φC31 -Mediated cassette exchange in Sf9 insect cells for stable expression.
FACS
Sf9
cassette exchange
insect cell line
φC31
Journal
Biotechnology journal
ISSN: 1860-7314
Titre abrégé: Biotechnol J
Pays: Germany
ID NLM: 101265833
Informations de publication
Date de publication:
Jul 2023
Jul 2023
Historique:
revised:
08
03
2023
received:
03
11
2022
accepted:
29
03
2023
medline:
10
7
2023
pubmed:
6
4
2023
entrez:
5
4
2023
Statut:
ppublish
Résumé
Insect cells, especially Sf9 cells, are commonly used in biomanufacturing due to their advantages in high expression levels and post-translational modification. However, the development of stable expression cell lines via random integration tended to be unstable. Site-specific integration (SSI) is an alternative strategy. In this study, a φC31 -mediated cassette exchange system in Sf9 cells was established for SSI. The tagging cassette with the reporter gene egfp was randomly inserted into the cell genome. Potential platform cell lines were obtained by fluorescence-activated cell sorting (FACS) and single-cell cloning. Platform cell lines were selected by assessing the fluorescence expression, stability, and growth kinetics of cell lines. The selected platform cell lines were co-transfected with the φC31-containing plasmid and the targeting cassette. Green-fluorescence-negative clones were screened by hygromycin resistance and FACS. The resulting cell clones exhibited the expression properties of the platform cell lines. The rapid development of cell lines for the production of influenza subunit vaccines by the cassette exchange system demonstrated that the system constituted a versatile and reusable platform for the production of various recombinant proteins. Overall, the φC31-mediated cassette exchange system in Sf9 cells has the potential to facilitate and accelerate biologics development.
Identifiants
pubmed: 37016558
doi: 10.1002/biot.202200557
doi:
Substances chimiques
Integrases
EC 2.7.7.-
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
e2200557Subventions
Organisme : Shanghai Municipal Education Commission
ID : 2021Sci&Tech03-28
Organisme : Fundamental Research Funds for the Central Universities
ID : 222201211716
Informations de copyright
© 2023 Wiley-VCH GmbH.
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