IRES-dependent second gene expression is significantly lower than cap-dependent first gene expression in a bicistronic vector.
The internal ribosome entry site (IRES) has been widely used to coexpress heterologous gene products by a message from a single promoter. However, little is known about the efficiency of IRES-dependent second gene expression in comparison with that of first gene expression. This study was undertaken to characterize the relative expression of IRES-dependent second gene in a bicistronic vector, which was derived from the 5' untranslated regions of the encephalomyocarditis virus (EMCV). IRES-dependent second gene expression was compared with cap-dependent first gene expression in several cultured cell lines and in mouse liver in vivo. The expression of the IRES-dependent second gene ranged from 6 to 100% (in most cases between 20 and 50%) that of the first gene. Second gene expression in a plasmid without the IRES was 0.1-0.8% (with some exceptions) that of the first gene. These findings have important implications for the use of IRES, i.e., care should be taken regarding the decreased capacity of IRES-dependent downstream gene expression as well as in determining which gene should be positioned as the first or second gene in a bicistronic vector.
Mizuguchi
H
H
Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, Tokyo, Japan. mizuguch@nihs.go.jp
Xu
Z
Z
Ishii-Watabe
A
A
Uchida
E
E
Hayakawa
T
T
D020121:5' Untranslated Regions; D000469:Alkaline Phosphatase / Q000235:genetics; D000818:Animals; D016466:CHO Cells; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D006224:Cricetinae; D004686:Encephalomyocarditis virus / Q000235:genetics; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005796:Genes / Q000235:genetics; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D007739:L Cells; D008099:Liver / Q000378:metabolism; D008156:Luciferases / Q000235:genetics; D051379:Mice; D010957:Plasmids / Q000235:genetics; D012315:RNA Caps / Q000235:genetics
D020121:5' Untranslated Regions; D012315:RNA Caps; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase; D000469:Alkaline Phosphatase