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

• Published in: Biochemical and biophysical research communications 2010-05, Vol.396 (2), p.353-358
• Main Authors: Magari, Masaki, Kanehiro, Yuichi, Todo, Kagefumi, Ikeda, Mika, Kanayama, Naoki, Ohmori, Hitoshi
• Format: Article
• Language: English
• Subjects: 60 APPLIED LIFE SCIENCES; AID; Animals; Antibodies, Monoclonal - genetics; B-Lymphocytes - immunology; Cell Line; Chicken B cell line DT40; CHICKENS; CYTIDINE; Cytidine Deaminase - genetics; Gene conversion; GENE MUTATIONS; GENES; Hypermutation; Immunoglobulin Variable Region - genetics; IMMUNOGLOBULINS; IN VITRO; MONOCLONAL ANTIBODIES; Monoclonal antibody; MUTANTS; MUTATION FREQUENCY; Peptide Library; Somatic Hypermutation, Immunoglobulin; TRANSCRIPTION
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2010.04.096

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.