Internal ribosome entry site of Rhopalosiphum padi virus is functional in mammalian cells and has cryptic promoter activity in baculovirus-infected Sf21 cells.
OBJECTIVE
To substantiate the in vitro translational studies of a cross-kingdom, internal ribosome entry site (IRES), the 5 untranslated region of the Rhopalosiphum padi virus (RhPV), can function in mammalian cells and act as a shuttle IRES between insect cells and mammalian cells.


METHODS
Cytomegalovirus (CMV) promoter-based bicistronic mammalian cell expression vectors, either in plasmids or baculovirus vectors, were generated. Plasmid transient transfection and baculovirus transduction assays were performed to test whether the RhPV IRES can mediate translation activity in versatile mammalian cell lines.


RESULTS
Both plasmids and recombinant baculoviruses containing the CMV promoter and the RhPV IRES can mediate bicistronic gene expression in mammalian cells. However, in the CMV promoter containing recombinant baculovirus-infected insect Sf21 cells, only the second cistron gene expression was observed. Northern blot analysis and a promoterless assay demonstrated that the RhPV IRES exhibited cryptic promoter activity in baculovirus-infected insect cells.


CONCLUSIONS
RhPV IRES can mediate gene expression in both insect cells and mammalian cells, and this characteristic of the RhPV IRES will facilitate the development of a bicistronic baculovirus gene therapy vectors.
Wu
Yi-jane
YJ
Department of Bioscience Technology, Chung Yuan Christian University, Chungli 320, Taiwan, China.
Teng
Chao-yi
CY
Chen
Yu-jie
YJ
Chen
Seng-chi
SC
Chen
Ying-ju
YJ
Lin
Yi-ting
YT
Wu
Tzong-yuan
TY
D000818:Animals; D016367:Baculoviridae / Q000502:physiology*; D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics; D010849:Picornaviridae; D010850:Picornaviridae Infections / Q000473:pathology / Q000821:virology*; D010957:Plasmids / Q000235:genetics; D012270:Ribosomes / Q000648:ultrastructure*; D018411:Spodoptera; D014161:Transduction, Genetic; D014162:Transfection