基因打靶TALEN技术-百替生物.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 screenPhenocopy(transient effect):RNAiMorpholino(MO)antisense oligonucleotidesReverse 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 Physiology2007-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 regionEach repeat contains of 33-35aaNumbers of repeats spread from 1.5 to 33.5,with the peak at 17.5,Group 1:validated via experiment,“With TAL effectors its 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 synthesisGolden gate assembly(Type IIs restriction enzyme),Golden Gate Shuffling,Based on Type IIs restriction enzymes,Cutting outside of their recognition site,Similar with Golden Gate,TALEN target site prediction,TALEN的应用：模式生物的基因敲除,In vitro Transcription,Microinjection,基于ES cel和同源重组的基因敲除,TALEN的基因敲除,2-6月,3月,3月,5-10天,3月,TALEN的应用：人类IPS干细胞的基因修饰,TALEN极大地提高了同源重组的效率,Nature biotechnology V29:731,将识别特异DNA序列的TALE与转录激活因子VP64 融合，表达的融合蛋白结合启动子附近的特异DNA序列，并通过VP64激活区域与Pol II 结合，激活基因的转录，提高了内源目标基因的表达。,TALEA-人工转录因子,TALEN has been applied in many species.,Thank you！,
