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

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 expre...

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Autores principales: Tschorn, Natalie, van Heuvel, Yasemin, Stitz, Jörn
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer US 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10352156/
https://www.ncbi.nlm.nih.gov/pubmed/36547824
http://dx.doi.org/10.1007/s12033-022-00642-6
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author Tschorn, Natalie
van Heuvel, Yasemin
Stitz, Jörn
author_facet Tschorn, Natalie
van Heuvel, Yasemin
Stitz, Jörn
author_sort Tschorn, Natalie
collection PubMed
description 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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spelling pubmed-103521562023-07-19 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 Mol Biotechnol Original Paper 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. Springer US 2022-12-22 2023 /pmc/articles/PMC10352156/ /pubmed/36547824 http://dx.doi.org/10.1007/s12033-022-00642-6 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Original Paper
Tschorn, Natalie
van Heuvel, Yasemin
Stitz, Jörn
Transgene Expression and Transposition Efficiency of Two-Component Sleeping Beauty Transposon Vector Systems Utilizing Plasmid or mRNA Encoding the Transposase
title Transgene Expression and Transposition Efficiency of Two-Component Sleeping Beauty Transposon Vector Systems Utilizing Plasmid or mRNA Encoding the Transposase
title_full Transgene Expression and Transposition Efficiency of Two-Component Sleeping Beauty Transposon Vector Systems Utilizing Plasmid or mRNA Encoding the Transposase
title_fullStr Transgene Expression and Transposition Efficiency of Two-Component Sleeping Beauty Transposon Vector Systems Utilizing Plasmid or mRNA Encoding the Transposase
title_full_unstemmed Transgene Expression and Transposition Efficiency of Two-Component Sleeping Beauty Transposon Vector Systems Utilizing Plasmid or mRNA Encoding the Transposase
title_short Transgene Expression and Transposition Efficiency of Two-Component Sleeping Beauty Transposon Vector Systems Utilizing Plasmid or mRNA Encoding the Transposase
title_sort transgene expression and transposition efficiency of two-component sleeping beauty transposon vector systems utilizing plasmid or mrna encoding the transposase
topic Original Paper
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10352156/
https://www.ncbi.nlm.nih.gov/pubmed/36547824
http://dx.doi.org/10.1007/s12033-022-00642-6
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