Investigation of promoter function in human and animal cells infected with human recombinant adenovirus expressing rotavirus antigen VP7sc.
Human adenovirus (Ad) vectors are being used increasingly for a variety of applications in vaccination and gene therapy. The ability of vectors to enter cells and the efficiency of promoters expressing the therapeutic gene or vaccine antigen are critical to the outcome of such experiments. To identify promoters which might be suitable for use under a variety of conditions we have investigated the expression of a rotavirus antigen, VP7sc, employing several commonly used promoters carried in E1-substituted Ad vectors both in cell types which support virus replication and in cells which do not. Although not all gene constructions were identical, wide variations in promoter function were evident even in human 293 cells which support virus replication. The simian virus type 40 (SV40) early and beta-actin promoters expressed poorly; the SV40 late promoter was somewhat better. The human IE94 cytomegalovirus (CMV) promoter and a modified Ad major late promoter were best, functioning equally well but with different kinetics. In other human cell lines the CMV promoter was more versatile, generally providing sustained expression at a significant level, in one case for at least 6 days. In addition, as mouse, rabbit and pig models of rotavirus infection are under investigation and VP7sc is a vaccine antigen, we also investigated the ability of the recombinant adenovirus to infect cells from these and other sources. VP7sc expression was detected in several heterologous cell types, illustrating the ubiquity of the human Ad receptor and the versatility of human Ad as vectors when suitable promoters are used.
Xu
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CSIRO Division of Biomolecular Engineering, NSW, Sydney, Australia.
Krougliak
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Prevec
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Graham
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Both
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GW
D000199:Actins / Q000235:genetics; D000260:Adenoviruses, Human / Q000235:genetics* / Q000502:physiology; D000818:Animals; D000956:Antigens, Viral / Q000096:biosynthesis* / Q000235:genetics; D001483:Base Sequence; D002213:Capsid / Q000096:biosynthesis* / Q000235:genetics; D036022:Capsid Proteins; D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics; D005347:Fibroblasts; D015967:Gene Expression Regulation, Viral / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D007668:Kidney; D008168:Lung; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012401:Rotavirus; D013539:Simian virus 40 / Q000235:genetics; D014779:Virus Replication
D000199:Actins; D000956:Antigens, Viral; D036022:Capsid Proteins; C050779:VP7 protein, Rotavirus