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. 2005 Nov 30;46(2-3):69–78. doi: 10.1007/s10616-005-2834-z

Application of a Reversible Immortalization System for the Generation of Proliferation-Controlled Cell Lines

Tobias May 1, Werner Lindenmaier 1, Dagmar Wirth 1, Peter P Mueller 1,✉
PMCID: PMC3449712  PMID: 19003263

Abstract

To employ physiological mechanisms to control cell growth primary cells were reversibly immortalized using the SV40 TAg. The cells showed a fibroblast-like morphology. When the expression of the TAg was turned off, the cells arrested in the G0/G1 cell cycle phase. The cell culture could be kept for over 1 week in the proliferation-controlled state while the growth arrest remained fully reversible. The regulation was highly efficacious in that the arrested cell population did not spontaneously resume growth, suggesting that in the absence of the immortalizing gene expression endogenous growth-control mechanisms can keep these cells in a viable state for a prolonged time. Recombinant protein expression increased in growth-controlled cells when compared to conventionally cultured cells. Analysis of a secreted pharmaceutical protein revealed high product integrity without any signs of degradation. Therefore, it is feasible to apply genetic regulation of cell immortalization to obtain proliferation-controlled cell lines and this technique may be of interest to generate novel biotechnological producer cells.

Keywords: Conditional immortalization, EPO, Proliferation, Recombinant protein expression, Regulated cell growth

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Glossary

DMEM

Dulbecco’s modified Eagle’s medium

Dox

doxycycline

eGFP

enhanced green fluorescent protein

EPO

recombinant human erythropoietin

FCS

fetal calf serum

IRES

internal ribosomal binding site

LTR

long terminal repeat

MEF

murine embryonic fibroblast cell

neo

Neomycin phosphotransferase

TAg

SV40 virus large T-antigen

w.t.

wild type

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