基因打靶TALEN技术-百替生物

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1、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Forwardgenetics:Chemicalmutagenesis(EMS,ENU)Insertionalmutagenesis(retrovirus,transposon)Genetrap(transposon)NaturalmutationscreenPhenocopy(transienteffect):RNAiMorpholino(MO)antisenseoligonucleotidesReversegenetics:Knockoutby

2、HR:viaplasmidinEScell(mouse)viarAAVGenetargetingbyZFNandTALENTheshortstoryofgenesilencing—themaincharacteristicsofshortRNAsShortinterferingRNAs—post-transcriptionalgenesilencingMorpholinostructureandfunctions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RNAiandMorpholino

3、: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TheNobelPrizeinPhysiology2007--thebeginningofaneweraingeneticsTheratesofrecombinationinmammaliancellstypicallyfallintherangeof10-6-10-5eventspercellpergenerationTALEfamilywasfirstidentifiedfrombacterialplantpathogensTALE：Transcription

4、Activator-LikeEffectorHrp-typeIIIsecretion(T3S)systemisessentialforpathogenicityofmostxanthomonads(黄单胞菌)FirstTALEgenewasclonedin1989BasicstructureofTALEfamilyproteinsThecoreofTALEisarepeatregionEachrepeatcontainsof33-35aaNumbersofrepeatsspreadfrom1.5to33.5,witht

5、hepeakat17.5Group1:validatedviaexperiment“WithTALeffectorsit’sreallyamazing.IfIseetheaminoacidsequenceoftheTALrepeat,andknowthevariableaminoacidresidues,Icanpredictwhatsequenceitwillbind.Conversely,seeingaDNAsequence,withfewexceptions,wecandesignaTALeffectorthat

6、recognizesthissequence.”TALEProteinDomainStructure(ScholzeHandBochJ,2011)(MahfouzMMandLiL,2011)PotentialapplicationsofTALEfactors(MahfouzMMandLiL,2011)TALEN(TALENuclease)MethodsforTALERepeatConstructionDenovosynthesisGoldengateassembly(TypeIIsrestrictionenzyme)

7、GoldenGateShufflingBasedonTypeIIsrestrictionenzymes,CuttingoutsideoftheirrecognitionsiteSimilarwithGoldenGateTALENtargetsitepredictionTALEN的应用：模式生物的基因敲除InvitroTranscriptionMicroinjection基于EScel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

8、源重组的效率NaturebiotechnologyV29:731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PolII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TALENhasbeenappliedinmanyspec