Second gene expression in bicistronic constructs using short synthetic intercistrons and viral IRES sequences

In this study, we describe the efficiency of second gene translation in bic istronic constructs containing either a short (36 bp) synthetic intercistro n or known internal ribosomal entry sites (IRES). Experiments were performe d using two different gene combinations: Herpes simplex virus-thymidine kin ase (HSV-TK) and neomycine (NEO) or human glucocerebrosidase (hGC) and a me thotrexate (MTX) resistant mutant dihydrofolate reductase (DHFR). We demons trate that upon transfection, second gene translation is efficient using ei ther an IRES or a 36-bp intercistron. Infection with retrovirus carrying th e TK and NEO genes linked via a 36-bp intercistron resulted in both G418(R) (NEO expression) and gancyclovir (GCV) sensitivity (TK expression), indica ting that both genes were expressed and thus that the genomic DNA and RNA o f this bicistronic construct were intact. Likewise, retrovirus carrying the hGC and mutant DHFR gene separated by a short intercistron was harvested f rom MTXR murine Psi CRE cells. However, infection of PA317 cells with this virus supernatant did not result in the presence of hGC enzyme activity in these murine cells. Proviral DNA and RNA analyses indicated that the hGC co ding region was lost from the original construct in the infected PA317 cell s. In contrast, retrovirus carrying the hGC and DHFR cDNAs was linked via a n IRES functioned as expected. Based on these results, we conclude that the efficiency of second gene translation using short synthetic intercistrons might prove useful in bicistronic constructs, depending on the gene combina tion used. (C) 1998 Elsevier Science B.V. All rights reserved.