BAC Modification through Serial or Simultaneous Use of CRELox Technology英文文献资料.docVIP

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BAC Modification through Serial or Simultaneous Use of CRELox Technology英文文献资料.doc

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BAC Modification through Serial or Simultaneous Use of CRELox Technology英文文献资料

HindawiPublishingCorporation JournalofBiomedicineandBiotechnology Volume2011,ArticleID924068,12pages doi:10.1155/2011/924068 ResearchArticle BACModi?cationthroughSerialorSimultaneousUseof CRE/LoxTechnology MarkParrish, JayUnruh, 1 1andRobbKrumlauf 1,2 1 2 KrumlaufLaboratory,StowersInstituteforMedicalResearch,100050thStreet,KansasCity,MO,64110,USA DepartmentofAnatomyandCellBiology,UniversityofKansasMedicalCenter,KansasCity,KS,66160,USA CorrespondenceshouldbeaddressedtoRobbKrumlauf,rek@stowers.org Received24July2010;Accepted6October2010 AcademicEditor:JamesBirchler Copyright?2011MarkParrishetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense, whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited. BacterialArti?cialChromosomes(BACs)arevitaltoolsinmousegenomicanalysesbecauseoftheirabilitytopropagatelarge inserts.Thesizeoftheseconstructs,however,preventstheuseofconventionalmolecularbiologytechniquesformodi?cation andmanipulation.TechniquessuchasrecombineeringandCre/Loxmethodologieshavethusbecomeheavilyrelieduponfor suchpurposes.Inthiswork,weinvestigatetheapplicabilityofLoxvariantsitesforserialand/orsimultaneousmanipulationsof BACs.WeshowthatLoxspacermutantsareveryspeci?c,andinvertedrepeatvariantsreduceLoxreactionratesthroughreducing thea?nityofCreforthesite,whileretainingsomefunctionality.Employingthesemethods,weproducedserialmodi?cations encompassingfourindependentchangeswhichgeneratedamouseHoxBBACwith?uorescentreporterproteinsinsertedinto fouradjacentHoxgenes.Wealsogeneratedspeci?c,simultaneousdeletionsusingcombinationsofspacervariantsandinverted repeatvariants.ThesetechniqueswillfacilitateBACmanipulationsandopenanewrepertoireofmethodsforBACandgenome manipulation. 1.Introduction fromthehostgenome[3,4].Inbothcases,expressionmust beextinguishedafterthetargetingeventtomaintainstability oftheresultantconstruct. Bacterial arti?cial chromosomes (BACs) are convenient vectors for ma