Reversible switching of immunoglobulin hypermutation machinery in a chicken B cell line

Naoki Kanayama, Kagefumi Todo, Michael Reth, Hitoshi Ohmori

Research output: Contribution to journalArticle

15 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)70-75
Number of pages6
JournalBiochemical and Biophysical Research Communications
Volume327
Issue number1
DOIs
Publication statusPublished - Feb 4 2005

Fingerprint

Machinery
Immunoglobulins
Chickens
B-Lymphocytes
Cells
Cell Line
Immunoglobulin Genes
Cell culture
Genes
Alleles
Puromycin
Mutation
Tamoxifen
AICDA (activation-induced cytidine deaminase)
Lymphoma
Screening
Equipment and Supplies
Antibodies
Enzymes

Keywords

  • Activation-induced cytidine deaminase
  • Antibody
  • DT40
  • Gene conversion
  • Hypermutation
  • Immunoglobulin gene

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Reversible switching of immunoglobulin hypermutation machinery in a chicken B cell line. / Kanayama, Naoki; Todo, Kagefumi; Reth, Michael; Ohmori, Hitoshi.

In: Biochemical and Biophysical Research Communications, Vol. 327, No. 1, 04.02.2005, p. 70-75.

Research output: Contribution to journalArticle

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