Cell Engineering Using Integrase and Recombinase Systems

Sone, Takefumi; Nishiumi, Fumiko; Yahata, Kazuhide; Sasaki, Yukari; Kishine, Hiroe; Andoh, Taichi; Inoue, Ken; Imamoto, Fumio; Thyagarajan, Bhaskar; Chesnut, Jonathan D.

doi:10.1002/9780470454923.ch21

Cited by 2 publications

(1 citation statement)

References 27 publications

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“…To mitigate the possibility of mutual transcriptional interference occurring between two of the same regulatory elements (EF-1α promoters), two tandem cHS4 insulators were inserted between the two reporter cDNAs (Yahata et al, 2007). In a similar experiment where cells transfected with the plasmid containing tandem three cDNA expression cassettes carrying H2B-SECFP, EB1-Venus and mCherry-β-tubulin, three fluorescently tagged proteins were produced at nearly equivalent levels by inserting an insulator cHS4 unit between the second and third downstream cDNAs (Sone et al, 2009) (Fig. 1B).…”

Section: Genomic Integration Sites In Helas3mentioning

confidence: 99%

Simultaneous Single Cell Stable Expression of 2-4 cDNAs in HeLaS3 Using .PHI.C31 Integrase System

Nishiumi

Sone

Kishine

et al. 2009

Cell Struct. Funct.

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ABSTRACT. An important consideration in the design of multigene delivery technology is the availability of suitable vectors to introduce multiple genes stably and stoichiometrically into living cells and co-express these genes efficiently. As a promising system for this purpose, we developed multi-cDNA expression constructs harboring two to three tandemly situated cDNAs in a single plasmid. The utility of this vector system is amplified by combining it with the φC31 recombinase system which mediates site-specific integration of the genes into naturally occurring chromosomal sequences. By analyzing 55 φC31-mediated integration events with five different constructs, each carrying one, two or three tandem cDNA expression cassettes, we identified 39 pseudo attP sites in the HeLaS3 chromosomes. All these sites share a common motif containing an inverted repeat and showing a similarity to the native φC31 attP. The 36 integration events represented 27 different pseudo attP sites, suggesting the possibility of duplicate integration of the multigene expression plasmids into different genomic loci in a single cell. We demonstrated successive introduction of two different multi-cDNA expression plasmids into definite chromosomal pseudo attP sites, attaining integration of four cDNAs of known genomic constitution at precise genomic loci of a single HeLaS3 cell. The expression levels of these several transgenes were enhanced and made equally stable and robust by inserting the cHS4 insulator between genes.

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Section: Genomic Integration Sites In Helas3mentioning

confidence: 99%

Simultaneous Single Cell Stable Expression of 2-4 cDNAs in HeLaS3 Using .PHI.C31 Integrase System

Nishiumi

Sone

Kishine

et al. 2009

Cell Struct. Funct.

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Methods for Constructing Clones for Protein Expression in Mammalian Cells

Sone

Imamoto

2011

Methods in Molecular Biology

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Multisite Gateway technology is a DNA cloning method based on in vitro site-specific recombination that is becoming increasingly popular because it allows quick and highly efficient assembly of multiple DNA fragments into a vector backbone. In the conventional Gateway Multisite strategy, cloning of multiple DNA fragments requires recombination of multiple entry clones with a single destination vector. The -limitation of this approach is that as the number of entry clones increases, the efficiency of the assembly reactions decreases due to difficulty in successfully recognizing multiple pairs of matched att signals simultaneously. To address this problem, we have devised methods to generate modular expression clones, modular entry clones, and modular destination vectors. These allow many DNA fragments to be -assembled stepwise into complex expression clones. We describe here how to construct these intermediate clones and vectors, and how to use these modules to construct expression clones comprising ten or more DNA -segments. These principles can be applied to make multicomponent DNAs for many applications.

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Cell Engineering Using Integrase and Recombinase Systems

Cited by 2 publications

References 27 publications

Simultaneous Single Cell Stable Expression of 2-4 cDNAs in HeLaS3 Using .PHI.C31 Integrase System

Simultaneous Single Cell Stable Expression of 2-4 cDNAs in HeLaS3 Using .PHI.C31 Integrase System

Methods for Constructing Clones for Protein Expression in Mammalian Cells

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