基因打靶-TALEN技術(shù)-百替生物

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

Chemicalmutagenesis(EMS,ENU)

Insertionalmutagenesis(retrovirus,transposon)

Genetrap(transposon)

Naturalmutationscreen

Phenocopy(transienteffect):

RNAi

Morpholino(MO)antisenseoligonucleotides

Reversegenetics:

KnockoutbyHR:viaplasmidinEScell(mouse)

viarAAV

GenetargetingbyZFNandTALEN

Theshortstoryofgenesilencing—themaincharacteristicsofshortRNAsShortinterferingRNAs—post-transcriptionalgenesilencingMorpholinostructureandfunctions細胞內(nèi)的穩(wěn)定性基因表達敲低的效率細胞毒性免疫應(yīng)答進入細胞的方式是否可遺傳RNAiandMorpholino:小鼠胚胎干細胞是基因剔除的必需媒介研究基因功能疾病模型TheNobelPrizeinPhysiology2007--thebeginningofaneweraingeneticsTheratesofrecombinationinmammaliancellstypicallyfallintherangeof10-6-10-5

eventspercellpergenerationTALEfamilywasfirstidentifiedfrombacterialplantpathogensTALE：TranscriptionActivator-LikeEffectorHrp-typeIIIsecretion(T3S)systemisessentialforpathogenicityofmostxanthomonads(黃單胞菌)FirstTALEgenewasclonedin1989BasicstructureofTALEfamilyproteinsThecoreofTALEisarepeatregion

Eachrepeatcontainsof33-35aa

Numbersofrepeatsspreadfrom1.5to33.5,withthepeakat17.5Group1:validatedviaexperiment“WithTALeffectorsit’sreallyamazing.IfIseetheaminoacidsequenceoftheTALrepeat,andknowthevariableaminoacidresidues,Icanpredictwhatsequenceitwillbind.Conversely,seeingaDNAsequence,withfewexceptions,wecandesignaTALeffectorthatrecognizesthissequence.”TALEProteinDomainStructure(ScholzeHandBochJ,2011)(MahfouzMMandLiL,2011)PotentialapplicationsofTALEfactors(MahfouzMMandLiL,2011)TALEN(TALENuclease)MethodsforTALERepeatConstructionDenovosynthesis

Goldengateassembly

(TypeIIsrestrictionenzyme)

GoldenGateShufflingBasedonTypeIIsrestrictionenzymes,CuttingoutsideoftheirrecognitionsiteSimilarwithGoldenGateTALENtargetsitepredictionTALEN的應(yīng)用：模式生物的基因敲除InvitroTranscriptionMicroinjection基于EScel和同源重組的基因敲除TALEN的基因敲除2-6月3月3月5-10天3月TALEN的應(yīng)用：人類IPS干細胞的基因修飾TALEN極大地提高了同源重組的效率NaturebiotechnologyV29:731

將識別特異DNA序列的TALE與轉(zhuǎn)錄激活因子VP64融合，表達的融合蛋白結(jié)合啟動子附近的特異D