Multi-gene gateway clone design for expression of multiple heterologous genes in living cells: conditional gene expression at near physiological levels.
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-1alpha 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 (CPalpha1 and CPbeta2), 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.
Yahata
Kazuhide
K
Department of Molecular Biology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
Kishine
Hiroe
H
Sone
Takefumi
T
Sasaki
Yukari
Y
Hotta
Junko
J
Chesnut
Jonathan D
JD
Okabe
Masaru
M
Imamoto
Fumio
F
D003001:Cloning, Molecular / Q000379:methods*; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D010957:Plasmids / Q000235:genetics*; D011995:Recombination, Genetic / Q000235:genetics*