Genetic manipulation of an exogenous non-immunoglobulin protein by gene conversion machinery in a chicken B cell line

Naoki Kanayama, Kagefumi Todo, Satoko Takahashi, Masaki Magari, Hitoshi Ohmori

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

During culture, a chicken B cell line DT40 spontaneously mutates immunoglobulin (Ig) genes by gene conversion, which involves activation-induced cytidine deaminase (AID)-dependent homologous recombination of the variable (V) region gene with upstream pseudo-V genes. To explore whether this mutation mechanism can target exogenous non-Ig genes, we generated DT40 lines that bears a gene conversion substrate comprising the green fluorescent protein (GFP) gene as a donor and the blue fluorescent protein (BFP) gene as an acceptor. A few percent of the initially BFP-expressing cells converted their fluorescence from blue to green after culture for 2-3 weeks when the substrate construct was integrated in the Ig light chain locus, but not in the ovalbumin locus. This was the result of AID-dependent and the GFP gene-templated gene conversion of the BFP gene, thereby leading to the introduction of various sizes of GFP-derived gene segment into the BFP gene. Thus, G/B construct may be used to visualize gene conversion events. After switching off AID expression in DT40 cells, the mutant clones were isolated stably and maintained with their mutations being fixed. Thus, the gene conversion machinery in DT40 cells will be a useful means to engineer non-Ig proteins by a type of DNA shuffling.

Original languageEnglish
Pages (from-to)1-9
Number of pages9
JournalNucleic acids research
Volume34
Issue number2
DOIs
Publication statusPublished - Feb 2006

ASJC Scopus subject areas

  • Genetics

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