A Multicassette Gateway Vector Set for High Throughput and Comparative Analyses in Ciona and Vertebrate Embryos

Roure, Agnès; Rothbächer, Ute; Robin, François; Kalmár, Éva; Ferone, Giustina; Lamy, C.; Missero, Caterina; Müller, Ferenc; Lemaire, Patrick

doi:10.1371/journal.pone.0000916

Cited by 120 publications

(122 citation statements)

References 51 publications

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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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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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“…A super core promoter, a synthetic intron (Arnold et al 2013), and a venus reporter gene (Roure et al 2007) were inserted between the KpnI and XbaI restriction sites of the pGL4.23 plasmid (Promega Catalog No. E8411).…”

Section: Cheq-seq Plasmid and Bait Designmentioning

confidence: 99%

Multiplex enhancer-reporter assays uncover unsophisticated TP53 enhancer logic

et al. 2016

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Transcription factors regulate their target genes by binding to regulatory regions in the genome. Although the binding preferences of TP53 are known, it remains unclear what distinguishes functional enhancers from nonfunctional binding. In addition, the genome is scattered with recognition sequences that remain unoccupied. Using two complementary techniques of multiplex enhancer-reporter assays, we discovered that functional enhancers could be discriminated from nonfunctional binding events by the occurrence of a single TP53 canonical motif. By combining machine learning with a meta-analysis of TP53 ChIP-seq data sets, we identified a core set of more than 1000 responsive enhancers in the human genome. This TP53 cistrome is invariably used between cell types and experimental conditions, whereas differences among experiments can be attributed to indirect nonfunctional binding events. Our data suggest that TP53 enhancers represent a class of unsophisticated cell-autonomous enhancers containing a single TP53 binding site, distinct from complex developmental enhancers that integrate signals from multiple transcription factors.

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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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A Multicassette Gateway Vector Set for High Throughput and Comparative Analyses in Ciona and Vertebrate Embryos

Cited by 120 publications

References 51 publications

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

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

Multiplex enhancer-reporter assays uncover unsophisticated TP53 enhancer logic

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

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