http://www.loc.gov/mods/v3 http://www.loc.gov/standards/mods/v3/mods-3-2.xsd
Transfer and expression of a retrovirus vector containing an interferon-alpha1 gene in interferon deficient cultured cells
The possibility of using retrovirus-mediated gene transfer to interferon-deficient cells as a model system for examining expression differences between members of the interferon-α gene family was investigated. A retrovirus vector containing an interferon-α1 gene was constricted and used to transfer this interferon gene to cultured cell lines bearing interferon gene deletions. An African green monkey genomic interferon-al gene was inserted into the cloning site of the retrovirus vector B2N and the orientation of the inserted gene was confirmed by DNA sequencing. B2N is a retrovirus vector designed for the expression of sequences under the control of an internal promoter supplied with the gene of interest. The interferon gene containing vector was packaged into infectious virions using the amphotropic packaging cell line ψ-CRIP. Recombinant virions were titered by infection of murine NIH/3T3 cells and titers ranged from 1 to 3.73x10^5 colony forming units/ml of virus supernatant. Delivery of correct proviral structure was confirmed by Southern blotting. Two interferon deficient cultured cell lines, the human chronic myelogenous leukemia K562 line, and the African green monkey Vero line, were successfully infected with the interferon gene containing vector as determined by Southern blotting. Clones of infected cells were isolated and analyzed for expression of vector sequences using cytopathic effect reduction bioassays, RNase protection assays, and Northern blotting. Expression of the transferred interferon gene was induced by infection with Bluetongue virus (BTV), a potent interferon inducing agent. Northern blotting showed expression of the vector at the RNA level. Quantitative changes in expression levels were seen in BTV-induced Vero cells. Attempts to demonstrate quantitative changes in expression over a time course after BTV induction showed variable expression, however. Cytopathic effect reduction assays showed no expression of biologically active interferon. These studies indicated that regulated expression of the interferon gene was altered by insertion of the gene into the retrovirus vector. Possible reasons for altered interferon expression include effects of the nearby viral promoter on interferon expression or effects of the host cell on expression patterns of the transferred gene.
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