细胞生物学-14转基因动物要点.ppt

第六章 基因打靶 (Gene targeting) 历史 1981 Evans and Kaufman建立了ES细胞。 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。 1988 Thomas et al在ES细胞中建立基于同源重组的基因打靶技术。 1989 Jaenisch建立b2-microglobulin-deficient基因剔除小鼠。 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。  Embryonic stem (ES) cells Early mouse development NM-2细胞的核型图 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 打靶载体的设计 选用同源DNA 同源臂的长度 正、负选择 Factors influencing targeting efficiency isogenic DNA (perfect homology) 10-25 fold size of region of homology exponential relationship robust screen!  Positive controls SELECTION MARKER GENES Breeding Chimeras (knock-out founder) Chimera - the founder germ-line transmission - usually the ES cells are derived from a 129 strain (agouti or white colour) and the ES cells are injected into a C57Bl/6 blastocyst (black). The more that the ES cells contribute to the genome of the mouse, the more the coat colour will be agouti. The chimera mouse is usually “tiger” striped. Breeding Chimeras (knock-out founder)cont Males that are 40% to 100% based on agouti coat colour should be bred Females should not be bred (low incidence of success) ES cells are male. Breed aggressively- rotate females through males cage. If the male produces more than 6 litters without transmitting, not likely to go germline and should be saced 基因打靶技术的局限性 胚胎致死 所有细胞都改变 功能代偿 冗余 单元与系统的关系 条件性打靶技术（conditional knockout） Cre/loxP recombinase FLP/FRT recombinase Inducible system Cre-Mediated Recombination - tandem repeats Cre-Mediated Recombination - inverted repeats Conditional knock-outs How to FLOX a gene Mate FLOXed mice with mice carrying a Cre transgene GENE TRAPPING USING EXPRESSION