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

Havenga, Menzo; Vogels, Ronald; Braakman, Eric; Kroos, Nathalie; Valerio, D.; Hagenbeek, Anton; Es, Helmuth H. G. van

doi:10.1016/s0378-1119(98)00453-3

Cited by 6 publications

(1 citation statement)

References 29 publications

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“…Shorter synthetic IRES elements have been reported, some as short as 30 nucleotides. 30,31 Moreover, shorter IRES elements may allow detailed investigation into the role of individual bases in the IRES element in the antifolate-induced translational regulation. Multiple genes can be placed in polycistronic vectors using shorter IRES elements to circumvent size restrictions in vectors (ie, DHFR-IRES-A-IRES-B-IRES-C-IRES-D).…”

Section: Discussionmentioning

confidence: 99%

Translational Modulation of Proteins Expressed from Bicistronic Vectors

et al. 2009

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Bicistronic vectors are useful tools for exogenous expression of two gene products from a single promoter element; however, reduced expression of protein from the second cistron compared with the first cistron is a common limitation to this approach. To overcome this limitation, we explored use of dihydrofolate reductase (DHFR) complementary DNA encoded in bicistronic vectors to induce a second protein of interest by methotrexate (MTX) treatment. Previous studies have demonstrated that levels of DHFR protein and DHFR fusion protein can be induced translationally following MTX treatment of cells. We demonstrated that in response to MTX treatment, DHFR partner protein in a bicistronic construct is induced for longer periods of time when compared with endogenous DHFR and DHFR fusion protein, in vitro and in vivo. Using rapamycin pretreatment followed by MTX treatment, we also devised a strategy to modulate levels of two proteins expressed from a bicistronic construct in a cap-independent manner. To our knowledge, this is the first report demonstrating that levels of proteins in DHFR-based bicistronic constructs can be induced and modulated using MTX and rapamycin treatment.

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Section: Discussionmentioning

confidence: 99%

Translational Modulation of Proteins Expressed from Bicistronic Vectors

et al. 2009

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Polyvalent Vectors for Coexpression of Multiple Genes

Müller¹,

Oumard²,

Wirth³

et al. 2001

Plasmids for Therapy and Vaccination

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IRES-Dependent Second Gene Expression Is Significantly Lower Than Cap-Dependent First Gene Expression in a Bicistronic Vector

et al. 2000

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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.

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

Cited by 6 publications

References 29 publications

Translational Modulation of Proteins Expressed from Bicistronic Vectors

Translational Modulation of Proteins Expressed from Bicistronic Vectors

Polyvalent Vectors for Coexpression of Multiple Genes

IRES-Dependent Second Gene Expression Is Significantly Lower Than Cap-Dependent First Gene Expression in a Bicistronic Vector

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