title: A quick and efficient method to generate mammalian stable cell lines based on a novel inducible alphavirus DNA/RNA layered system.
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.
authors.affiliation: 3P Biopharmaceuticals S.L., Polígono Mocholí, C/Mocholí 2, 31110, Noain, Spain.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Aranda
authors.forename: Jaione
authors.initials: J
authors.lastname: Bezunartea
authors.forename: Erkuden
authors.initials: E
authors.lastname: Casales
authors.forename: Juan R
authors.initials: JR
authors.lastname: Rodriguez-Madoz
authors.forename: Esther
authors.initials: E
authors.lastname: Larrea
authors.forename: Jesus
authors.initials: J
authors.lastname: Prieto
authors.forename: Cristian
authors.initials: C
authors.lastname: Smerdou
mesh_terms: D000524:Alphavirus / Q000235:genetics*; D000818:Animals; D018929:Cell Culture Techniques; D002460:Cell Line / Q000166:cytology / Q000378:metabolism* / Q000821:virology; D003001:Cloning, Molecular / Q000379:methods*; D006224:Cricetinae; D016207:Cytokines / Q000235:genetics; D004247:DNA / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D016678:Genome; D049452:Green Fluorescent Proteins / Q000235:genetics*; D056945:Hep G2 Cells; D006801:Humans; D053683:Oncostatin M / Q000235:genetics; D012313:RNA / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D019076:Transgenes
chemical_list: D016207:Cytokines; D011994:Recombinant Proteins; D053683:Oncostatin M; D049452:Green Fluorescent Proteins; D012313:RNA; D004247:DNA; C092073:cardiotrophin 1