A versatile nonviral vector system for tetracycline-dependent one-step conditional induction of transgene expression.
In this study, we describe a novel self-contained, nonviral vector system for the rapid development of tetracycline (Tet)-inducible transgene expression systems in mammalian cell lines. To avoid multiple rounds of clonal selection for the establishment of stably transfected cell clones, as is necessary with conventional systems, we constructed a multicomplementary DNA(cDNA) expression vector that enables both one-step targeted genomic integration and conditional induction of transgene expression. This vector system consists of several modules including a Tet-inducible promoter directing the expression of a transgene and two Tet repressor expression units placed in tandem on a single vector. The cell clones, generated using a one-step phiC31 integrase-mediated chromosomal integration of the multi-cDNA expression construct, showed a stable and robust expression with high induction rates upon addition of doxycycline inducer in five different cell lines tested. By using this system, we show c-Src-induced cell transformation and anticancer cell therapy for this transformation in cultured fibroblast cells. The results show a rapid production and accumulation of target protein on addition of the inducer starting from extremely low background levels and reduction to background levels in a matter of days after the inducer was withdrawn from the culture medium.
Inoue
K
K
Department of Molecular Biology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.
Sone
T
T
Oneyama
C
C
Nishiumi
F
F
Kishine
H
H
Sasaki
Y
Y
Andoh
T
T
Okada
M
M
Chesnut
J D
JD
Imamoto
F
F
D002460:Cell Line; D002461:Cell Line, Transformed; D018076:DNA, Complementary; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D013752:Tetracycline / Q000494:pharmacology*; D019076:Transgenes
D018076:DNA, Complementary; D013752:Tetracycline