《分子生物学》第12次课基因打靶和遗传修饰动物模型.pptx

基因打靶和遗传修饰动物模型;Introduction;Introduction;Introduction;概 述;概 述 ：随着基因敲除技术的发展，除了同源重组外，新 的原理和技术也逐渐被应用，比较成功的有ZFN、 TALEN、CRISPR/Cas9、The NgAgo–gDNA system ，它们同样可以达到基因敲除的目的。;The BIG Breakthroughs;The BIG Breakthroughs;实现基因敲除的多种原理和方法;同源重组;;;;利用同源重组构建基因敲除动物模型的基本步骤;1. Vector construction;1. Vector construction;1. Vector construction;1. Vector construction;：Isolated from a pre-implantation embryos From inner cell mass of blastula stage embryo Cells are undifferentiated 挺Cells are pluripotent 挺Able to differentiate into many (all?) different cell types in embryo.;：Isolated from a pre-implantation embryos grown in culture need cells to divide but not differentiate longer time in culture = more differentiation.;2. Embryonic Stem Cell Isolation;3. Homologous recombination;：Gene Targeting Technology 挺 Cells take up exogenous DNA Frequency is very low Can be induced to higher frequency (chemical, electrical, injection) 挺 In Dividing Cells: Some DNA incorporated into genome 鸟 Random integration — rare event 鸟 Homologous Recombination — even more rare;：Random Integration: 挺 DNA incorporated anywhere in genome (not targeted) 、 copy number can be very high. 、 disrupts the endogenous DNA at insertion site. 、 transgenic animals are usually random integrations.;driven by the DNA sequences (targeted) rare (1000× less frequent than random) increased efficiency: isogenic DNA longer stretches of homology results in: endogenous DNA replaced with exogenous specific - targeted copy number = 1;3. Homologous recombination;3. Homologous recombination;3. Homologous recombination;3. Homologous recombination;3. Homologous recombination;3. Homologous recombination;3. Homologous recombination;3. Homologous recombination;3. Homologous recombination;4. Selection of recombinant ES cells;4. Selection of recombinant ES cells;;;;4. Selection of recombinant ES cells;Positive and negative selection of recombinant ES cells;Positive and negative selection of recombinant ES cells;Cre Mediated Recombination;Targeting