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Nucleic Acids Research 2006 34(2):e10; doi:10.1093/nar/gnj013
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Published online 18 January 2006

© The Author 2006. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oxfordjournals.org

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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 and Hitoshi Ohmori*

Department of Biotechnology, Okayama University Tsushima-Naka, 3-1-1, Okayama 700-8530, Japan

*To whom correspondence should be addressed. Tel/Fax: +81 86 251 8197; Email: hit2224{at}cc.okayama-u.ac.jp

Received December 12, 2005. Revised January 9, 2006. Accepted January 9, 2006.

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.

Correspondence may also be addressed to Naoki Kanayama. Email: nkanayam{at}cc.okayama-u.ac.jp


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