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A quick and efficient method to generate mammalian stable cell lines based on a novel inducible alphavirus DNA/RNA layered system.
- Source :
-
Cellular and molecular life sciences : CMLS [Cell Mol Life Sci] 2014 Dec; Vol. 71 (23), pp. 4637-51. Date of Electronic Publication: 2014 May 03. - Publication Year :
- 2014
-
Abstract
- We report a new method to generate high-expressing mammalian cell lines in a quick and efficient way. For that purpose, we developed a master cell line (MCL) containing an inducible alphavirus vector expressing GFP integrated into the genome. In the MCL, recombinant RNA levels increased >4,600-fold after induction, due to a doxycycline-dependent RNA amplification loop. The MCL maintained inducibility and expression during 50 passages, being more efficient for protein expression than a conventional cell line. To generate new cell lines, mutant LoxP sites were inserted into the MCL, allowing transgene and selection gene exchange by Cre-directed recombination, leading to quick generation of inducible cell lines expressing proteins of therapeutic interest, like human cardiotrophin-1 and oncostatin-M at several mg/l/24 h. These proteins contained posttranslational modifications, showed bioactivity, and were efficiently purified. Remarkably, this system allowed production of toxic proteins, like oncostatin-M, since cells able to express it could be grown to the desired amount before induction. These cell lines were easily adapted to growth in suspension, making this methodology very attractive for therapeutic protein production.
- Subjects :
- Animals
Cell Culture Techniques
Cell Line cytology
Cell Line virology
Cricetinae
Cytokines genetics
DNA genetics
Genome
Hep G2 Cells
Humans
Oncostatin M genetics
RNA genetics
Recombinant Proteins genetics
Alphavirus genetics
Cell Line metabolism
Cloning, Molecular methods
Genetic Vectors genetics
Green Fluorescent Proteins genetics
Transgenes
Subjects
Details
- Language :
- English
- ISSN :
- 1420-9071
- Volume :
- 71
- Issue :
- 23
- Database :
- MEDLINE
- Journal :
- Cellular and molecular life sciences : CMLS
- Publication Type :
- Academic Journal
- Accession number :
- 24794511
- Full Text :
- https://doi.org/10.1007/s00018-014-1631-2