Reversible switching of immunoglobulin hypermutation machinery in a chicken B cell line.
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.
Kanayama
Naoki
N
Department of Biotechnology, Okayama University, Tsushima-Naka 3-1-1, Okayama 700-8530, Japan.
Todo
Kagefumi
K
Reth
Michael
M
Ohmori
Hitoshi
H
D000818:Animals; D001287:Attachment Sites, Microbiological / Q000235:genetics; D001402:B-Lymphocytes / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D002645:Chickens / Q000235:genetics* / Q000276:immunology; D003564:Cytidine Deaminase / Q000235:genetics* / Q000378:metabolism*; D004351:Drug Resistance / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D006915:Hydroxytestosterones / Q000494:pharmacology; D007136:Immunoglobulins / Q000235:genetics*; D011691:Puromycin / Q000494:pharmacology; D027041:Somatic Hypermutation, Immunoglobulin / Q000235:genetics*; D013629:Tamoxifen / Q000031:analogs & derivatives* / Q000494:pharmacology; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
D006915:Hydroxytestosterones; D007136:Immunoglobulins; D013629:Tamoxifen; D049452:Green Fluorescent Proteins; C016601:afimoxifene; C032278:4,17 beta-dihydroxy-4-androstene-3-one; D011691:Puromycin; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D003564:Cytidine Deaminase