Floxing - Wikipedia

In genetics, floxing refers to the sandwiching of a DNA sequence between two lox P sites. ... Therefore, Cre-lox system can be used in mice to manipulate gene ... Floxing FromWikipedia,thefreeencyclopedia Jumptonavigation Jumptosearch SandwichingofaDNAsequencebetweentwoloxPsites ThisfiguredepictshowFloxingisusedinscientificresearchforspatialandtemporalcontrolofgeneexpression. Ingenetics,floxingreferstothesandwichingofaDNAsequence(whichisthensaidtobefloxed)betweentwoloxPsites.Thetermsareconstructeduponthephrase"flanking/flankedbyLoxP".RecombinationbetweenLoxPsitesiscatalysedbyCrerecombinase.Floxingageneallowsittobedeleted(knockedout),translocatedorinvertedinaprocesscalledCre-Loxrecombination. [1]Thefloxingofgenesisessentialinthedevelopmentofscientificmodelsystemsasitallowsresearcherstohavespatialandtemporalalterationofgeneexpression.[2]Moreover,animalssuchasmicecanbeusedasmodelstostudyhumandisease.Therefore,Cre-loxsystemcanbeusedinmicetomanipulategeneexpressioninordertostudyhumandiseasesanddrugdevelopment.[3]Forexample,usingtheCre-loxsystem,researcherscanstudyoncogenesandtumorsuppressorgenesandtheirroleindevelopmentandprogressionofcancerinmicemodels.[4] Contents 1Usesinresearch 1.1Mechanismofdeletion 1.2Mechanismofinversion 1.3Mechanismoftranslocation 2Commonapplicationsinresearch 3References Usesinresearch[edit] Floxingageneallowsittobedeleted(knockedout),[5][6]translocatedorinserted[7](throughvariousmechanismsinCre-Loxrecombination). Thefloxingofgenesisessentialinthedevelopmentofscientificmodelsystemsasitallowsspatialandtemporalalterationofgeneexpression.Inlayman'sterms,thegenecanbeknocked-out(inactivated)inaspecifictissueinvivo,atanytimechosenbythescientist.Thescientistcanthenevaluatetheeffectsoftheknocked-outgeneandidentifythegene’snormalfunction[8].Thisisdifferentfromhavingthegeneabsentstartingfromconception,wherebyinactivationorlossofgenesthatareessentialforthedevelopmentoftheorganismmayinterferewiththenormalfunctionofcellsandpreventtheproductionofviableoffspring.[9] Mechanismofdeletion[edit] AmodelexperimentingeneticsusingtheCre-loxsystem:theprematurestopsequencepresentinfloxedmiceisremovedonlyfromcellsthatexpressCrerecombinasewhenthemicearebredtogether. Deletioneventsareusefulforperforminggeneeditingexperimentsthroughpreciselyeditingoutsegmentsoforevenwholegenes.DeletionrequiresfloxingofthesegmentofinterestwithloxPsiteswhichfacethesamedirection.TheCrerecombinasewilldetecttheunidirectionalloxPsitesandexcisethefloxedsegmentofDNA.[10]ThesuccessfullyeditedclonescanbeselectedusingaselectionmarkerwhichcanberemovedusingthesameCre-loxPsystem.[10]ThesamemechanismcanbeusedtocreateconditionalallelesbyintroducinganFRT/Flpsitewhichaccomplishesthesamemechanismbutwithadifferentenzyme. Mechanismofinversion[edit] Inversioneventsareusefulformaintainingtheamountofgeneticmaterial.Theinvertedgenesarenotoftenassociatedwithabnormalphenotypes,meaningtheinvertedgenesaregenerallyviable.[11]Cre-loxPrecombinationthatresultininsertionrequiresloxPsitestofloxthegeneofinterest,withtheloxPsitesorientedtowardseachother.ByundergoingCrerecombination,theregionfloxedbytheloxPsiteswillbecomeinverted,[12]thisprocessisnotpermanentandcanbereversed.[13]  Mechanismoftranslocation[edit] TranslocationeventsoccurwhentheloxPsitesfloxgenesontwodifferentDNAmoleculesinaunidirectionalorientation.CrerecombinaseisthenusedtogenerateatranslocationbetweenthetwoDNAmolecules,exchangingthegeneticmaterialfromoneDNAmoleculetotheotherformingasimultaneoustranslocationofbothfloxedgenes.[14][15] Commonapplicationsinresearch[edit] Cardiomyocytes(heartmuscletissue)havebeenshowntoexpressatypeofCrerecombinasethatishighlyspecifictocardiomyocytesandcanbeusedbyresearcherstoperformhighlyefficientrecombinations.ThisisachievedbyusingatypeofCrewhoseexpressionisdrivenbythe α {\displaystyle\alpha} -myosinheavychainpromoter( α {\displaystyle\alpha} -MyHC).Theserecombinationsarecapableofdisruptinggenesinamannerthatisspecifictoonlyhearttissueinvivoandallowsforthecreationofconditionalknockoutsoftheheartmostlyforuseascontrols.[16] Forexample,usingtheCrerecombinasewiththe α {\displaystyle\alpha} -MyHCpromotercausesthefloxedgenetobeinactivatedintheheartalone.Further,theseknockoutscanbeinducible.Inseveralmousestudies,tamoxifenisusedtoinducetheCrerecombinase.[17][18]Inthiscase,Crerecombinaseisfusedtoaportionofthemouseestrogenreceptor(ER)whichcontainsamutationwithinitsligandbindingdomain(LBD).Themutationrendersthereceptorinactive,whichleadstoincorrectlocalizationthroughitsinteractionswithchaperoneproteinssuchasheatshockprotein70and90(Hsp70andHsp90).TamoxifenbindstoCre-ERanddisruptsitsinteractionswiththechaperones,whichallowstheCre-ERfusionproteintoenterthenucleusandperformrecombinationonthefloxedgene.[19][20]Additionally,Crerecombinasecanbeinducedbyheatwhenunderthecontrolofspecificheatshockelements(HSEs).[21][22] References[edit] ^ NagyA(February2000)."Crerecombinase:theuniversalreagentforgenometailoring".Genesis.26(2):99–109.doi:10.1002/(SICI)1526-968X(200002)26:2<99::AID-GENE1>3.0.CO;2-B.PMID 10686599.S2CID 2916710. ^HayashiS,McMahonAP(April2002)."Efficientrecombinationindiversetissuesbyatamoxifen-inducibleformofCre:atoolfortemporallyregulatedgeneactivation/inactivationinthemouse".DevelopmentalBiology.244(2):305–18.doi:10.1006/dbio.2002.0597.PMID 11944939. ^Mousegenetics :methodsandprotocols.Singh,ShreeRam,,Coppola,Vincenzo.NewYork,NY.ISBN 9781493912155.OCLC 885338722.{{citebook}}:CS1maint:others(link) ^GreenJE,RiedT(2012).GeneticallyEngineeredMiceforCancerResearch.doi:10.1007/978-0-387-69805-2.ISBN 978-0-387-69803-8. ^FriedelRH,WurstW,WefersB,KühnR(2011)."Generatingconditionalknockoutmice".TransgenicMouseMethodsandProtocols.MethodsinMolecularBiology.Vol. 693.pp. 205–31.doi:10.1007/978-1-60761-974-1_12.ISBN 978-1-60761-973-4.PMID 21080282. ^SakamotoK,GurumurthyCB,WagnerK(2014),SinghSR,CoppolaV(eds.),"GenerationofConditionalKnockoutMice",MouseGenetics,SpringerNewYork,vol. 1194,pp. 21–35,doi:10.1007/978-1-4939-1215-5_2,ISBN 9781493912148,PMID 25064096 ^ImutaY,KiyonariH,JangCW,BehringerRR,SasakiH(March2013)."Generationofknock-inmicethatexpressnuclearenhancedgreenfluorescentproteinandtamoxifen-inducibleCrerecombinaseinthenotochordfromFoxa2andTloci".Genesis.51(3):210–8.doi:10.1002/dvg.22376.PMC 3632256.PMID 23359409. ^HallB,LimayeA,KulkarniAB(September2009)."Overview:generationofgeneknockoutmice".CurrentProtocolsinCellBiology.Chapter19:Unit19.1219.12.1–17.doi:10.1002/0471143030.cb1912s44.PMC 2782548.PMID 19731224. ^RodriguesJV,ShakhnovichEI(2019-08-01)."AdaptationtomutationalinactivationofanessentialE.coligeneconvergestoanaccessiblesuboptimalfitnesspeak".bioRxiv:552240.doi:10.1101/552240. ^abSchwenkF,BaronU,RajewskyK(December1995)."Acre-transgenicmousestrainfortheubiquitousdeletionofloxP-flankedgenesegmentsincludingdeletioningermcells".NucleicAcidsResearch.23(24):5080–1.doi:10.1093/nar/23.24.5080.PMC 307516.PMID 8559668. ^GriffithsAJ,MillerJH,SuzukiDT,LewontinRC,GelbartWM(2000)."Inversions".AnIntroductiontoGeneticAnalysis.7thEdition. ^XuJ,ZhuY(August2018)."Arapidinvitromethodtoflipbackthedouble-floxedinvertedopenreadingframeinaplasmid".BMCBiotechnology.18(1):52.doi:10.1186/s12896-018-0462-x.PMC 6119287.PMID 30170595. ^OberdoerfferP,OtipobyKL,MaruyamaM,RajewskyK(November2003)."UnidirectionalCre-mediatedgeneticinversioninmiceusingthemutantloxPpairlox66/lox71".NucleicAcidsResearch.31(22):140e–140.doi:10.1093/nar/gng140.PMC 275577.PMID 14602933. ^XuJ,ZhuY(August2018)."Arapidinvitromethodtoflipbackthedouble-floxedinvertedopenreadingframeinaplasmid".BMCBiotechnology.18(1):52.doi:10.1186/s12896-018-0462-x.PMC 6119287.PMID 30170595. ^GriffithsAJ,MillerJH,SuzukiDT,LewontinRC,GelbartWM(2000)."Translocations".AnIntroductiontoGeneticAnalysis(7th ed.). ^PugachEK,RichmondPA,AzofeifaJG,DowellRD,LeinwandLA(September2015)."ProlongedCreexpressiondrivenbytheα-myosinheavychainpromotercanbecardiotoxic".JournalofMolecularandCellularCardiology.86:54–61.doi:10.1016/j.yjmcc.2015.06.019.PMC 4558343.PMID 26141530. ^HayashiS,McMahonAP(April2002)."Efficientrecombinationindiversetissuesbyatamoxifen-inducibleformofCre:atoolfortemporallyregulatedgeneactivation/inactivationinthemouse".DevelopmentalBiology.244(2):305–18.doi:10.1006/dbio.2002.0597.PMID 11944939. ^HayashiS,McMahonAP(April2002)."Efficientrecombinationindiversetissuesbyatamoxifen-inducibleformofCre:atoolfortemporallyregulatedgeneactivation/inactivationinthemouse".DevelopmentalBiology.244(2):305–18.doi:10.1006/dbio.2002.0597.PMID 11944939. ^DanielianPS,MuccinoD,RowitchDH,MichaelSK,McMahonAP(December1998)."Modificationofgeneactivityinmouseembryosinuterobyatamoxifen-inducibleformofCrerecombinase".CurrentBiology.8(24):1323–6.doi:10.1016/s0960-9822(07)00562-3.PMID 9843687. ^Transgenesistechniques :principlesandprotocols.Clarke,AlanR.(2nd ed.).Totowa,NJ:HumanaPress.2002.ISBN 9781592591787.OCLC 50175106.{{citebook}}:CS1maint:others(link) ^Cancerandzebrafish :mechanisms,techniques,andmodels.Langenau,DavidM.Switzerland.ISBN 9783319306544.OCLC 949668674.{{citebook}}:CS1maint:others(link) ^KobayashiK,KameiY,KinoshitaM,CzernyT,TanakaM(January2013)."Aheat-inducibleCRE/LOXPgeneinductionsysteminmedaka".Genesis.51(1):59–67.doi:10.1002/dvg.22348.PMID 23019184. Retrievedfrom"https://en.wikipedia.org/w/index.php?title=Floxing&oldid=1084383571" Categories:DNAGeneticstechniquesHiddencategories:CS1maint:othersCS1:longvolumevalueArticleswithshortdescriptionShortdescriptionmatchesWikidata Navigationmenu Personaltools NotloggedinTalkContributionsCreateaccountLogin Namespaces ArticleTalk English Views ReadEditViewhistory More Search Navigation MainpageContentsCurrenteventsRandomarticleAboutWikipediaContactusDonate Contribute HelpLearntoeditCommunityportalRecentchangesUploadfile Tools WhatlinkshereRelatedchangesUploadfileSpecialpagesPermanentlinkPageinformationCitethispageWikidataitem Print/export DownloadasPDFPrintableversion Languages Addlinks