO RD AA 4O B SAN 自0aA
NM-2细胞的核型图
Stem cell cultures Mouse embryonic fibroblast cells: Mitotically inactivated by irradiation or mitomycin C ES cells: Medium with 15% FCS 1000u/mILIF CO2:7-10% LiF (leukaemia inhibitory factor): Maintains stem cells in an undifferentiated state
Stem cell cultures Mouse Embryonic Fibroblast Cells: Mitotically inactivated by irradiation or mitomycin C ES cells: Medium with 15% FCS 1000u/ml LIF CO2 : 7-10% LIF (leukaemia inhibitory factor) ： Maintains stem cells in an undifferentiated state
ES Cells differentiation Drug or Growth factors Blood Blood euro Muscle Ⅴ essels Development Cells Cells
ES Cells Differentiation Tissues Muscle Cells Drug or Growth factors Bone Cells Blood Cells Blood Vessels Neuronal Development