基因打靶_TALEN技术-百替生物课件.ppt

基因打靶 ——TALEN 正向遗传学 VS 反向遗传学 目前常用的遗传学技术 Forward genetics:Chemical mutagenesis (EMS,ENU)Insertional mutagenesis(retrovirus, transposon)Gene trap(transposon)Natural mutation screen Phenocopy (transient effect):RNAiMorpholino(MO) antisense oligonucleotides Reverse genetics:Knock out by HR: via plasmid in ES cell (mouse) via rAAV Gene targeting by ZFN and TALEN The short story of gene silencing — the main characteristics of short RNAs Short interfering RNAs — post-transcriptional gene silencing Morpholino structure and functions 细胞内的稳定性 基因表达敲低的效率 细胞毒性 免疫应答 进入细胞的方式 是否可遗传 RNAi and Morpholino: 小鼠胚胎干细胞是基因剔除的必需媒介 研究基因功能 疾病模型 The Nobel Prize in Physiology 2007-- the beginning of a new era in genetics The rates of recombination in mammalian cells typically fall in the range of 10-6 -10-5 events per cell per generation TALE family was first identified from bacterial plant pathogens TALE：Transcription Activator-Like Effector Hrp-type III secretion (T3S) system is essential for pathogenicity of most xanthomonads(黄单胞菌) First TALE gene was cloned in 1989 Basic structure of TALE family proteins The core of TALE is a repeat region Each repeat contains of 33-35aa Numbers of repeats spread from 1.5 to 33.5, with the peak at 17.5 Group 1: validated via experiment “With TAL effectors it’s really amazing. If I see the amino acid sequence of the TAL repeat, and know the variable amino acid residues, I can predict what sequence it will bind. Conversely, seeing a DNA sequence, with few exceptions, we can design a TAL effector that recognizes this sequence.” TALE Protein Domain Structure (Scholze H and Boch J, 2011) (Mahfouz MM and Li L, 2011) Potential applications of TALE factors (Mahfouz MM and Li L, 2011) TALEN(TALE Nuclease) Methods for TALE Repeat Construction De novo synthesis Golden gate assembly (Type IIs restriction enzyme) Golden Gate Shuffling Based on Type IIs restriction enzymes, Cutting outside of their recognition site Similar with Golden