Enhancement of hypermutation frequency in the chicken B cell line DT40 for efficient diversification of the antibody repertoire.
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-SWDeltaC, which conditionally expresses a C-terminus-truncated AID mutant lacking the nuclear export signal. The transcription level of the mutant AID gene in DT40-SWDeltaC 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-SWDeltaC cells, although the protein might be highly susceptible to degradation. In DT40-SWDeltaC 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-SWDeltaC cells may be useful for constructing Ab libraries for efficient screening of mAbs in vitro.
Magari
Masaki
M
Department of Bioscience and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-Naka, Okayama 700-8530, Japan.
Kanehiro
Yuichi
Y
Todo
Kagefumi
K
Ikeda
Mika
M
Kanayama
Naoki
N
Ohmori
Hitoshi
H
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics*; D001402:B-Lymphocytes / Q000276:immunology*; D002460:Cell Line; D002645:Chickens; D003564:Cytidine Deaminase / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D019151:Peptide Library; D027041:Somatic Hypermutation, Immunoglobulin; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D003564:Cytidine Deaminase