TY - JOUR
T1 - Reversible switching of immunoglobulin hypermutation machinery in a chicken B cell line
AU - Kanayama, Naoki
AU - Todo, Kagefumi
AU - Reth, Michael
AU - Ohmori, Hitoshi
N1 - Funding Information:
We thank Dr. H. Arakawa (GSF National Research Center for Environment and Health, Neuherverg-Munich, Germany) for kindly providing pLoxPuro, pLoxBsr, and pExpress, and Dr. J. Miyazaki (Osaka University Medical School, Japan) for generously giving pCAGGS. This work was supported by Grants-in-Aid from The Ministry of Education, Science, Sports and Culture of Japan to H.O. (12450334 and 16360414) and N.K. (15760589), and The Sumitomo Foundation grant to N.K.
PY - 2005/2/4
Y1 - 2005/2/4
N2 - A chicken B lymphoma line, DT40, hypermutates immunoglobulin (Ig) genes spontaneously during culture. Thus, cultured DT 40 cells constitute a useful Ig library for screening antibodies (Abs) in vitro. To fix desirable Ig mutants by stopping hypermutation or to resume mutation for further improvement of Ab affinity, activation-induced cytidine deaminase (AID), a key enzyme responsible for the Ig mutation machinery, must be switched on or off. To this end, we generated a DT40 line whose one AID allele was disrupted, and the other allele was replaced by the loxP-flanked AID construct. In this engineered cell line designated as DT40-SW, AID expression could be switched reversibly by tamoxifen-regulated Cre recombinase. Devices were also introduced to discriminate between the "AID-ON" and the "AID-OFF" cells by GFP expression and puromycin resistance, respectively. Starting from a single DT40-SW cell, Ig gene repertoire was efficiently diversified during culture only when AID expression was on.
AB - A chicken B lymphoma line, DT40, hypermutates immunoglobulin (Ig) genes spontaneously during culture. Thus, cultured DT 40 cells constitute a useful Ig library for screening antibodies (Abs) in vitro. To fix desirable Ig mutants by stopping hypermutation or to resume mutation for further improvement of Ab affinity, activation-induced cytidine deaminase (AID), a key enzyme responsible for the Ig mutation machinery, must be switched on or off. To this end, we generated a DT40 line whose one AID allele was disrupted, and the other allele was replaced by the loxP-flanked AID construct. In this engineered cell line designated as DT40-SW, AID expression could be switched reversibly by tamoxifen-regulated Cre recombinase. Devices were also introduced to discriminate between the "AID-ON" and the "AID-OFF" cells by GFP expression and puromycin resistance, respectively. Starting from a single DT40-SW cell, Ig gene repertoire was efficiently diversified during culture only when AID expression was on.
KW - Activation-induced cytidine deaminase
KW - Antibody
KW - DT40
KW - Gene conversion
KW - Hypermutation
KW - Immunoglobulin gene
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U2 - 10.1016/j.bbrc.2004.11.143
DO - 10.1016/j.bbrc.2004.11.143
M3 - Article
C2 - 15629431
AN - SCOPUS:11144234004
SN - 0006-291X
VL - 327
SP - 70
EP - 75
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 1
ER -