研究生实验技术导论与细胞培养.ppt

显微注射 病毒转基因的方法： Adenovirus Retrovirus Lenti-virus Viral Vectors Retrovirus: stable gene transfer, dividing cells only Adenovirus: transient gene expression, dividing and nondividing cells Lentivirus: transient or stable gene expression, high efficiency, dividing and nondividing cells Baculovirus and vaccinia virus: high level protein production in insect cells Lentivirus Jak GHR GH membrane inactive active +1 OPEN Light mRNA 0f Luc Protein CAR GHR JAK2 JAK2 GH Yp Yp Firefly Luciferase STAT5 GHR JAK2 JAK2 Yp Yp Yp Yp GH Ad-Reporter (Luc) infection 5’ 3’ TTC-NNN-GAA Luc Luciferin STAT5 Yp Yp Light Luciferase reporter assay 1. Conform promoter of regulatory elements of the gene 2. Detecting a specific protein working on promoter of the target gene 3. Detecting if the two proteins working together on the target gene Protein or proteins working on the promoter Protein 1 Protein 2 Protein or proteins working on the promoter Protein 1 Protein 2 Protein or proteins working on the promoter Protein 1 Protein 2 Protein or proteins working on the promoter Protein 1 Protein 2 Protein or proteins working on the promoter Protein 1 Protein 2 Protein or proteins working on the promoter Protein 1 Protein 2 D. 酵母双杂交用于筛选相互作用的蛋白质 GFP， another report gene Monitor plamid existence, therefore, monitor the transfection rate. Monitor gene expression Detecting the location of a protein by making GFP fusion protein GFP fusion protein Expression of H2B-GFP before and after UV-B-induced apoptosis. A431 cells were stably transfected with H2B-GFP fusion protein (K/H2B-GFP). H2B-GFP is expressed only within the nuclei. In the right panel an apoptotic K/H2B-GFP cell is shown after UV-B irradiation. The “Blebs and bodies forming” phase is shown. Noticeably, redistribution of SLE self antigens are among the remarkable changes that occur during apoptosis. Images were collected in real time by laser confocal microscopy. 把外源基因引入细胞的方法 1.脂质体法（转染) 2.电转移法 (转染） 3. 显微注射（转染） 4.病毒感染法 Protein A. Introduc