Controlled growth and production in mammalian cell culture

• Research unit: EU RFP
• Project number: 95.0683

Key words (English)

Mammalian cell culture; controlled proliferation; genetic engineering

Alternative project number (English)

EU project number: BIO4CT950291

Research programs (English)

EU-programme: 4. Frame Research Programme - 4.1 Biotechnology

Short description (English)

See abstract

Partners and International Organizations (English)

GBf, Braunschweig (D)

Abstract (English)

Based on internal ribosomal entry sites (IRES) of picornaviral origin we constructed a novel family of mammalian expression vectors. pQuattro vectors contain quattrocistronic artificial eukaryotic operons which link, in a single transcript, the simultaneous and coordinated as well as adjustable expression of up to three independent genes of interest to ˆa terminal neomycin (neo) resistance marker. Due to the strict genetic linkage of the transgenes and the terminal selection marker, this genetic configuration enables, by the selection on neomycin, multigene metabolic engineering of mammalian cells in a single step (one-step metabolic engineering). Furthermore, selection on the terminal cistron of multicistronic expression units enforces cocistronic expression of all upstream encoded genes and maximizes genetic integrity of the eucaryotic operon in stable mammalian cell lines, since clones harboring damaged multicistronic expression units become neomycin-sensitive and are automatically counterselected (autoselection).
Controlled proliferation bioprocesses how a great enhancement of heterologous protein production. This novel technology has been implemented here using a multicistronic expression unit encoding the product gene and cytostatic cell-cycle arresting gene (p27) under control of a single tetracycline-repressible (tet off) promoter. The strict genetic linkage of both genes allows the dissection of the production process into a non-productive phase (dicistronic expression unit repressed) followed by a proliferation-inhibited production phase (dicistronic expression unit induced) when the cells have reached an optimal cell density. Based on rapid degradation of the external repressible agents tetracycline (tet) and doxycycline (dox) in the cell culture medium, we developed a self-regulated process for transition from the growth phase to the production phase in a fashion which is dependent only on the starting cell population and the initial concentration of the tetracyclifles. With this process, no change in medium is required in order to accomplish the transition from growth to production phase. This Tet SWITCHprocess is readily adaptable to a variety of industrial processes designed for production of difficult-to-express Drotein pharmaceuticals.

References in databases (English)

Swiss Database: Euro-DB of the
State Secretariat for Education and Research
Hallwylstrasse 4
CH-3003 Berne, Switzerland
Tel. [phone]
Swiss Project-Number: 95.0683