Transgene Expression and Transposition Efficiency of Two-Component Sleeping Beauty Transposon Vector Systems Utilizing Plasmid or mRNA Encoding the Transposase

Publikation: Beitrag in FachzeitschriftArtikelForschungPeer-Review

Autorschaft

  • Natalie Tschorn
  • Yasemin van Heuvel
  • Jörn Stitz

Organisationseinheiten

Externe Organisationen

  • Technische Hochschule Köln
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Details

OriginalspracheEnglisch
Seiten (von - bis)1327-1335
Seitenumfang9
FachzeitschriftMolecular Biotechnology
Jahrgang65
Ausgabenummer8
Frühes Online-Datum22 Dez. 2022
PublikationsstatusVeröffentlicht - Aug. 2023

Abstract

The use of two-component transposon plasmid vector systems, namely, a transposase construct and a donor vector carrying the gene of interest (GOI) can accelerate the development of recombinant cell lines. However, the undesired stable transfection of the transposase construct and the sustained expression of the enzyme can cause genetic instability due to the re-mobilization of the previously transposed donor vectors. Using a Sleeping Beauty-derived vector system, we established three recombinant cell pools and demonstrate stable integration of the transposase construct and sustained expression of the transposase over a period of 48 days. To provide an alternative approach, transcripts of the transposase gene were generated in vitro and co-transfected with donor vector plasmid at different ratios and mediating high GOI copy number integrations and expression levels. We anticipate that the use of transposase mRNA will foster further improvements in future cell line development processes.

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Transgene Expression and Transposition Efficiency of Two-Component Sleeping Beauty Transposon Vector Systems Utilizing Plasmid or mRNA Encoding the Transposase. / Tschorn, Natalie; van Heuvel, Yasemin; Stitz, Jörn.
in: Molecular Biotechnology, Jahrgang 65, Nr. 8, 08.2023, S. 1327-1335.

Publikation: Beitrag in FachzeitschriftArtikelForschungPeer-Review

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title = "Transgene Expression and Transposition Efficiency of Two-Component Sleeping Beauty Transposon Vector Systems Utilizing Plasmid or mRNA Encoding the Transposase",
abstract = "The use of two-component transposon plasmid vector systems, namely, a transposase construct and a donor vector carrying the gene of interest (GOI) can accelerate the development of recombinant cell lines. However, the undesired stable transfection of the transposase construct and the sustained expression of the enzyme can cause genetic instability due to the re-mobilization of the previously transposed donor vectors. Using a Sleeping Beauty-derived vector system, we established three recombinant cell pools and demonstrate stable integration of the transposase construct and sustained expression of the transposase over a period of 48 days. To provide an alternative approach, transcripts of the transposase gene were generated in vitro and co-transfected with donor vector plasmid at different ratios and mediating high GOI copy number integrations and expression levels. We anticipate that the use of transposase mRNA will foster further improvements in future cell line development processes.",
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author = "Natalie Tschorn and {van Heuvel}, Yasemin and J{\"o}rn Stitz",
note = "Funding Information: We are indebted to Stefanie Schatz for expert technical support. This work was supported by the German Federal Ministry of Education and Research, funding program Forschung an Fachhochschulen, contract number 13FH242PX6 to JS. ",
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AU - Tschorn, Natalie

AU - van Heuvel, Yasemin

AU - Stitz, Jörn

N1 - Funding Information: We are indebted to Stefanie Schatz for expert technical support. This work was supported by the German Federal Ministry of Education and Research, funding program Forschung an Fachhochschulen, contract number 13FH242PX6 to JS.

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N2 - The use of two-component transposon plasmid vector systems, namely, a transposase construct and a donor vector carrying the gene of interest (GOI) can accelerate the development of recombinant cell lines. However, the undesired stable transfection of the transposase construct and the sustained expression of the enzyme can cause genetic instability due to the re-mobilization of the previously transposed donor vectors. Using a Sleeping Beauty-derived vector system, we established three recombinant cell pools and demonstrate stable integration of the transposase construct and sustained expression of the transposase over a period of 48 days. To provide an alternative approach, transcripts of the transposase gene were generated in vitro and co-transfected with donor vector plasmid at different ratios and mediating high GOI copy number integrations and expression levels. We anticipate that the use of transposase mRNA will foster further improvements in future cell line development processes.

AB - The use of two-component transposon plasmid vector systems, namely, a transposase construct and a donor vector carrying the gene of interest (GOI) can accelerate the development of recombinant cell lines. However, the undesired stable transfection of the transposase construct and the sustained expression of the enzyme can cause genetic instability due to the re-mobilization of the previously transposed donor vectors. Using a Sleeping Beauty-derived vector system, we established three recombinant cell pools and demonstrate stable integration of the transposase construct and sustained expression of the transposase over a period of 48 days. To provide an alternative approach, transcripts of the transposase gene were generated in vitro and co-transfected with donor vector plasmid at different ratios and mediating high GOI copy number integrations and expression levels. We anticipate that the use of transposase mRNA will foster further improvements in future cell line development processes.

KW - mRNA transfection

KW - Producer cell line

KW - Sleeping Beauty

KW - Transgene expression

KW - Transposition

KW - Transposon vector

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