High-titer bicistronic retroviral vectors employing foot-and-mouth disease virus internal ribosome entry site.
Bicistronic retroviral vectors were constructed containing the foot-and-mouth disease virus (FMDV) internal ribosome entry site (IRES) followed by the coding region of beta-galactosidase (beta-gal) or therapeutic genes, with the selectable neomycin phosphotransferase gene under the control of the viral long terminal repeat (LTR) promoter. LNFX, a vector with a multiple cloning site 3' to foot-and-mouth disease virus IRES, was used to construct vectors encoding rat erythropoietin (EP), rat granulocyte colony-stimulating factor (G-CSF), human adenosine deaminase (ADA) and beta-gal. In transduced primary rat vascular smooth muscle cells the cytokines were expressed at high levels, similar to those obtained from vectors employing the viral LTR promoter. LNFZ, a vector encoding beta-gal, had a 10-fold increase in titer over that of LNPoZ, a comparable vector containing the poliovirus (Po) internal ribosome entry site. Primary canine vascular smooth muscle cells infected with LNFZ and LNPoZ expressed similar activities of beta-gal and neomycin phosphotransferase (NPT). Overall, these vectors had titers between 10(6) and 2 x 10(7) c.f.u./ml, indicating that foot-and-mouth disease virus IRES provides high-titer bicistronic vectors with high-level two gene expression.
Ramesh
N
N
Department of Pediatrics, University of Washington School of Medicine Seattle 98195, USA.
Kim
S T
ST
Wei
M Q
MQ
Khalighi
M
M
Osborne
W R
WR
D016475:3T3 Cells; D000243:Adenosine Deaminase / Q000235:genetics; D000818:Animals; D005537:Aphthovirus / Q000235:genetics*; D002478:Cells, Cultured; D004285:Dogs; D004921:Erythropoietin / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D016179:Granulocyte Colony-Stimulating Factor / Q000235:genetics; D006801:Humans; D051379:Mice; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism; D017955:Poliovirus / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D051381:Rats; D012190:Retroviridae / Q000235:genetics* / Q000254:growth & development; D012270:Ribosomes; D014161:Transduction, Genetic; D001616:beta-Galactosidase / Q000235:genetics
D012367:RNA, Viral; D004921:Erythropoietin; D016179:Granulocyte Colony-Stimulating Factor; D001616:beta-Galactosidase; D000243:Adenosine Deaminase