Multi-gene Gateway clone design for expression of multiple heterologous genes in living cells: Conditional gene expression at near physiological levels

被引:32
作者
Yahata, K
Kishine, H
Sone, T
Sasaki, Y
Hotta, J
Chesnut, JD
Okabe, M
Imamoto, F
机构
[1] Osaka Univ, Microbial Dis Res Inst, Dept Mol Biol, Suita, Osaka 5650871, Japan
[2] Invitrogen Corp, Carlsbad, CA USA
[3] Osaka Univ, Fac Pharmaceut Sci, Dept Expt Genome Res, Suita, Osaka 5650871, Japan
[4] Osaka Univ, Fac Pharmaceut Sci, Dept Bacterial Infect, Suita, Osaka 5650871, Japan
关键词
multisite Gateway cloning; multi-gene expression clone; simultaneous introduction of multiple cDNAs; low expression promoters; controllable expression of transgenes;
D O I
10.1016/j.jbiotec.2005.02.020
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
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.
引用
收藏
页码:123 / 134
页数:12
相关论文
共 22 条
[1]   SEQUENCE-ANALYSIS AND CHROMOSOMAL LOCALIZATION OF HUMAN CAP-Z - CONSERVED RESIDUES WITHIN THE ACTIN-BINDING DOMAIN MAY LINK CAP-Z TO GELSOLIN/SEVERIN AND PROFILIN PROTEIN FAMILIES [J].
BARRONCASELLA, EA ;
TORRES, MA ;
SCHERER, SW ;
HENG, HHQ ;
TSUI, LC ;
CASELLA, JF .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1995, 270 (37) :21472-21479
[2]   THE F-PLASMID CCDB PROTEIN INDUCES EFFICIENT ATP-DEPENDENT DNA CLEAVAGE BY GYRASE [J].
BERNARD, P ;
KEZDY, KE ;
VANMELDEREN, L ;
STEYAERT, J ;
WYNS, L ;
PATO, ML ;
HIGGINS, PN ;
COUTURIER, M .
JOURNAL OF MOLECULAR BIOLOGY, 1993, 234 (03) :534-541
[3]   A VERY STRONG ENHANCER IS LOCATED UPSTREAM OF AN IMMEDIATE EARLY GENE OF HUMAN CYTOMEGALO-VIRUS [J].
BOSHART, M ;
WEBER, F ;
JAHN, G ;
DORSCHHASLER, K ;
FLECKENSTEIN, B ;
SCHAFFNER, W .
CELL, 1985, 41 (02) :521-530
[4]   A monomeric red fluorescent protein [J].
Campbell, RE ;
Tour, O ;
Palmer, AE ;
Steinbach, PA ;
Baird, GS ;
Zacharias, DA ;
Tsien, RY .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2002, 99 (12) :7877-7882
[5]   A 5' ELEMENT OF THE CHICKEN BETA-GLOBIN DOMAIN SERVES AS AN INSULATOR IN HUMAN ERYTHROID-CELLS AND PROTECTS AGAINST POSITION EFFECT IN DROSOPHILA [J].
CHUNG, JH ;
WHITELEY, M ;
FELSENFELD, G .
CELL, 1993, 74 (03) :505-514
[6]   FACS-optimized mutants of the green fluorescent protein (GFP) [J].
Cormack, BP ;
Valdivia, RH ;
Falkow, S .
GENE, 1996, 173 (01) :33-38
[7]   Site-specific chromosomal integration in mammalian cells: Highly efficient CRE recombinase-mediated cassette exchange [J].
Feng, YQ ;
Seibler, J ;
Alami, R ;
Eisen, A ;
Westerman, KA ;
Leboulch, P ;
Fiering, S ;
Bouhassira, EE .
JOURNAL OF MOLECULAR BIOLOGY, 1999, 292 (04) :779-785
[8]  
FURUKAWA Y, 1994, J BIOL CHEM, V269, P26249
[9]   Insulators prevent transcriptional interference between two promoters in a double gene construct for transgenesis [J].
Hasegawa, K ;
Nakatsuji, N .
FEBS LETTERS, 2002, 520 (1-3) :47-52
[10]  
HWANG A, 1995, J BIOL CHEM, V270, P28419