Expression of Transgenes Targeted to the Gt(ROSA)26Sor Locus Is Orientation Dependent 英文参考文献.docVIP

ExpressionofTransgenesTargetedtotheGt(ROSA)26Sor LocusIsOrientationDependent DouglasStrathdee1,2*,HelenIbbotson2,SethG.N.Grant1,2 1TheWellcomeTrustSangerInstitute,WellcomeTrustGenomeCampus,Hinxton,Cambridge,UnitedKingdom,2CentreforNeuroscienceResearch, UniversityofEdinburgh,Edinburgh,Scotland Background.Targetingtransgenestoachosenlocationinthegenomehasanumberofadvantages.AsinglecopyoftheDNA construct can be inserted by targeting into regions of chromatin that allow the desired developmental and tissue-specific expression of the transgene. Methodology. In order to develop a reliable system for reproducibly expressing trangenes it wasdecidedtoinsertconstructsattheGt(ROSA)26Sorlocus.Acytomegalovirus(CMV)promoterwasusedtodriveexpression of the Tetracycline (tet) transcriptional activator, rtTA2s-M2, and test the effectiveness of using the ROSA26 locus to allow transgene expression. The tet operator construct was inserted into one allele of ROSA26 and a tet responder construct controlling expression of EGFP was inserted into the other allele. Conclusions. Expression of the targeted transgenes was shown to be affected by both the presence of selectable marker cassettes and by the orientation of the transgenes with respect to the endogenous ROSA26 promoter. These results suggest that transcriptional interference from the endogenous gene promoter or from promoters in the selectable marker cassettes may be affecting transgene expression at thelocus.Additionallywehavebeenabletodeterminetheoptimalorientationfortransgeneexpression atthe ROSA26 locus. Citation: StrathdeeD,IbbotsonH,GrantSGN(2006)ExpressionofTransgenesTargetedtotheGt(ROSA)26SorLocusIsOrientationDependent.PLoS ONE1(1):e4.doi:10.1371/journal.pone.0000004 INTRODUCTION a single copy is be introduced which avoids problems associated withalargemulticopyarray. Spatial and temporal control of gene expression represents an extremelypowerfultoolfortheanalysisofgenefunctionandthe eventsunderlyingcomplexbiological