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Rapid generation of specific antibodies by enhanced homologous recombination

Nature Biotechnology volume 23, pages 731–735 (2005)Cite this article

Abstract

In the chicken immune system, gene conversion, a type of homologous recombination, primarily contributes to diversification of the immunoglobulin gene1,2. Here, we report on the rapid generation of specific monoclonal antibodies using the chicken DT40 B-cell line undergoing gene conversion. We discovered that the gene conversion frequency at the immunoglobulin locus is increased by treating DT40 cells with a histone deacetylase inhibitor, trichostatin A (TSA)3, thereby generating diversity at the immunoglobulin locus in the majority of treated cells. This indicates that TSA treatment accelerates the autonomous diversification of surface IgMs on DT40 cells. We took advantage of this effect to select DT40 cells producing specific antibodies with antigen-conjugated magnetic beads. This autonomously diversifying library (ADLib) selection system enables the quick establishment (∼1 week from a diversifying library) of various clones producing monoclonal IgMs with enough specificity and affinity for immunological assays, and is applicable to various biotechnologies including rational protein design.

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Figure 1: Histone modification of IgLC in DT40 cells and the effect of TSA.
Figure 2: Effect of TSA treatment on gene conversion frequency at the IgLC locus.
Figure 3: Selection of specific antibodies against human IgG.
Figure 4: Selection of a streptavidin-specific antibody and the variation of VL sequence of selected clones producing IgMs specific to various antigens.

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Acknowledgements

We thank T. Yamada, K. Hirota and K. Okuhara for helpful advice and discussion, H. Sasanuma for helpful advice on antibody selection, Y. Ichikawa and R. Nakazawa for DNA sequencing, and M. Kawasaki for advice on Kd determination by Biacore. This work was supported by grants from the CREST (Core Research for Evolutional Science and Technology) program of JST (Japan Science and Technology Agency); REDS (Rational Evolutionary Design of Advanced Biomolecules, Saitama); 'Bioarchitect Research Program' of RIKEN; the Ministry of Education, Science, Culture, & Sports, Japan.

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Authors and Affiliations

  1. Genetic Dynamics Research Unit, RIKEN Discovery Research Institute, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan

    Hidetaka Seo, Mieko Masuoka & Kunihiro Ohta

  2. REDS Group (Saitama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence, JST), Saitama Small Enterprise Promotion Corporation, 3-12-18 Kamiaoki, Kawaguchi, Saitama, 333-0844, Japan

    Hidetaka Seo & Mieko Masuoka

  3. Department of Biology, Faculty of Science, Yamaguchi University, 1677-1 Ohaza Yoshida, Yamaguchi-shi, Yamaguchi, 753-8512, Japan

    Hiromu Murofushi

  4. Radiation Genetics, Faculty of Medicine, Kyoto University, Yoshida Konoe, Sakyo-ku, Kyoto, 606-8315, Japan

    Shunichi Takeda

  5. Laboratory of Cellular & Molecular Biology, RIKEN Discovery Research Institute, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan

    Takehiko Shibata

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  1. Hidetaka Seo
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Correspondence to Kunihiro Ohta.

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The authors are applying for patents that are based on this research. Some authors are founders and stockholders of Chiome Bioscience, a private company in Tokyo.

Supplementary information

Supplementary Fig. 1

Histone modification of IgLC in DT40 cells and the effect of TSA on IgLC expression. (PDF 1180 kb)

Supplementary Fig. 2

Effect of TSA treatment on gene conversion frequency at the IgLC locus and AID transcription. (PDF 473 kb)

Supplementary Fig. 3

Selection of specific antibodies against rabbit IgG. (PDF 391 kb)

Supplementary Fig. 4

Variation of the nucleotide and predicted amino acid sequences of VL and VH of selected clones producing IgM specific to various antigens. (PDF 241 kb)

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Seo, H., Masuoka, M., Murofushi, H. et al. Rapid generation of specific antibodies by enhanced homologous recombination. Nat Biotechnol 23, 731–735 (2005). https://doi.org/10.1038/nbt1092

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