Novel bicistronic retroviral vector expressing @entity2638 synthetase and the @entity21651 ( @entity21651 ) protects cells from XXXX -effluxed drugs and alkylating agents.

@entity2638 @entity5272 @entity21651 @entity9925 @entity19 @entity25808 @entity8310 @entity2041 @entity238
We have constructed two retroviral vectors, one expressing @entity21651 ( @entity21651 ) alone (SF91MRP) and the other expressing @entity21651 and @entity2638 synthetase (gamma-GCS), the rate-limiting enzyme of @entity2041 biosynthesis ( @entity25808 ). We have utilized the hybrid FMEV (Friend mink cell focus-forming/ @entity19 embryonic stem cell virus) backbone, previously shown to be efficient in early hematopoietic cells, even when coexpressing two distinct genes. In SF91GCS- @entity25808 , the cDNAs were combined via an internal ribosomal entry site (IRES) sequence from @entity9925 , resulting in a bicistronic mRNA produced via the long terminal repeat (LTR). Producer @entity238 -eco clones were established by trans-infection with @entity8310 virus glycoprotein (VSV-G)-pseudotyped retroviral supernatants. Drug-resistant producer clones were subsequently selected with antimony @entity5272 , a nonmutagenic @entity21651 substrate. By RNA slot-blot and transduction of 3T3 fibroblasts, titers of both SF91MRP and @entity25808 were found to be greater than 10(6) viral particles/ml. The correct viral integration in the genome was established by Southern blotting. By flow cytometry, both @entity21651 and bicistronic clones showed an increase in expression of the @entity21651 protein. The bicistronic producer clones, as well as 3T3 cells transduced with @entity25808 , presented an increase in intracellular @entity2041 levels, compared with the parental counterparts. Producer cells, 3T3 fibroblasts transduced with either SF91MRP or @entity25808 , and primary @entity19 myeloid progenitor cells transduced with @entity25808 were resistant to @entity21651 -effluxed drugs. However, only bicistronic producers, 3T3 fibroblasts transduced with @entity25808 , and primary @entity19 myeloid progenitor cells transduced with @entity25808 were also resistant to alkylating agents. We conclude that the retrovirus @entity25808 has features that make it a suitable vector to induce bone marrow resistance to multiple classes of chemotherapeutic agents. The strategy of coexpressing gamma-GCS and @entity21651 may help to design an effective in vivo selection for various clinical protocols of gene therapy.
