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

Yahata, Kazuhide; Kishine, Hiroe; Sone, Takefumi; Sasaki, Yukari; Hotta, Junko; Chesnut, Jonathan D.; Okabe, Masaru; Imamoto, Fumio

doi:10.1016/j.jbiotec.2005.02.020

Cited by 36 publications

(38 citation statements)

References 21 publications

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“…[8][9][10][11][12] Construction of multiple functional DNA elements in tandem on a single plasmid was performed by stepwise LR and BP reactions as indicated in Figure 1b. All functional elements required for Tet regulation were placed on one contiguous DNA fragment of 12 340 bp between att1 and att2 Gateway recombination signals.…”

Section: Resultsmentioning

confidence: 99%

“…For this purpose, two full-length cDNAs encoding TetR were cloned downstream from TetO 2 -controlling GOI on a single plasmid (Figure 1), using Multisite Gateway technology [8][9][10][11][12] to assemble 11 DNA fragments. Using a fC31 integrase-mediated recombination system, we constructed stably transfected cells carrying the Tet-inducible expression cassette in a onestep procedure.…”

Section: Discussionmentioning

confidence: 99%

“…An explanation for this could be that three tandem strong CMV promoters situated closely to each other on a vector may lead to a stronger interference between the promoters, although the insulators exist between them, as the insulator effect is shown to be dependent on the strength of the promoters. 11 Regarding the configuration of the expression elements in pBKT2 (Figure 1a), two cHS4 insulators situated between two different promoters are necessary for a well-balanced expression of those three cDNA units.…”

Section: Discussionmentioning

confidence: 99%

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A versatile nonviral vector system for tetracycline-dependent one-step conditional induction of transgene expression

et al. 2009

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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 fC31 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.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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A versatile nonviral vector system for tetracycline-dependent one-step conditional induction of transgene expression

et al. 2009

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“…On the other hand, such a negative effect was not observed for the multisite Gateway vector R4pGWB series, which has attB sequences in the transcribed region, suggesting that the differences in vector systems and/or attB sequences between the pDEST and R4pGWB series may be involved in the negative effect of the attB sequences. Although studies in several eukaryotes have reported that the additional sequences from the Gateway system did not interfere with the biological activity of a cloned fragment, including subcellular localization or tissue specificity, there has been no description about any effect on gene expression (Curtis and Grossniklaus 2003;Nakagawa et al 2007a, b;Roure et al 2007;Yahata et al 2005). Our results demonstrated that the Gateway linker sequences did not interfere with expected biological function, but could reduce gene expression depending on vector systems.…”

Section: Discussionmentioning

confidence: 49%

Novel vector systems to accelerate functional analysis of transcription factors using chimeric repressor gene-silencing technology (CRES-T)

Oshima

Mitsuda

Nakata

et al. 2011

Plant Biotechnology

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Chimeric repressor gene-silencing technology (CRES-T) is a powerful tool that has recently been developed for the functional analysis of plant transcription factors and for the genetic manipulation of plant traits. For CRES-T, a transcription factor is converted to a strong repressor by fusion with an SRDX repression domain, which is then expressed in plants to induce a loss-of-function phenotype. However, the traditional CRES-T vectors are inconvenient for gene cloning and promoter exchange. In this study, we developed new CRES-T vectors that are efficient and convenient to use by employing the Gateway system, a new vector backbone and a terminator derived from the heat shock protein 18.2 (HSP) gene. Our test experiments revealed that the CRES-T vector containing the Gateway linker sequence within the transcribed region showed reduced efficiency of CRES-T when compared with the traditional CRES-T vector. However, the HSP terminator compensated for the negative effect of the Gateway sequence and improved the efficiency of CRES-T in all cases tested and resulted in the highest efficiency achieved to date. We found that the HSP terminator increased transcription efficiency or transcript stability; in contrast, these factors were negatively affected by the Gateway linker sequence in our vector system. We, therefore, propose that the appropriate CRES-T vector should be chosen depending on situations and purposes.

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“…However, if the expression of a cDNA under a particular promoter is intended, the cDNA and the promoter should be connected first and then cloned into an entry vector, because only a single DNA fragment can be cloned into existing Gateway-compatible binary vector systems, including ImpGWB by the Gateway LR reaction. In this study, we developed a series of novel vectors, R4pGWB, in which the attR1 site of the ImpGWBs was replaced with the attR4 sequence, 12,13) which enables one-step tripartite LR reaction among the vector, a promoter, and a cDNA.…”

mentioning

confidence: 99%

Development of R4 Gateway Binary Vectors (R4pGWB) Enabling High-Throughput Promoter Swapping for Plant Research

Nakagawa

Nakamura

Tanaka

et al. 2008

Bioscience, Biotechnology, and Biochemistry

125

117

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We developed a new series of Gateway binary vectors, R4pGWBs, that are plant transformation vectors designed for one-step construction of chimeric genes between any promoter and any cDNA. The structure of R4pGWBs is almost the same as the promoterless type of improved pGWBs (ImpGWBs), except that the attR1 site is replaced with attR4, which enables tripartite recombination of these vectors with promoter-and cDNA-entry clones. While ImpGWBs are suitable for promoter analysis and constitutive expression of cDNAs in higher plants, R4pGWBs have a great advantage in expressing a cDNA under the regulation of desired promoters.

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

Cited by 36 publications

References 21 publications

A versatile nonviral vector system for tetracycline-dependent one-step conditional induction of transgene expression

A versatile nonviral vector system for tetracycline-dependent one-step conditional induction of transgene expression

Novel vector systems to accelerate functional analysis of transcription factors using chimeric repressor gene-silencing technology (CRES-T)

Development of R4 Gateway Binary Vectors (R4pGWB) Enabling High-Throughput Promoter Swapping for Plant Research

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