基因打靶talen技术-百替生物

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1. Forward genetics:Chemical mutagenesis (EMS,ENU)Insertional mutagenesis(retrovirus, transposon)Gene trap(transposon)Natural mutation screen2. Phenocopy (transient effect):RNAiMorpholino(MO) antisense oligonucleotides3. Reverse genetics:Knock out by HR: via plasmid in ES cell (mouse)via rAAV Gene targeting by ZFN and TALENThe short story of gene silencing the main characteristics of short RNAsShort interfering RNAs post-transcriptional gene silencingMorpholino 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小鼠胚胎干细胞是基因剔除的必需媒介l研究基因功能l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 generationTALE family was first identified from bacterial plant pathogensTALE： Transcription Activator-Like EffectorHrp-type III secretion (T3S) system is essential for pathogenicity of most xanthomonads(黄单胞菌 )First TALE gene was cloned in 1989Basic structure of TALE family proteinsThe 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 17.5Group 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 Constructionu De novo synthesisuGolden gate assembly(Type IIs restriction enzyme)Golden Gate ShufflingBased on Type IIs restriction enzymes,Cutting outside of their recognition siteSimilar with Golden Gate TALEN target site predictionTALEN的应用：模式生物的基因敲除In vitro TranscriptionMicroinjection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