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Eukaryotic expression system operating on the basis of Flp recombinase activity

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Wicher K. B., Jura J., Kowanetz M., Lorenowicz M., Rafalska I., Szybalski W., Bereta M.

Biotechnologia

1999

issue 1

144-152

Eukaryotic expression vectors are usually used for basic research and gene therapy. The major drawback of all vectors is a nonspecific leakage of the message which significantly hampers the use of vectors for the expression of cytotoxic proteins or enzymes. Production of proteins in eukariotic cells would be facilitated by the use of very strong inducible promoters that are well controlled. Such expression systems are presently not available because eukaryotic inducible promoters are usually weak. We designed eukaryotic inducible expression system in which strong expression of reporter gene was achieved. Our system is based on the activity of yeast site-specific recombinase Flp which recognizes two FRT sequences and inverses the orientation of the DNA fragment present between them. When placed in reverse orientation in relation to the promoter, a gene of interest may not be expressed, whereas inversion of such a gene gives full expression. We introduced the gene of interest between FRT sequences and used Flp for the inversion of the gene from reverse to forward orientation. This way, a strong constitutive promoter switches to the inducible mode while preserving its strong promoting activity.

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1 Biotechnologia

1 1999

1 Bereta M.

1 Jura J.

1 Kowanetz M.

1 Lorenowicz M.

1 Rafalska I.

1 Szybalski W.

1 Wicher K. B.

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Eukaryotic expression system operating on the basis of Flp recombinase activity