Details
| Original language | English |
|---|---|
| Pages (from-to) | 1327-1335 |
| Number of pages | 9 |
| Journal | Molecular Biotechnology |
| Volume | 65 |
| Issue number | 8 |
| Early online date | 22 Dec 2022 |
| Publication status | Published - 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.
Keywords
- mRNA transfection, Producer cell line, Sleeping Beauty, Transgene expression, Transposition, Transposon vector
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Biotechnology
- Chemical Engineering(all)
- Bioengineering
- Biochemistry, Genetics and Molecular Biology(all)
- Biochemistry
- Immunology and Microbiology(all)
- Applied Microbiology and Biotechnology
- Biochemistry, Genetics and Molecular Biology(all)
- Molecular Biology
Cite this
- Standard
- Harvard
- Apa
- Vancouver
- BibTeX
- RIS
In: Molecular Biotechnology, Vol. 65, No. 8, 08.2023, p. 1327-1335.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Transgene Expression and Transposition Efficiency of Two-Component Sleeping Beauty Transposon Vector Systems Utilizing Plasmid or mRNA Encoding the Transposase
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.
PY - 2023/8
Y1 - 2023/8
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
UR - http://www.scopus.com/inward/record.url?scp=85144639460&partnerID=8YFLogxK
U2 - 10.1007/s12033-022-00642-6
DO - 10.1007/s12033-022-00642-6
M3 - Article
C2 - 36547824
AN - SCOPUS:85144639460
VL - 65
SP - 1327
EP - 1335
JO - Molecular Biotechnology
JF - Molecular Biotechnology
SN - 1073-6085
IS - 8
ER -