Multi-gene gateway clone design for expression of multiple heterologous genes in living cells: Modular construction of multiple cDNA expression elements using recombinant cloning

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

doi:10.1016/j.jbiotec.2008.06.006

Cited by 22 publications

(21 citation statements)

References 20 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%

“…10,12 The expression clones pBKT1, pBKT2 and pBKT3 were constructed by stepwise LR and BP reactions with one destination vector and six or eight types of entry clones (Figure 1b, Supplementary Table 1). Construction of expression clones harboring c-Src cDNA and Cbp cDNA, pBKT2-c-Src and pBKT2-Cbp, respectively, was carried out by an LR reaction with one destination vector and nine types of entry clones shown in Supplementary Tetracycline-inducible system for versatile use K Inoue et al PCR of pEF5/FRT/V5-DEST (Invitrogen Corp.).…”

Section: Plasmid Constructionmentioning

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: Plasmid Constructionmentioning

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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“…The in vivo assembly methods described to date have proceeded with efficiencies that are simply too low (≤10 2 colonies) to be useful for the one-pot assembly of collections of pathways. Even after modification and optimization, most in vitro assembly techniques based on restriction digestion and ligation or on enzymatic recombination typically display profound losses in cloning efficiency for ≥3 fragments (≤10 3 colonies per reaction) (37,38). Several particularly efficient in vitro approaches (8), notably in vitro recombination (12)(13)(14), could theoretically generate libraries of ∼10 4 .…”

Section: Discussionmentioning

confidence: 99%

Reiterative Recombination for the in vivo assembly of libraries of multigene pathways

Wingler

Cornish

2011

Proc. Natl. Acad. Sci. U.S.A.

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The increasing sophistication of synthetic biology is creating a demand for robust, broadly accessible methodology for constructing multigene pathways inside of the cell. Due to the difficulty of rationally designing pathways that function as desired in vivo, there is a further need to assemble libraries of pathways in parallel, in order to facilitate the combinatorial optimization of performance. While some in vitro DNA assembly methods can theoretically make libraries of pathways, these techniques are resource intensive and inherently require additional techniques to move the DNA back into cells. All previously reported in vivo assembly techniques have been low yielding, generating only tens to hundreds of constructs at a time. Here, we develop “Reiterative Recombination,” a robust method for building multigene pathways directly in the yeast chromosome. Due to its use of endonuclease-induced homologous recombination in conjunction with recyclable markers, Reiterative Recombination provides a highly efficient, technically simple strategy for sequentially assembling an indefinite number of DNA constructs at a defined locus. In this work, we describe the design and construction of the first Reiterative Recombination system in Saccharomyces cerevisiae , and we show that it can be used to assemble multigene constructs. We further demonstrate that Reiterative Recombination can construct large mock libraries of at least 10 4 biosynthetic pathways. We anticipate that our system’s simplicity and high efficiency will make it a broadly accessible technology for pathway construction and render it a valuable tool for optimizing pathways in vivo.

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“…One improvement to the current strategy would be the delivery of multiple reprogramming factors within the context of a single vector. Construction of multiple functional DNA elements in tandem on a single plasmid has been performed by stepwise Gateway recombination reactions using Multisite Gateway technology (Life Technologies, Carlsbad, USA) (Cheo et al, 2004;Inoue et al, 2009;Sasaki et al, 2004;Sone and Imamoto, 2011;Sone et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

Generation of iPS Cells Using a BacMam Multigene Expression System

Takata

Kishine

Sone

et al. 2011

Cell Struct. Funct.

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Multi-gene gateway clone design for expression of multiple heterologous genes in living cells: Modular construction of multiple cDNA expression elements using recombinant cloning

Cited by 22 publications

References 20 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

Reiterative Recombination for the in vivo assembly of libraries of multigene pathways

Generation of iPS Cells Using a BacMam Multigene Expression System

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