We report the construction of two types of Rous sarcoma virus (RSV)-based replication-competent avian retrovirus vectors, IR1 and IR2 to express an exogenous gene at a very high level. In these vectors, the internal ribosomal entry site (IRES) derived from encephalomyocarditis virus (EMCV) was inserted between the env gene and an exogenous gene. The IR1 vector retains the splicing acceptor site that is present in the downstream of the env gene while the IR2 vector lacks it. Using a v-fos mutant (v-fos-CD3) as an example of exogenous genes, we show here that both IR1 and IR2 vectors expressed the gene product, CD3, at expression levels 5- and 8-fold higher than that of their parental vector without IRES, respectively. These vectors were moderately stable and kept a high-level expression of CD3 for at least three passages through the cells. Analysis of viral transcripts indicate that exogenous genes carried by both IR vectors were translated exclusively from the IRES that is present in all the species of the viral transcripts. High-level expression of exogenous genes was also observed in the case of the Hoxa-13 gene in the IR1 vector or the fra-2 gene in the IR2 vector, indicating that the extremely high-level expression characteristic of these vectors is applicable to several exogenous genes.
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