Adenovirus vector designed for expression of toxic proteins.
To construct recombinant adenoviruses expressing biologically active proteins may be impossible, or result in a significant reduction in virus yield, if the protein expressed has an inhibitory effect on virus replication or cellular growth. To overcome this problem, we previously designed adenovirus vectors expressing foreign proteins from inducible promoters. However, during our work with a replication-deficient virus expressing the ASF/SF2 splicing factor from a progesterone antagonist-inducible gene cassette, we discovered that ASF/SF2 was expressed at a significant level in the 293 producer cell line, even in the absence of inducer. 293 cells code for adenovirus E1A and E1B proteins and thus support the growth of E1-deficient adenoviruses. Here we show that this background ASF/SF2 expression results from a low level of E1A-mediated transactivation of the basal promoter driving transgene expression. To overcome the problem of leaky expression, we reconstructed a novel gene cassette that combines an inducible promoter and a Lac repressor protein-based block to reduce transcriptional elongation. We show that this novel vector system dramatically reduced background transgene expression and therefore should be useful for the rescue and propagation of high-titer stocks of recombinant adenoviruses expressing toxic proteins.
Edholm
D
D
Department of Medical Biochemistry and Microbiology, BMC, Uppsala University, SE-751 23 Uppsala, Sweden.
Molin
M
M
Bajak
E
E
Akusjärvi
G
G
D000256:Adenoviridae / Q000235:genetics* / Q000502:physiology; D017500:Adenovirus E1A Proteins / Q000378:metabolism*; D002460:Cell Line; D005822:Genetic Vectors; D009687:Nuclear Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D050783:Proto-Oncogene Proteins c-ets; D012326:RNA Splicing; D016601:RNA-Binding Proteins; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D000068103:Serine-Arginine Splicing Factors; D015534:Trans-Activators; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014779:Virus Replication
D017500:Adenovirus E1A Proteins; C079853:ETV4 protein, human; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D000068103:Serine-Arginine Splicing Factors