, Volume 53, Issue 1–3, pp 65–73 | Cite as

Targeted genetic modification of cell lines for recombinant protein production

  • Niall Barron
  • Olga Piskareva
  • Mohan Muniyappa
NICB special issue


Considerable increases in productivity have been achieved in biopharmaceutical production processes over the last two decades. Much of this has been a result of improvements in media formulation and process development. Though advances have been made in cell line development, there remains considerable opportunity for improvement in this area. The wealth of transcriptional and proteomic data being generated currently hold the promise of specific molecular interventions to improve the performance of production cell lines in the bioreactor. Achieving this—particularly for multi-gene modification—will require specific, targeted and controlled genetic manipulation of these cells. This review considers some of the current and potential future techniques that might be employed to realise this goal.


Targeted Homologous recombination Recombinases Site-specific Inducible Transgene Recombinant protein CHO 



Work in this laboratory is supported by funding from Science Foundation Ireland and Enterprise Ireland.


  1. Abremski K, Hoess R (1984) Bacteriophage P1 site-specific recombination. Purification and properties of the Cre recombinase protein. J Biol Chem 259:1509–1514Google Scholar
  2. Abremski K, Wierzbicki A, Frommer B, Hoess RH (1986) Bacteriophage P1 Cre-loxP site-specific recombination. Site-specific DNA topoisomerase activity of the Cre recombination protein. J Biol Chem 261:391–396Google Scholar
  3. Bhat K, McBurney MW, Hamada H (1988) Functional cloning of mouse chromosomal loci specifically active in embryonal carcinoma stem cells. Mol Cell Biol 8:3251–3259Google Scholar
  4. Bibikova M, Carroll D, Segal DJ, Trautman JK, Smith J, Kim YG, Chandrasegaran S (2001) Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Mol Cell Biol 21:289–297CrossRefGoogle Scholar
  5. Chamberlain JR, Schwarze U, Wang PR, Hirata RK, Hankenson KD, Pace JM, Underwood RA, Song KM, Sussman M, Byers PH, Russell DW (2004) Gene targeting in stem cells from individuals with osteogenesis imperfecta. Science 303:1198–1201CrossRefGoogle Scholar
  6. Coates CJ, Kaminski JM, Summers JB, Segal DJ, Miller AD, Kolb AF (2005) Site-directed genome modification: derivatives of DNA-modifying enzymes as targeting tools. Trends Biotechnol 23:407–409 ReviewCrossRefGoogle Scholar
  7. Cohen-Tannoudji M, Robine S, Choulika A, Pinto D, El Marjou F, Babinet C, Louvard D, Jaisser F (1998) I-SceI-induced gene replacement at a natural locus in embryonic stem cells. Mol Cell Biol 18:1444–1448Google Scholar
  8. Culver KW, Hsieh WT, Huyen Y, Chen V, Liu J, Khripine Y, Khorlin A (1999) Correction of chromosomal point mutations in human cells with bifunctional oligonucleotides. Nat Biotechnol 17:989–993 Google Scholar
  9. Derouazi M, Martinet D, Besuchet Schmutz N, Flaction R, Wicht M, Bertschinger M, Hacker DL, Beckmann JS, Wurm FM (2006) Genetic characterization of CHO production host DG44 and derivative recombinant cell lines, Biochem Biophys Res Commun 340:1069–1077CrossRefGoogle Scholar
  10. Durai S, Mani M, Kandavelou K, Wu J, Porteus MH, Chandrasegaran S (2005) Zinc-finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells. Nucleic Acids Res 33:5978–5990CrossRefGoogle Scholar
  11. Ferlini A, Muntoni F (1998) The 5’ region of intron 11 of the dystrophin gene contains target sequences for mobile elements and three overlapping ORFs. Biochem Biophys Res Commun 242:401–406CrossRefGoogle Scholar
  12. Figueroa B, Chen SL, Oyler GA et al (2004) Aven and Bcl-X-L enhance protection against apoptosis for mammalian cells exposed to various culture conditions. Biotechnol Bioeng 85:589–600CrossRefGoogle Scholar
  13. Ghosh SS, Gopinath P, Ramesh A (2006) Adenoviral vectors: a promising tool for genetherapy. Appl Biochem Biotechnol 133:9–29CrossRefGoogle Scholar
  14. Giovannangeli C, Thuong NT, Helene C (1992) Oligodeoxynucleotide-directed photo-induced cross-linking of HIV proviral DNA via triple-helix formation. Nucleic Acids Res 20:4275–4281CrossRefGoogle Scholar
  15. Gonzalez-Nicolini V, Fussenegger M (2005) A novel binary adenovirus-based dual-regulated expression system for independent transcription control of two different transgenes. J Gene Med 7:1573–1585CrossRefGoogle Scholar
  16. Groth AC, Olivares EC, Thyagarajan B, Calos MP (2000) A phage integrase directs efficient site-specific integration in human cells. Proc Natl Acad Sci USA 97:5995–6000CrossRefGoogle Scholar
  17. Hartenbach S, Fussenegger M (2005) Autoregulated, bidirectional and multicistronic gas-inducible mammalian as well as lentiviral expression vectors. J Biotech 120:83–98CrossRefGoogle Scholar
  18. Hasty P, Rivera-Perez J, Bradley A (1991) The length of homology required for gene targeting in embryonic stem cells. Mol Cell Biol 11:5586–5591Google Scholar
  19. Helene C (1991) The anti-gene strategy: control of gene expression by triplex-forming-oligonucleotides. Anticancer Drug Des 6:569–584Google Scholar
  20. Hirata R, Chamberlain J, Dong R, Russell DW (2002) Targeted transgene insertion into human chromosomes by adeno-associated virus vectors. Nat Biotechnol 20:735–738CrossRefGoogle Scholar
  21. Hoess RH, Abremski K (1984) Interaction of the bacteriophage P1 recombinase Cre with the recombining site loxP. Proc Natl Acad Sci USA 81:1026–1029CrossRefGoogle Scholar
  22. Kaufman CD, Zayed H, Miskey C, Walisko O, Izsvak Z (2004) Sleeping Beauty transposable element: evolution, regulation and genetic applications. Curr Issues Mol Biol 6:43–55Google Scholar
  23. Kilby NJ, Snaith MR, Murray JA (1993) Site-specific recombinases: tools for genome engineering. Trends Genet 9:413–421CrossRefGoogle Scholar
  24. Koduri RK, Miller JT, Thammana P (2001) An efficient homologous recombination vector pTV(I) contains a hot spot for increased recombinant protein expression in Chinese hamster ovary cells. Gene 280:87–95CrossRefGoogle Scholar
  25. Kotin RM, Siniscalco M, Samulski RJ, Zhu XD, Hunter L, Laughlin CA, McLaughlin S, Muzyczka N, Rocchi M, Berns KI (1990) Site-specific integration by adeno-associated virus. Proc Natl Acad Sci USA 87:2211–2215CrossRefGoogle Scholar
  26. Kramer BP, Fussenegger M (2005) Transgene control engineering in mammalian cells. Methods Mol Biol 308:123–143Google Scholar
  27. Lee G, Saito I (1998) Role of nucleotide sequences of loxP spacer region in Cre-mediated recombination. Gene 216:55–65CrossRefGoogle Scholar
  28. Liu XY, Gu JF (2006) Targeting gene-virotherapy of cancer. Cell Res 16:25–30CrossRefGoogle Scholar
  29. Malphettes L, Fussenegger M (2006) Improved transgene expression fine-tuning in mammalian cells using a novel transcription-translation network. J Biotechnol 124:732–746Google Scholar
  30. Mandell JG, Barbas CF III (2006) Zinc Finger Tools: custom DNA-binding domains for transcription factors and nucleases. Nucleic Acids Res 34:W516–W523CrossRefGoogle Scholar
  31. May T, Hauser H, Wirth D (2006) Current status of transcriptional regulation systems. Cytotechnology 50:109–119CrossRefGoogle Scholar
  32. Mazur X, Fussenegger M, Renner WA et al (1998a) Higher productivity of growth-arrested Chinese hamster ovary cells expressing the cyclin-dependent kinase inhibitor p27. Biotechnol Progr 14:705–713CrossRefGoogle Scholar
  33. Mazur X, Eppenberger HM, Bailey JE et al (1999b) A novel autoregulated proliferation-controlled production process using recombinant CHO cells. Biotechnol Bioengg 65:144–150CrossRefGoogle Scholar
  34. Meents H, Enenkel B, Eppenberger HM, Werner RG, Fussenegger M (2002) Cell survival, and mitochondria number in CHO-DG44 grown in suspension and serum-free media. Biotechnol Bioeng 80Google Scholar
  35. Miller DG, Wang PR, Petek LM, Hirata RK, Sands MS, Russell DW (2006) Gene targeting in vivo by adeno-associated virus vectors. Nat Biotechnol 24:1022–10226CrossRefGoogle Scholar
  36. Pavlou AK (2004) The market of therapeutic recombinant proteins to 2010. J Comm Biotechnol 10:363–367(5)CrossRefGoogle Scholar
  37. Perkins BD, Wilson JH, Wensel TG, Vasquez KM (1998) Triplex targets in the human. Biochemistry 37:11315–11322CrossRefGoogle Scholar
  38. Rouet P, Smih F, Jasin M (1994) Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells. Proc Natl Acad Sci USA 91:6064–6068CrossRefGoogle Scholar
  39. Sauer B (1998) Inducible gene targeting in mice using the Cre/lox system. Methods 14:381–392 CrossRefGoogle Scholar
  40. Seibler J, Bode J (1997) Double-reciprocal crossover mediated by FLP-recombinase: a concept and an assay. Biochemistry 36:1740–1747CrossRefGoogle Scholar
  41. Smales CM, Dinnis DM, Stansfield SH, Alete D, Saga EA, Birch JR, Racher AJ, Marshall CT, James DC (2004) Comparative proteomic analysis of GS-NSO murine myeloma cell lines with varying recombinant monoclonal antibody production rate. Biotechnol Bioeng 88:474–488CrossRefGoogle Scholar
  42. Smithies O, Gregg RG, Boggs SS, Koralewski MA, Kucherlapati RS (1985) Insertion of DNA sequences into the human chromosomal beta-globin locus by homologous recombination. Nature 317:230–234CrossRefGoogle Scholar
  43. Soares MB, Schon E, Efstratiadis A (1985) Rat LINE1: the origin and evolution of a family of long interspersed middle repetitive DNA elements. J Mol Evol 22:117–133CrossRefGoogle Scholar
  44. Sorrell DA, Kolb AF (2005) Targeted modification of mammalian genomes. Biotechnol Adv 23:431–469CrossRefGoogle Scholar
  45. Sutter NB, Scalzo D, Fiering S, Groudine M, Martin DI (2003) Chromatin insulation by a transcriptional activator. Proc Natl Acad Sci USA 100:1105–1110CrossRefGoogle Scholar
  46. Te Riele H, Maandag ER, Berns A (1992) Highly efficient gene targeting in embryonic stem cells through homologous recombination with isogenic DNA constructs. Proc Natl Acad Sci USA 89:5128–5132CrossRefGoogle Scholar
  47. Thorpe HM, Smith MC (1998) In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family. Proc Natl Acad Sci USA 95:5505–5510CrossRefGoogle Scholar
  48. Vasquez KM, Marburger K, Intody Z, Wilson JH (2001) Manipulating the mammalian genome by homologous recombination. Proc Natl Acad Sci USA 98:8403–8410CrossRefGoogle Scholar
  49. Ward MA, Abramow-Newerly W, Roder JC (1993) Effect of vector topology on homologous recombination at the CHO aprt locus. Somat Cell Mol Genet 19:257–264CrossRefGoogle Scholar
  50. Weber W, Rimann M, Spielmann M, Keller B, Daoud-El Baba M, Aubel D, Weber CC, Fussenegger M (2004) Gas-inducible transgene expression in mammalian cells and mice. Nat Biotechnol 22:1440–1444 CrossRefGoogle Scholar
  51. Wlaschin KF, Seth G, Hu W-S (2006) Hu WSToward genomic cell culture engineering. Cytotechnology 50:121–140CrossRefGoogle Scholar
  52. Wong NSC, Yap MGS, (2006) Wang DICEnhancing recombinant glycoprotein sialylation through CMP-sialic acid transporter over expression in chinese hamster ovary cells. Biotechnol Bioeng 93:1005–1016CrossRefGoogle Scholar
  53. Wurm FM (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol. 22:1393–1398Google Scholar
  54. Wurm FM, Petropoulos CJ (1994) Plasmid integration, amplification and cytogenetics in CHO cells: questions and comments. Biologicals 22:95–102CrossRefGoogle Scholar
  55. Yamane-Ohnuki N, Kinoshita S, Inoue-Urakubo M, Kusunoki M, Iida S, Nakano R, Wakitani M, Niwa R, Sakurada M, Uchida K, Shitara K, Satoh M (2004) Establishment of FUT8 knockout Chinese hamster ovary cells: an ideal host cell line for producing completely defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity. Biotechnol Bioeng 87:614–622CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2007

Authors and Affiliations

  1. 1.National Institute for Cellular Biotechnology, Dublin City UniversityDublinIreland

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