TY - JOUR
T1 - Genetic manipulation of an exogenous non-immunoglobulin protein by gene conversion machinery in a chicken B cell line
AU - Kanayama, Naoki
AU - Todo, Kagefumi
AU - Takahashi, Satoko
AU - Magari, Masaki
AU - Ohmori, Hitoshi
N1 - Funding Information:
The authors thank Dr Michael Reth (Max-Planck-Institute for Immunology) for kindly providing the tamoxifen-regulated Cre recombinase system and Dr Hiroshi Arakawa (GSF National Research Center for Environment and Health, Neuherverg-Munich, Germany) for pLoxBsr. This work was supported by Grants-in-Aid from The Ministry of Education, Science, Sports and Culture of Japan to H.O. and N.K. K.T. was supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists. Funding to pay the Open Access publication charges for this article was provided by Grants-in-Aid from The Ministry of Education, Science, Sports and Culture of Japan.
PY - 2006/2
Y1 - 2006/2
N2 - 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.
AB - 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.
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U2 - 10.1093/nar/gnj013
DO - 10.1093/nar/gnj013
M3 - Article
C2 - 16421270
AN - SCOPUS:32644480287
SN - 0305-1048
VL - 34
SP - 1
EP - 9
JO - Nucleic acids research
JF - Nucleic acids research
IS - 2
ER -