基因打靶_TALEN技術(shù)-百替生物

1、基因打靶 TALEN正向遺傳學(xué)正向遺傳學(xué) VS VS 反向遺傳學(xué)反向遺傳學(xué)目前常用的遺傳學(xué)技術(shù) 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

2、plasmid in ES cell (mouse) via rAAV Gene targeting by ZFN and TALENGene 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細(xì)胞內(nèi)的穩(wěn)定性細(xì)胞內(nèi)的穩(wěn)定性基因表達(dá)敲低的效率基因表達(dá)敲低的效率細(xì)胞毒性細(xì)胞

3、毒性免疫應(yīng)答免疫應(yīng)答進(jìn)入細(xì)胞的方式進(jìn)入細(xì)胞的方式是否可遺傳是否可遺傳RNAi and Morpholino:l研究基因功能研究基因功能l疾病模型疾病模型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

4、 from bacterial plant pathogensTALE family was first identified from bacterial plant pathogensTALE：Transcription Activator-Like EffectorHrp-type III secretion (T3S) system is essential for pathogenicity of most xanthomonads(黃單胞菌)First TALE gene was cloned in 1989First TALE gene was cloned in 1989Bas

5、ic structure of TALE family Basic structure of TALE family proteinsproteinsThe 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 experimentGroup 1: validated via experiment“With TAL effectors its real

6、ly 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 StructureTALE P

7、rotein Domain Structure(Scholze H and Boch J, 2011)(Mahfouz MM and Li L, 2011)Potential applications of TALE Potential applications of TALE factorsfactors(Mahfouz MM and Li L, 2011)TALEN(TALE Nuclease)TALEN(TALE Nuclease)Methods for TALE Repeat ConstructionMethods for TALE Repeat Constructionu De no

8、vo synthesisuGolden gate assembly (Type IIs restriction enzyme)Golden Gate ShufflingGolden Gate ShufflingBased on Type IIs restriction enzymes,Cutting outside of their recognition siteSimilar with Golden Gate TALEN target site predictionTALEN target site predictionTALENTALEN的應(yīng)用：模式生物的基因敲除的應(yīng)用：模式生物的基因敲除In vitro TranscriptionMicroinjection基于基于ES cel和同源重組的基因敲除和同源重組的基因敲除TALEN的基因敲除的基因敲除2-6月3月3月5-10天3月TALENTALEN的應(yīng)用：人類的應(yīng)用：人類IPSIPS干細(xì)胞的基因修飾干細(xì)胞的基因修飾TALEN極大地提高了同源重組的效率Nature biotechnology V29:731 將識別特異DNA序列的TALE與轉(zhuǎn)錄激活因子VP64 融合，表達(dá)的融