RRC ID 1522
Author Kanayama N, Todo K, Reth M, Ohmori H.
Title Reversible switching of immunoglobulin hypermutation machinery in a chicken B cell line.
Journal Biochem Biophys Res Commun
Abstract 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.
Volume 327(1)
Pages 70-5
Published 2005-2-4
DOI 10.1016/j.bbrc.2004.11.143
PII S0006-291X(04)02753-6
PMID 15629431
MeSH Animals Attachment Sites, Microbiological / genetics B-Lymphocytes / metabolism* B-Lymphocytes / pathology Cell Line, Tumor Chickens / genetics* Chickens / immunology Cytidine Deaminase / genetics* Cytidine Deaminase / metabolism* Drug Resistance / genetics Green Fluorescent Proteins / genetics Hydroxytestosterones / pharmacology Immunoglobulins / genetics* Puromycin / pharmacology Somatic Hypermutation, Immunoglobulin / genetics* Tamoxifen / analogs & derivatives* Tamoxifen / pharmacology
IF 2.985
Times Cited 13
Human and Animal Cells DT40(RCB1464)