A gas-inducible expression system in HEK.EBNA cells applied to controlled proliferation studies by expression of p27(Kip1).
We describe an efficient inducible gene expression system in HEK.EBNA cells, a well-established cell system for the rapid transient expression of research-tool proteins. The transgene control system of choice is the novel acetaldehyde-inducible regulation (AIR) technology, which has been shown to modulate transgene levels following exposure of cells to acetaldehyde. For application in HEK.EBNA cells, AlcR transactivator plasmids were constructed and co-expressed with the secreted alkaline phosphatase (SEAP) gene under the control of a chimeric mammalian promoter (P(AIR)) for acetaldehyde-regulated expression. Several highly inducible transactivator cell lines were established. Adjustable transgene induction by gaseous acetaldehyde led to high induction levels and tight repression in transient expression trials and in stably transfected HEK.EBNA cell lines. Thus, the AIR technology can be used for inducible expression of any desired recombinant protein in HEK.EBNA cells. A possible application for inducible gene expression is a controlled proliferation strategy. Clonal HEK.EBNA cell lines, expressing the fungal transactivator protein AlcR, were engineered for gas-adjustable expression of the cell-cycle regulator p27(Kip1). We show that expression of p27(Kip1) via transient or stable transfection led to a G1-phase specific growth arrest of HEK.EBNA cells. Furthermore, production pools engineered for gas-adjustable expression of p27(Kip1) and constitutive expression of SEAP showed enhanced productive capacity.
Werner
Nicola Susann
NS
Novartis Institutes for BioMedical Research, Discovery Technologies/Biomolecules Production, WSJ 506.3.04, CH-4002 Basel, Switzerland.
Weber
Wilfried
W
Fussenegger
Martin
M
Geisse
Sabine
S
D000079:Acetaldehyde / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000235:genetics* / Q000378:metabolism; D019149:Bioreactors; D002460:Cell Line; D019309:Epstein-Barr Virus Nuclear Antigens / Q000378:metabolism; D016193:G1 Phase / Q000502:physiology*; D005786:Gene Expression Regulation; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000196:embryology; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism*; D019076:Transgenes / Q000502:physiology; D014764:Viral Proteins / Q000378:metabolism
C402641:EBNA-2 protein, Human herpesvirus 4; D019309:Epstein-Barr Virus Nuclear Antigens; D018809:Proliferating Cell Nuclear Antigen; D011994:Recombinant Proteins; D014764:Viral Proteins; C413374:p27 antigen; D000469:Alkaline Phosphatase; D000079:Acetaldehyde