T7 RNA聚合酶与报告基因共递送表达研究.pdfVIP

Cytoexpressionofareportergenebyco-

deliveryofT7RNApolymeraseandT7promoter

sequencewithcationicliposomes

XiangGaoandLeafHuang*

LaboratoryofDrugTargeting,DepartmentofPharmacology,UniversityofPittsburgh

SchoolofMedicine,Pittsburgh,PA15261,USA

ReceivedMarch8,1993;AcceptedApril25,1993

ExpressionofbacteriophageT7RNApolymeraseinmliancellscanefficientlydrive

thetranscriptionofaforeigngenecontrolledbytheT7promoter(Elroy-Stelnetal.,Proc.

Natl.Acad.Sci.USA.86,6126-6130,1989).Wehavetestedthehypothesisthatpurified

T7RNApolymerasecanbeco-deliveredintomliancellstogetherwithareporter

gene(chloramphenicolacetyltransferase,CAT)controlledbytheT7promoter(pT7-

EMC-CAT)usingDC-cholcationicliposomes.Indeed,significantlevelofCATactivitywas

observedinhumanlungadenocarcinoma(A549-1)cellswhichhadbeenincubatedwith

acomplexofT7RNApolymerase,pT7-EMC-CATandDC-cholcationicliposomes.

TheexpressionwasspecificinthatT3RNApolymerasecouldnotrecetheT7RNA

polymerase,andthatco-deliveredT7RNApolymerasedidnotenhancetheexpression

ofaCATgenecontrolledbytheSV40earlypromoter.Thesystemwasoptimizedin

termsofenzyme,andliposomeconcentrations.Timecourseexperimentindicated

thattheexpressionoftheT7systemwasabout8-10hourssoonerthantheSV40system,

consistentwiththenotionthatT7RNApolymerasedoesnotenterintothenucleusand

thetranscriptiontakesceinthecytosmofthetransfectedcells.Theexpressionof

theT7systemwastransient;itdeclinedafter30hoursposttransfection,probablydue

toturnoverofthephageenzymeinthemliancells.Theexpressionsystem

describedhereshouldbeusefulforgenetransferexperimentswhichrequireafastbut

transientexpressionofaforeigngene.