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Title: Multi-gene Gateway clone design for expression of multiple heterologous genes in living cells: Conditional gene expression at near physiological levels
Authors: Yahata, K ×
Kishine, H
Sone, T
Sasaki, Yutaka
Hotta, Junichi
Chesnut, JD
Okabe, M
Imamoto, F #
Issue Date: Jan-2005
Publisher: Elsevier BV
Series Title: Journal of Biotechnology vol:118 issue:2 pages:123-134
Abstract: Using Multisite Gateway five-DNA-fragment constructs vectors that enable expression of two tandemly situated cDNAs on a single plasmid were developed. Heterologous protein production in cells was achieved by modulating respective cDNA expression to pre-determined and different levels. Optimization of cDNA expression at near physiological protein levels was achieved using promoters from four cell cycle-dependent genes. In comparison with conventionally available promoters, EF-1 alpha or CMV, the promoters used in this study were able to modulate cDNA expression levels over a magnitude of approximately 10 or 100-fold, respectively. In transiently transfected cells, two different proteins (CP alpha 1 and CP beta 2), which form a heterodimer, each labeled with a different-colored fluorescent protein, were successfully synthesized at pre-determined levels from their respective cDNAs. The above vectors were designed to contain an FRT/Flp recombination site for integration onto chromosomes and for establishment of stable clones in HeLa cells by site-specific recombination. In the stable transformant cells produced only about 4% of the protein production levels measured in the transiently transformed cells. The biological significance of these observations is discussed. (C) 2005 Elsevier B.V. All rights reserved.
ISSN: 0168-1656
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Molecular Imaging and Photonics
× corresponding author
# (joint) last author

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