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

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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.

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Abbreviations

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

References

  • S.H. Ali J.A. DeCaprio (2001) ArticleTitleCellular transformation by SV40 large T antigen: interaction with host proteins Semin. Cancer Biol. 11 15–23 Occurrence Handle10.1006/scbi.2000.0342 Occurrence Handle11243895 Occurrence Handle1:CAS:528:DC%2BD3MXhslSntLc%3D

    Article  PubMed  CAS  Google Scholar 

  • D.C. Andersen L. Krummen (2002) ArticleTitleRecombinant protein expression for therapeutic applications Curr. Opin. Biotechnol. 13 117–123 Occurrence Handle10.1016/S0958-1669(02)00300-2 Occurrence Handle11950561 Occurrence Handle1:CAS:528:DC%2BD38Xis1Clt7s%3D

    Article  PubMed  CAS  Google Scholar 

  • U. Baron S. Freundlieb M. Gossen H. Bujard (1995) ArticleTitleCo-regulation of two gene activities by tetracycline via a bidirectional promoter Nucleic Acids Res. 23 3605–3606 Occurrence Handle7567477 Occurrence Handle1:CAS:528:DyaK2MXotlKgtr4%3D

    PubMed  CAS  Google Scholar 

  • J.X. Bi J. Shuttleworth M. Al-Rubeai (2004) ArticleTitleUncoupling of cell growth and proliferation results in enhancement of productivity in p21CIP1-arrested CHO cells Biotechnol. Bioeng. 85 741–749 Occurrence Handle10.1002/bit.20025 Occurrence Handle14991652 Occurrence Handle1:CAS:528:DC%2BD2cXitV2gu7o%3D

    Article  PubMed  CAS  Google Scholar 

  • D. Chilov C. Fux H. Joch M. Fussenegger (2003) ArticleTitleIdentification of a novel proliferation-inducing determinant using lentiviral expression cloning Nucleic Acids Res. 31 1–7 Occurrence Handle10.1093/nar/gng115

    Article  Google Scholar 

  • F. Colbère-Garapin F. Horodniceanu P. Khourilsky A.C. Garapin (1981) ArticleTitleA new dominant hybrid selection marker for higher eucaryotic cells J. Mol. Biol. 150 1–13 Occurrence Handle6271971

    PubMed  Google Scholar 

  • J. Davies L. Jiang L.Z. Pan M.J. LaBarre D. Anderson M. Reff (2001) ArticleTitleExpression of GnTIII in a recombinant anti-CD20 CHO production cell line: Expression of antibodies with altered glycoforms leads to an increase in ADCC through higher affinity for FC gamma RIII Biotechnol. Bioeng. 74 288–294 Occurrence Handle10.1002/bit.1119 Occurrence Handle11410853 Occurrence Handle1:CAS:528:DC%2BD3MXlt1Gisr0%3D

    Article  PubMed  CAS  Google Scholar 

  • F.J. Fallaux A. Bout I. Velde Particleder D.J. Wollenberg Particleden K.M. Hehir J. Keegan C. Auger S.J. Cramer H. Ormondt Particlevan A.J. Eb Particleder D. Valerio R.C. Hoeben (1998) ArticleTitleNew helper cells and matched early region 1-deleted adenovirus vectors prevent generation of replication-competent adenoviruses Hum. Gene Ther. 9 1909–1917 Occurrence Handle9741429 Occurrence Handle1:CAS:528:DyaK1cXlslKnsL4%3D

    PubMed  CAS  Google Scholar 

  • M.R. Fibi P. Hermentin J.U. Pauly L. Lauffer G. Zettlmeissl (1995) ArticleTitleN- and O-glycosylation muteins of recombinant human erythropoietin secreted from BHK-21 cells Blood 85 1229–1236 Occurrence Handle7858253 Occurrence Handle1:CAS:528:DyaK2MXjvFenurg%3D

    PubMed  CAS  Google Scholar 

  • M.B. Fogolin R. Wagner M. Etcheverrigaray R. Kratje (2004) ArticleTitleImpact of temperature reduction and expression of yeast pyruvate carboxylase on hGM-CSF-producing CHO cells J. Biotechnol. 109 179–191 Occurrence Handle15063626 Occurrence Handle1:CAS:528:DC%2BD2cXivVKmsb8%3D

    PubMed  CAS  Google Scholar 

  • M. Fussenegger J.E. Bailey H. Hauser P.P. Mueller (1999) ArticleTitleGenetic optimization of recombinant glycoprotein production by mammalian cells Trends Biotechnol. 17 35–42 Occurrence Handle10.1016/S0167-7799(98)01248-7 Occurrence Handle10098276 Occurrence Handle1:CAS:528:DyaK1MXlt1KjtrY%3D

    Article  PubMed  CAS  Google Scholar 

  • M. Fussenegger S. Schlatter D. Datwyler X. Mazur J.E. Bailey (1998) ArticleTitleControlled proliferation by multigene metabolic engineering enhances the productivity of Chinese hamster ovary cells Nat. Biotechnol. 16 468–472 Occurrence Handle10.1038/nbt0598-468 Occurrence Handle9592397 Occurrence Handle1:CAS:528:DyaK1cXjtV2jtbo%3D

    Article  PubMed  CAS  Google Scholar 

  • M. Gawlitzek H.S. Conradt R. Wagner (1995) ArticleTitleEffect of different cell culture conditions on the polypeptide integrity and N-glycosylation of a recombinant model glycoprotein Biotechnol. Bioeng. 46 536–544 Occurrence Handle10.1002/bit.260460606 Occurrence Handle1:CAS:528:DyaK2MXlslKjtbs%3D

    Article  CAS  Google Scholar 

  • M. Gawlitzek U. Valley R. Wagner (1998) ArticleTitleAmmonium ion/glucosamine dependent increase of oligosaccharide complexity in recombinant glycoproteins secreted from cultivated BHK-21 cells Biotechnol. Bioeng. 57 518–528 Occurrence Handle10.1002/(SICI)1097-0290(19980305)57:5<518::AID-BIT3>3.0.CO;2-J Occurrence Handle10099230 Occurrence Handle1:CAS:528:DyaK1cXnslSjtw%3D%3D

    Article  PubMed  CAS  Google Scholar 

  • C. Geserick H. Bonarius L. Kongerslev H. Hauser P.P. Mueller (2000) ArticleTitleEnhanced productivity during controlled proliferation of BHK cells in continuously perfused bioreactors Biotechnol. Bioeng. 69 266–274 Occurrence Handle10.1002/1097-0290(20000805)69:3<266::AID-BIT4>3.0.CO;2-Z Occurrence Handle10861406 Occurrence Handle1:CAS:528:DC%2BD3cXlt1WqsLk%3D

    Article  PubMed  CAS  Google Scholar 

  • E. Grabenhorst A. Hoffmann M. Nimtz G. Zettlmeissl H.S. Conradt (1995) ArticleTitleConstruction of stable BHK-21 cells coexpressing human secretory glycoproteins and human Gal(beta 1–4)GlcNAc-R alpha 2,6-sialyltransferase alpha 2,6-linked NeuAc is preferentially attached to the Gal(beta 1–4)GlcNAc(beta 1–2)Man(alpha 1–3)-branch of diantennary oligosaccharides from secreted recombinant beta-trace protein Eur. J. Biochem. 232 718–725 Occurrence Handle10.1111/j.1432-1033.1995.718zz.x Occurrence Handle7588709 Occurrence Handle1:CAS:528:DyaK2MXosFOgtL8%3D

    Article  PubMed  CAS  Google Scholar 

  • E. Grabenhorst P. Schlenke S. Pohl M. Nimtz H.S. Conradt (1999) ArticleTitleGenetic engineering of recombinant glycoproteins and the glycosylation pathway in mammalian host cells Glycoconjugate J. 16 81–97 Occurrence Handle10.1023/A:1026466408042 Occurrence Handle1:CAS:528:DyaK1MXntFSjsr4%3D

    Article  CAS  Google Scholar 

  • F.L. Graham L. Prevec (1991) Manipulation of adenovirus vector E.J. Murray (Eds) Gene Transfer and Expression Protocols Humana Press Clifton, NJ 109s–129s

    Google Scholar 

  • N. Ibarra S. Watanabe J.X. Bi J. Shuttleworth M. Al-Rubeai (2003) ArticleTitleModulation of cell cycle for enhancement of antibody productivity in perfusion culture of NS0 cells Biotechnol. Prog. 19 224–228 Occurrence Handle10.1021/bp025589f Occurrence Handle12573029 Occurrence Handle1:CAS:528:DC%2BD38XpsFWmtb8%3D

    Article  PubMed  CAS  Google Scholar 

  • N. Irani A.J. Beccaria R. Wagner (2002) ArticleTitleExpression of recombinant cytoplasmic yeast pyruvate carboxylase for the improvement of the production of human erythropoietin by recombinant BHK-21 cells J. Biotechnol. 93 269–282 Occurrence Handle10.1016/S0168-1656(01)00409-6 Occurrence Handle11755990 Occurrence Handle1:CAS:528:DC%2BD38XhvVen

    Article  PubMed  CAS  Google Scholar 

  • N. Irani M. Wirth J. Heuvel ParticleDen R. Wagner (1999) ArticleTitleImprovement of the primary metabolism of cell cultures by introducing a new cytoplasmic pyruvate carboxylase reaction Biotechnol. Bioeng. 66 238–246 Occurrence Handle10.1002/(SICI)1097-0290(1999)66:4<238::AID-BIT5>3.0.CO;2-6 Occurrence Handle10578094 Occurrence Handle1:CAS:528:DyaK1MXotFalsr4%3D

    Article  PubMed  CAS  Google Scholar 

  • P.S. Jat P.A. Sharp (1989) ArticleTitleCell lines established by a temperature-sensitive simian virus 40 large-T-antigen gene are growth restricted at the nonpermissive temperature Mol. Cell. Biol. 9 1672–1681 Occurrence Handle2542774 Occurrence Handle1:STN:280:BiaB287nvFM%3D

    PubMed  CAS  Google Scholar 

  • H. Kaufmann X. Mazur R. Marone J.E. Bailey M. Fussenegger (2001) ArticleTitleComparative analysis of two controlled proliferation strategies regarding product quality influence on tetracycline-regulated gene expression and productivity Biotechnol. Bioeng. 72 592–602 Occurrence Handle10.1002/1097-0290(20010320)72:6<592::AID-BIT1024>3.0.CO;2-J Occurrence Handle11460250 Occurrence Handle1:CAS:528:DC%2BD3MXhs12gsbw%3D

    Article  PubMed  CAS  Google Scholar 

  • U.K. Laemmli (1970) ArticleTitleCleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227 680–685 Occurrence Handle10.1038/227680a0 Occurrence Handle5432063 Occurrence Handle1:CAS:528:DC%2BD3MXlsFags7s%3D

    Article  PubMed  CAS  Google Scholar 

  • E.A. Lidington R.M. Rao F.M. Marelli-Berg P.S. Jat D.O. Haskard J.C. Mason (2002) ArticleTitleConditional immortalization of growth factor-responsive cardiac endothelial cells from H- 2K(b)-tsA58 mice Am. J. Physiol. Cell Physiol. 282 C67–C74 Occurrence Handle11742799 Occurrence Handle1:CAS:528:DC%2BD38XlsVKnsg%3D%3D

    PubMed  CAS  Google Scholar 

  • D.R. Lloyd P. Holmes L.P. Jackson A.N. Emery M. Al-Rubeai (2000) ArticleTitleRelationship between cell sizecell cycle and specific recombinant protein productivity Cytotechnology 34 59–70 Occurrence Handle10.1023/A:1008103730027 Occurrence Handle1:CAS:528:DC%2BD3cXosVSnsLw%3D

    Article  CAS  Google Scholar 

  • T. May H. Hauser D. Wirth (2004) ArticleTitleTranscriptional control of SV40 T-antigen expression allows a complete reversion of immortalization Nucleic Acids Res. 32 5529–5538 Occurrence Handle15486202 Occurrence Handle1:CAS:528:DC%2BD2cXovVOiurs%3D

    PubMed  CAS  Google Scholar 

  • X. Mazur H.M. Eppenberger J.E. Bailey M. Fussenegger (1999) ArticleTitleA novel autoregulated proliferation-controlled production process using recombinant CHO cells Biotechnol. Bioeng. 65 144–150 Occurrence Handle10.1002/(SICI)1097-0290(19991020)65:2<144::AID-BIT3>3.0.CO;2-Q Occurrence Handle10458734 Occurrence Handle1:CAS:528:DyaK1MXmtVKrt7g%3D

    Article  PubMed  CAS  Google Scholar 

  • H. Meents B. Enenkel H.M. Eppenberger R.G. Werner M. Fussenegger (2002a) ArticleTitleImpact of coexpression and coamplification of sICAM and antiapoptosis determinants bcl-2/bcl-x(L) on productivity cell survival and mitochondria number in CHO-DG44 grown in suspension and serum-free media Biotechnol. Bioeng. 80 706–716 Occurrence Handle10.1002/bit.10449 Occurrence Handle1:CAS:528:DC%2BD38XovVOhu7w%3D

    Article  CAS  Google Scholar 

  • H. Meents B. Enenkel R.G. Werner M. Fussenegger (2002b) ArticleTitlep27Kip1-mediated controlled proliferation technology increases constitutive sICAM production in CHO-DUKX adapted for growth in suspension and serum-free media Biotechnol. Bioeng. 79 619–627 Occurrence Handle10.1002/bit.10322 Occurrence Handle1:CAS:528:DC%2BD38XmslSkt7o%3D

    Article  CAS  Google Scholar 

  • N. Mittereder K.L. March B.C. Trapnell (1996) ArticleTitleEvaluation of the concentration and bioactivity of adenovirus vectors for gene therapy J. Virol. 70 7498–7509 Occurrence Handle8892868 Occurrence Handle1:CAS:528:DyaK28Xmt1Glt7o%3D

    PubMed  CAS  Google Scholar 

  • L. Monaco A. Marc A. Eon-Duval G. Acerbis G. Distefano D. Lamotte J.M. Engasser M. Soria N. Jenkins (1996) ArticleTitleGenetic engineering of 2,6-sialyltransferase in recombinant CHO cells and its effects on the sialylation of recombinant interferon-gamma Cytotechnology 22 197–203 Occurrence Handle10.1007/BF00353939 Occurrence Handle1:CAS:528:DyaK1cXislels78%3D

    Article  CAS  Google Scholar 

  • M. Nimtz W. Martin V. Wray K.D. Klöppel J. Augustin H.S. Conradt (1993) ArticleTitleStructures of sialylated oligosaccharides of human erythropoietin expressed in recombinant BHK-21 cells Eur. J. Biochem. 213 39–56 Occurrence Handle10.1111/j.1432-1033.1993.tb17732.x Occurrence Handle8477709 Occurrence Handle1:CAS:528:DyaK3sXksVWitLs%3D

    Article  PubMed  CAS  Google Scholar 

  • M. Obinata (2001) ArticleTitlePossible applications of conditionally immortalized tissue cell lines with differentiation functions Biochem. Biophys. Res. Commun. 286 667–672 Occurrence Handle10.1006/bbrc.2001.5247 Occurrence Handle11520047 Occurrence Handle1:CAS:528:DC%2BD3MXmtFyru7w%3D

    Article  PubMed  CAS  Google Scholar 

  • M.J. O’Hare J. Bond C. Clarke Y. Takeuchi A.J. Atherton C. Berry J. Moody A.R. Silver D.C. Davies A.E. Alsop A.M. Neville P.S. Jat (2001) ArticleTitleConditional immortalization of freshly isolated human mammary fibroblasts and endothelial cells Proc. Natl. Acad. Sci. USA 98 646–651 Occurrence Handle11209060 Occurrence Handle1:CAS:528:DC%2BD3MXnslKmsg%3D%3D

    PubMed  CAS  Google Scholar 

  • D.T. Rutkowski R.J. Kaufman (2004) ArticleTitleA trip to the ER: coping with stress Trends Cell Biol. 14 20–28 Occurrence Handle10.1016/j.tcb.2003.11.001 Occurrence Handle14729177 Occurrence Handle1:CAS:528:DC%2BD2cXjtFegsw%3D%3D

    Article  PubMed  CAS  Google Scholar 

  • J. Sambrook E.F. Fritsch T. Maniatis (1989) Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory Cold Spring HarborNY

    Google Scholar 

  • S. Schlatter J.E. Bailey M. Fussenegger (2001) ArticleTitleNovel surface tagging technology for selection of complex proliferation-controlled mammalian cell phenotypes Biotechnol. Bioeng. 75 597–606 Occurrence Handle10.1002/bit.1189 Occurrence Handle11745136 Occurrence Handle1:CAS:528:DC%2BD3MXptVGkt74%3D

    Article  PubMed  CAS  Google Scholar 

  • P. Schlenke E. Grabenhorst M. Nimtz H.S. Conradt (1999) ArticleTitleConstruction and characterization of stably transfected BHK-21 cells with human-type sialylation characteristics Cytotechnology 30 17–25 Occurrence Handle10.1023/A:1008049603947 Occurrence Handle1:CAS:528:DyaK1MXjsl2lu7o%3D

    Article  CAS  Google Scholar 

  • K. Schroeder S. Koschmieder O.G. Ottmann D. Hoelzer H. Hauser P.P. Mueller (2002) ArticleTitleCoordination of cell growth in cocultures by a genetic proliferation control system Biotechnol. Bioeng. 78 346–352 Occurrence Handle10.1002/bit.10196 Occurrence Handle11920451 Occurrence Handle1:CAS:528:DC%2BD38XjslSns7w%3D

    Article  PubMed  CAS  Google Scholar 

  • T. Sekiguchi T. Hunter (1998) ArticleTitleInduction of growth arrest and cell death by overexpression of the cyclin-Cdk inhibitor p21 in hamster BHK21 cells Oncogene 16 369–380 Occurrence Handle10.1038/sj.onc.1201539 Occurrence Handle9467962 Occurrence Handle1:CAS:528:DyaK1cXhtVagtL4%3D

    Article  PubMed  CAS  Google Scholar 

  • M. Takeuchi N. Inoue T.W. Strickland M. Kubota M. Wada R. Shimizu S. Hoshi H. Kozutsumi S. Takasaki A. Kobota (1989) ArticleTitleRelationship between sugar chain structure and biological activity of recombinant human erythropoietin produced in Chinese hamster ovary cells Proc. Natl. Acad. Sci. USA 86 7819–7822 Occurrence Handle2813359 Occurrence Handle1:CAS:528:DyaL1MXmt1GnsL4%3D

    PubMed  CAS  Google Scholar 

  • P. Umaña J. Jean-Mairet R. Moudry H. Amstutz J.E. Bailey (1999) ArticleTitleEngineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity Nat. Biotechnol. 17 176–180 Occurrence Handle10052355

    PubMed  Google Scholar 

  • S. Urlinger U. Baron M. Thellmann M.T. Hasan H. Bujard W. Hillen (2000) ArticleTitleExploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range sensitivity Proc. Natl. Acad. Sci. USA 97 7963–7968 Occurrence Handle10.1073/pnas.130192197 Occurrence Handle10859354 Occurrence Handle1:CAS:528:DC%2BD3cXkvFCnu7o%3D

    Article  PubMed  CAS  Google Scholar 

  • E. Verhoeyen H. Hauser D. Wirth (2001) ArticleTitleEvaluation of retroviral vector design in defined chromosomal loci by Flp-mediated cassette replacement Hum. Gene Ther. 12 933–944 Occurrence Handle10.1089/104303401750195890 Occurrence Handle11387058 Occurrence Handle1:CAS:528:DC%2BD3MXktVWhtrc%3D

    Article  PubMed  CAS  Google Scholar 

  • S. Watanabe J. Shuttleworth M. Al-Rubeai (2002) ArticleTitleRegulation of cell cycle and productivity in NS0 cells by the over-expression of p21CIP1 Biotechnol. Bioeng. 77 1–7 Occurrence Handle10.1002/bit.10112 Occurrence Handle11745168 Occurrence Handle1:CAS:528:DC%2BD38XktlaksA%3D%3D

    Article  PubMed  CAS  Google Scholar 

  • Westerman K.A. and Leboulch P. 1996. Reversible immortalization of mammalian cells mediated by retroviral transfer and site-specific recombination. Proc. Natl. Acad. Sci. USA. 8971–8976.

  • C. Wiethe K. Dittmar T. Doan W. Lindenmaier R. Tindle (2003) ArticleTitleProvision of 4–1BB ligand enhances effector and memory CTL responses generated by immunization with dendritic cells expressing a human tumor-associated antigen J. Immunol. 170 2912–2922 Occurrence Handle12626542 Occurrence Handle1:CAS:528:DC%2BD3sXhslOku7c%3D

    PubMed  CAS  Google Scholar 

  • K. Yamaguchi K. Akai G. Kawanishi M. Ueda S. Masuda R. Sasaki (1991) ArticleTitleEffects of site-directed removal of N-glycosylation sites in human erythropoietin on its production and biological properties J. Biol. Chem. 30 20434–20439

    Google Scholar 

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  1. Department of Gene Regulation and Differentiation, GBF – National, Research Center for Biotechnology, Mascheroder Weg 1, D-38124, Braunschweig, Germany

    Tobias May, Werner Lindenmaier, Dagmar Wirth & Peter P. Mueller

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  1. Tobias May
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  2. Werner Lindenmaier
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  3. Dagmar Wirth
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  4. Peter P. Mueller
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Correspondence to Peter P. Mueller.

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May, T., Lindenmaier, W., Wirth, D. et al. Application of a Reversible Immortalization System for the Generation of Proliferation-Controlled Cell Lines. Cytotechnology 46, 69–78 (2004). https://doi.org/10.1007/s10616-005-2834-z

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