基因打靶TALEN技術(shù)-百替生物課件

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

ShortinterferingRNAs—post-transcriptionalgenesilencingMorpholinostructureandfunctions細(xì)胞內(nèi)的穩(wěn)定性基因表達(dá)敲低的效率細(xì)胞毒性免疫應(yīng)答進(jìn)入細(xì)胞的方式是否可遺傳RNAiandMorpholino:TheNobelPrizeinPhysiology2007--thebeginningofaneweraingeneticsTheratesofrecombinationinmammaliancellstypicallyfallintherangeof10-6-10-5

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

Eachrepeatcontainsof33-35aa

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

Goldengateassembly

(TypeIIsrestrictionenzyme)

SimilarwithGoldenGateTALENtargetsitepredictionTALEN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

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