Development of an All-in-One Lentiviral Vector System Based on the Original TetR for the Easy Generation of Tet-ON Cell Lines 英文参考文献.docVIP

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Development of an All-in-One Lentiviral Vector System Based on the Original TetR for the Easy Generation of Tet-ON Cell Lines 英文参考文献.doc

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Development of an All-in-One Lentiviral Vector System Based on the Original TetR for the Easy Generation of Tet-ON Cell Lines 英文参考文献

DevelopmentofanAll-in-OneLentiviralVectorSystem BasedontheOriginalTetRfortheEasyGenerationof Tet-ONCellLines KarimBenabdellah¤,Marie′nCobo¤,PilarMun?oz¤,MiguelG.Toscano¤,FranciscoMartin*¤ AndalusianStemCellBank,CentrodeInvestigacionesBiome′dicas, UniversidaddeGranada,ParqueTecnolo′gicoCienciasdelaSalud,Granada,Spain Abstract Lentiviralvectors(LVs)areconsideredoneofthemostpromisingvehiclestoefficientlydelivergeneticinformationforbasic researchandgenetherapyapproaches.CombiningLVswithdrug-inducibleexpressionsystemsshouldallowtightcontrol oftransgeneexpressionwithminimalsideeffectonrelevanttargetcells.Anewdoxycycline-regulatedsystembasedonthe originalTetRrepressorwasdevelopedin1998asanalternativetotheTetR-VP16chimeras(tTAandrtTA)toavoidsecondary effects due to theexpression of transactivator domains. However, previously described TetR-based systems required cell cloning and/or antibiotic selection of tetracycline-responsive cells in order to achieve good regulation. In the present manuscriptwehaveconstructedadualTet-ONsystembasedontwolentiviralvectors,oneexpressingtheTetRthroughthe spleen focus forming virus (SFFV) promoter (STetR) and a second expressing eGFP through the regulatable CMV-TetO promoter (CTetOE). Using these vectors we have demonstrated that the TetR repressor, contrary to the reverse transactivator (rtTA), can be expressed in excess to bind and modulate a high number of TetO operons. We have also showedthatthisdualvectorsystemcangenerateregulatablebulkcelllines(expressinghighlevelsofTetR)thatareableto modulatetransgeneexpressioneitherbyvaryingdoxycyclineconcentrationand/orbyvaryingtheamountofCTetOEvector genomespercell.Basedontheseresultswehavedevelopedanewall-in-onelentiviralvector(CEST)drivingtheexpression ofTetRthroughtheSFFVpromoterandtheexpressionofeGFPthroughthedoxycycline-responsiveCMV-TetOoperon.This vector efficiently produced Tet-ON regulatable immortalized (293T) and primary (human mesenchymal stem cells and human primary fibroblasts) c