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Construction of plasmid bicistronic vectors and their application for in vitro transfection

100%

Kucharzewska F., Zagorska A., Leja J., Jazwa A., Gozdecka M., Jozkowicz A., Dulak J.

Biotechnologia

2007

issue 3

66-81

Internal ribosome entry site (IRES) sequences, which stand for the basic element of cap 5'-independent translation, are currently widely used to coexpress heterologous genes from one plasmid. In this study construction of four bicistronic plasmids containing IRES and application of these vectors for transfection of in vitro cultured cells were described. The obtained data show that constructed bicistronic plasmids are very efficient in vitro in terms of simultaneous expression of fibroblast growth factor-4 (FGF-4) and vascular endothelial growth factor (VEGF) or one of these factors and green fluorescent protein (GFP) from one plasmid. Interestingly, expression of two genes, although simultaneous, is not equal. It has turned out that IRES-dependent mRNA translation is less efficient than cap 5'-dependent translation of the first gene, which should be taken into account during construction of bicistronic plasmids.

Regulation of gene expression in plasmid vectors: doxocyclin-dependent and hypoxia-regulated systems

84%

Golda S., Kucharzewska P., Cisowski J., Florczyk U., Zagorska A., Jazwa A., Loboda A., Jozkowicz A., Dulak J.

Biotechnologia

2007

issue 3

82-97

Regulation of gene expression in gene therapy is crucial for obtaining the therapeutic effects, thanks to limitation of transgene activity to the selected cells in a given time. In this paper we have focused on plasmid expression systems regulated by doxycycline or hypoxia. We have described in details the structure, regulatory elements and biological applications of 1) the modified, commercially available Tet-On system, expressing doxycycline-controlled b-galactosidase and, 2) hypoxia-activated FGF-4/VEGF expression plasmid containing the hypoxia responsive sequence. The presented expression systems can also be used in viral vectors, enabling not only regulated, but also high and long-term expression of transgenes.

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2 Biotechnologia

2 2007

2 Dulak J.

2 Jazwa A.

2 Jozkowicz A.

2 Zagorska A.

1 Cisowski J.

1 Florczyk U.

1 Golda S.

1 Gozdecka M.

1 Kucharzewska F.

1 Kucharzewska P.

1 Leja J.

1 Loboda A.

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Construction of plasmid bicistronic vectors and their application for in vitro transfection

Regulation of gene expression in plasmid vectors: doxocyclin-dependent and hypoxia-regulated systems