Production of Human or Humanized Antibodies in Mice

  • Brice Laffleur
  • Virginie Pascal
  • Christophe Sirac
  • Michel CognéEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 901)


Mice are widely available laboratory animals that can easily be used for the production of antibodies against a broad range of antigens, using well-defined immunization protocols. Such an approach allows optimal in vivo affinity maturation of the humoral response. In addition, high-affinity antibodies arising in this context can readily be further characterized and produced as monoclonals after immortalizing and selecting specific antibody-producing cells through hybridoma derivation. Using such conventional strategies combined with mice that are either genetically engineered to carry humanized immunoglobulin (Ig) genes or engrafted with a human immune system, it is thus easy to obtain and immortalize clones that produce either fully human Ig or antibodies associating variable (V) domains with selected antigen specificities to customized human-like constant regions, with defined effector functions. In some instances, where there is a need for in vivo functional assays of a single antibody with a known specificity, it might be of interest to transiently express that gene in mice by in vivo gene transfer. This approach allows a rapid functional assay. More commonly, mice are used to obtain a diversified repertoire of antibody specificities after immunization by producing antibody molecules in the mouse B cell lineage from mouse strains with transgene Ig genes which are of human, humanized, or chimeric origin. After in vivo maturation of the immune response, this will lead to the secretion of antibodies with optimized antigen binding sites, associated to the desired human constant domains. This chapter focuses on two simple methods: (1) to obtain such humanized Ig mice and (2) to transiently express a human Ig gene in mice using hydrodynamics-based transfection.

Key words

B cells ES cells Genetic engineered mice Homologous recombination Humanized mice Humanized antibodies Hydrodynamics-based transfection Immunoglobulin 


  1. 1.
    Kohler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495–497PubMedCrossRefGoogle Scholar
  2. 2.
    Green LL, Hardy MC, Maynard-Currie CE et al (1994) Antigen-specific human monoclonal antibodies from mice engineered with human Ig heavy and light chain YACs. Nat Genet 7:13–21PubMedCrossRefGoogle Scholar
  3. 3.
    Green LL, Jakobovits A (1998) Regulation of B cell development by variable gene complexity in mice reconstituted with human immunoglobulin yeast artificial chromosomes. J Exp Med 188: 483–495PubMedCrossRefGoogle Scholar
  4. 4.
    Kuroiwa Y, Tomizuka K, Shinohara T et al (2000) Manipulation of human minichromosomes to carry greater than megabase-sized chromosome inserts. Nat Biotechnol 18: 1086–1090PubMedCrossRefGoogle Scholar
  5. 5.
    Le Provost F, Lillico S, Passet B et al (2010) Zinc finger nuclease technology heralds a new era in mammalian transgenesis. Trends Biotechnol 28:134–141PubMedCrossRefGoogle Scholar
  6. 6.
    Mendez MJ, Green LL, Corvalan JR et al (1997) Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice. Nat Genet 15:146–156PubMedCrossRefGoogle Scholar
  7. 7.
    Popov AV, Zou X, Xian J et al (1999) A human immunoglobulin lambda locus is similarly well expressed in mice and humans. J Exp Med 189: 1611–1620PubMedCrossRefGoogle Scholar
  8. 8.
    Tomizuka K, Shinohara T, Yoshida H et al (2000) Double trans-chromosomic mice: maintenance of two individual human chromosome fragments containing Ig heavy and kappa loci and expression of fully human antibodies. Proc Natl Acad Sci U S A 97:722–727PubMedCrossRefGoogle Scholar
  9. 9.
    Zou YR, Muller W, Gu H et al (1994) Cre-loxP-mediated gene replacement: a mouse strain producing humanized antibodies. Curr Biol 4:1099–1103PubMedCrossRefGoogle Scholar
  10. 10.
    Ishida I, Tomizuka K, Yoshida H et al (2002) Production of human monoclonal and polyclonal antibodies in TransChromo animals. Cloning Stem Cells 4:91–102PubMedCrossRefGoogle Scholar
  11. 11.
    Lonberg N, Taylor LD, Harding FA et al (1994) Antigen-specific human antibodies from mice comprising four distinct genetic modifications. Nature 368:856–859PubMedCrossRefGoogle Scholar
  12. 12.
    Jakobovits A, Amado RG, Yang X et al (2007) From XenoMouse technology to panitumumab, the first fully human antibody product from transgenic mice. Nat Biotechnol 25:1134–1143PubMedCrossRefGoogle Scholar
  13. 13.
    Duchez S, Amin R, Cogne N et al (2010) Premature replacement of mu with alpha immunoglobulin chains impairs lymphopoiesis and mucosal homing but promotes plasma cell maturation. Proc Natl Acad Sci U S A 107:3064–3069PubMedCrossRefGoogle Scholar
  14. 14.
    Kiefer JC (2011) Primer and interviews: advances in targeted gene modification. Dev Dyn 240:2688–2696CrossRefGoogle Scholar
  15. 15.
    Menoret S, Iscache AL, Tesson L et al (2010) Characterization of immunoglobulin heavy chain knockout rats. Eur J Immunol 40: 2932–2941PubMedCrossRefGoogle Scholar
  16. 16.
    Flisikowska T, Thorey IS, Offner S et al (2011) Efficient immunoglobulin gene disruption and targeted replacement in rabbit using zinc finger nucleases. PLoS One 6:e21045PubMedCrossRefGoogle Scholar
  17. 17.
    Li H, Haurigot V, Doyon Y et al (2011) In vivo genome editing restores haemostasis in a mouse model of haemophilia. Nature 475:217–221PubMedCrossRefGoogle Scholar
  18. 18.
    Liu F, Song Y, Liu D (1999) Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Ther 6:1258–1266PubMedCrossRefGoogle Scholar
  19. 19.
    Song YK, Liu F, Zhang G et al (2002) Hydrodynamics-based transfection: simple and efficient method for introducing and expressing transgenes in animals by intravenous injection of DNA. Methods Enzymol 346:92–105PubMedCrossRefGoogle Scholar
  20. 20.
    Pinaud E, Khamlichi AA, Le Morvan C et al (2001) Localization of the 3′ IgH locus elements that effect long-distance regulation of class switch recombination. Immunity 15:187–199PubMedCrossRefGoogle Scholar
  21. 21.
    Nagy A, Gertsenstein M, Vintersten K et al (2004) In: Cuddihy J (ed) Manipulating the mouse embryo, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  22. 22.
    Zuckier LS, Chang CJ, Scharff MD et al (1998) Chimeric human-mouse IgG antibodies with shuffled constant region exons demonstrate that multiple domains contribute to in vivo half-life. Cancer Res 58:3905–3908PubMedGoogle Scholar
  23. 23.
    Wilber A, Linehan JL, Tian X et al (2007) Efficient and stable transgene expression in human embryonic stem cells using transposon-mediated gene transfer. Stem Cells 25:2919–2927PubMedCrossRefGoogle Scholar
  24. 24.
    Mortensen RM, Zubiaur M, Neer EJ et al (1991) Embryonic stem cells lacking a functional inhibitory G-protein subunit (alpha i2) produced by gene targeting of both alleles. Proc Natl Acad Sci U S A 88:7036–7040PubMedCrossRefGoogle Scholar
  25. 25.
    Taniguchi M, Sanbo M, Watanabe S et al (1998) Efficient production of Cre-mediated site-directed recombinants through the utilization of the puromycin resistance gene, pac: a transient gene-integration marker for ES cells. Nucleic Acids Res 26:679–680PubMedCrossRefGoogle Scholar
  26. 26.
    McCarrick JW 3rd, Parnes JR, Seong RH et al (1993) Positive-negative selection gene targeting with the diphtheria toxin A-chain gene in mouse embryonic stem cells. Transgenic Res 2:183–190PubMedCrossRefGoogle Scholar
  27. 27.
    Yagi T, Nada S, Watanabe N et al (1993) A novel negative selection for homologous recombinants using diphtheria toxin A fragment gene. Anal Biochem 214:77–86PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Brice Laffleur
    • 1
  • Virginie Pascal
    • 1
  • Christophe Sirac
    • 1
  • Michel Cogné
    • 1
    Email author
  1. 1.CNRS UMR6101, Contrôle des Réponses Immunes B et LymphoproliférationsUniversité de LimogesLimogesFrance

Personalised recommendations