利用小鼠動物模型研究基因调节与病理发育.pptVIP

Mouse Model for Gene Regulation Studies Course Materials Introduction to Gene Regulations Introduction to mouse models Introduction to transgenic techniques Examples: VEGF gene regulation and pathologic development Transgenic Technology Part 1： Basis of classic transgenics Part 2： Gene Targeting Part 3： Applications What’s transgenic？ Narrow Definition：Artifacial insertion of DNA fragment into genome Broad Definition：Artifacial modification of genome, including insertion, mutation and deletion Importance of Transgenic Technology Basic Research Gene regulation，promoter function Gene expression tracing（Knockout，Gene Trap） Cell tracing，tumor cell labeling Functional study，embryonic, developmental and pathological Commercial Protein product Low cost Disease model Organ donor Strain improvement Gene Therapy Common Techniques Used for Making Transgenic animals Most Comkon ：Pronuclei DNA injection，random insertion，low predictability，variable Sperm associated DNA transduction，low repetibility Transposons, not common Viral infection：High efficiency，less random，limited DNA size，safety consern Embryonic stem cell／Blastocyst microinjection：Gene targeting, only for mice Nuclear transpalntation/animal cloning／only way to generate targeted modification. High cost and low efficiency 真核基因结构与调节 真核基因结构：启动子(promoter)，外显子(exon)，内含子(intron)，polyA信号, 活化信号（enhancers), 沉默信号（silencers），封闭区(Insulators) 转录因子(transcription factors)，活化因子(activators)，抑制因子(inhibitors) 真核mRNA结构：Cap， 5’非编码区(5’-UT)， 编码区(coding region)，3’非编码区(3’-UT)， polyA 组织专一性调节(tissue-specific) 发育阶段调节(developmental) 诱导性调节(inducible) 转录后调节: mRNA剪接和修饰， mRNA的稳定性，蛋白质合成效率，蛋白质半衰期 Transgenic Construct Selection for promoters cDNA Poly A Introns and insulators Construct design Promoter Tissue-specific：研究基因调节，启动子的功能，或调节其它基因的表达。如果用于调节其它基因，一定要查询启动子在转基因动物应用方面的文献，了解启动子的完整性 High expression：用于高表达某个基因，调节其它基因，或高表达后蛋白的生产 Universal expression：显示基因细胞标记，基因调节研究 Inducible：用于基因调节，有毒蛋白表达，致命基因的可逆调控等 Conbination：可诱导，高表达，组织专一，构成基因调节系统，用于调节基因或高表达蛋