Enhancement of hypermutation frequency in the chicken B cell line DT40 for efficient diversification of the antibody repertoire

Masaki Magari, Yuichi Kanehiro, Kagefumi Todo, Mika Ikeda, Naoki Kanayama, Hitoshi Ohmori

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Chicken B cell line DT40 continuously accumulates mutations in the immunoglobulin variable region (IgV) gene by gene conversion and point mutation, both of which are mediated by activation-induced cytidine deaminase (AID), thereby producing an antibody (Ab) library that is useful for screening monoclonal Abs (mAbs) in vitro. We previously generated an engineered DT40 line named DT40-SW, whose AID expression can be reversibly switched on or off, and developed an in vitro Ab generation system using DT40-SW cells. To efficiently create an Ab library with sufficient diversity, higher hypermutation frequency is advantageous. To this end, we generated a novel cell line DT40-SWΔC, which conditionally expresses a C-terminus-truncated AID mutant lacking the nuclear export signal. The transcription level of the mutant AID gene in DT40-SWΔC cells was similar to that of the wild-type gene in DT40-SW cells. However, the protein level of the truncated AID mutant was less than that of the wild type. The mutant protein was enriched in the nuclei of DT40-SWΔC cells, although the protein might be highly susceptible to degradation. In DT40-SWΔC cells, both gene conversion and point mutation occurred in the IgV gene with over threefold higher frequency than in DT40-SW cells, suggesting that a lower level of the mutant AID protein was sufficient to increase mutation frequency. Thus, DT40-SWΔC cells may be useful for constructing Ab libraries for efficient screening of mAbs in vitro.

Original languageEnglish
Pages (from-to)353-358
Number of pages6
JournalBiochemical and Biophysical Research Communications
Volume396
Issue number2
DOIs
Publication statusPublished - May 28 2010

Fingerprint

Chickens
B-Lymphocytes
Cells
Cell Line
Antibodies
Genes
Immunoglobulin Variable Region
Gene Conversion
Point Mutation
Screening
Nuclear Export Signals
Proteins
Mutant Proteins
Transcription
Mutation Rate
AICDA (activation-induced cytidine deaminase)
Degradation
Mutation
In Vitro Techniques

Keywords

  • AID
  • Chicken B cell line DT40
  • Gene conversion
  • Hypermutation
  • Monoclonal antibody

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Cell Biology
  • Molecular Biology

Cite this

Enhancement of hypermutation frequency in the chicken B cell line DT40 for efficient diversification of the antibody repertoire. / Magari, Masaki; Kanehiro, Yuichi; Todo, Kagefumi; Ikeda, Mika; Kanayama, Naoki; Ohmori, Hitoshi.

In: Biochemical and Biophysical Research Communications, Vol. 396, No. 2, 28.05.2010, p. 353-358.

Research output: Contribution to journalArticle

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