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1、GenesandgenomesConstructionofmammalianartificialchromosomes:prospectsfordefininganoptimalcentromereDirkSchindelhauerSummaryTworeportshaveshownthatmammalianartificialchromosomes(MAC)canbeconstructedfromclonedhumancentromereDNAandtelomererepeats,provingtheprinc
2、iplethatchromosomescanformfromnakedDNAmoleculestransfectedintohumancells.TheMACsweremitoticallystable,lowcopynumberandboundantibodiesassociatedwithactivecentromeres.Asasteptowardsecond-generationMACs,yeastandbacterialcloningsystemswillhavetobeadaptedtoachieve
3、largeMACconstructshavingacentromere,twotelomeres,andgenomiccopiesofmammaliangenes.AvailableconstructiontechniquesarediscussedalongwithanewP1artificialchromosome(PAC)-derivedtelomerevector(pTAT)thatcanbejoinedtootherPACsinvitro,avoidingacloningstepduringwhichl
4、argerepetitivearraysoftenrearrange.ThePACsystemcanbeusedasaroutetofurtherdefinetheoptimalDNAelementsrequiredforefficientMACformation,toinvesti-gatetheexpressionofgenesonMACs,andpossiblytodevelopefficientMAC-deliveryprotocols.BioEssays1999;21:76–83.r1999JohnWi
5、ley&Sons,Inc.IntroductionintegratedatrandompositionsintothehostchromosomesofThesystematicintroductionofgenesintomammaliancellscellculturesandtransgenicanimals.Evenso,thechallengeshasbeenanelusivegoal.Initialtrieswerethwartedbytheofavoidingchangesofthehostgeno
6、mewhilestillmaintain-lackofcloningtoolsforverylargeDNA.SubsequentcDNA-ingstablysegregatinglowcopynumberDNAelementsduringbasedtechniquesprovedusefulinavarietyofapplicationsinmitosis,butnotmeiosis(forgermlineexclusioninhumans)culturedcellsandforsomeapplications
7、inanimalsandwerenotmet.humans;however,theirlackofcontrolovertheexactlevelTofulfilltheserequirements,mammalianartificialchromo-anddurationofgeneexpressionremainedalimitingfactor.Tosomes(MACs)areneeded.Wecoulddothisashasbeenpermitregulatedexpression,geneswouldnee
8、dtobeintro-donewithYACsinyeast—i.e.,justputagene,acentromere,ducedinaformreflectinggenesonnaturalchromosomes.andanoriginofreplicationtogetherandaddtelomerestotheOnlyrecentlyhavethefirstmode
