|Budget Amount *help
¥3,600,000 (Direct Cost : ¥3,600,000)
Fiscal Year 2003 : ¥1,500,000 (Direct Cost : ¥1,500,000)
Fiscal Year 2002 : ¥2,100,000 (Direct Cost : ¥2,100,000)
In the chicken B-cell line DT40, gene conversion, a type of homologous recombination, primarily contributes to diversification of the immunoglobulin (Ig) gene. We have studied roles of chromatin structure in the regulation of this gene conversion in the chicken DT40 cell lines. We found that recombination-active Ig locus exhibits much higher levels of histone acetylation. Treatment of DT40 cells with histone deacetylase inhibitor Tricostatin A (TSA) markedly induced gene conversion frequency and transcriptional activity at recombination-active allele, but observed elevation of histone acetylation levels in both recombination-active and inactive-alleles, suggesting links between recombination activation and transcriotional activity.
Using DT40 cells harboring an artificial chromosomal recombination marker including partial GFP sequence, complete CFP sequence combined with inducible Tet promoter, 3' enhancer, and a matrix attachment region sequence, we further examined relationship between transcriptional activity and gene conversion frequency. After transcriptional activation, gene conversion events between partial GFP and CFP sequences were induced; thereby generating GFP-type fluorescence was detected in a small portion of the DT40 cells. Thus, it is suggested that gene conversion may be activated coupled to some transcription-related mechanisms.
In addition, using the effects by TSA on gene conversion, we developed a rapid and flexible system to produce specific monoclonal antibodies using the DT40 cells undergoing activated gene conversion. The selection system (ADLib: Autonomously Diversifying Library) enables quick establishment (〜one week from a diversifying library) of various clones producing monoclonal IgMs with enough specificity and affinity for immunological assays, and is applicable to various biotechnologies including rational protein design.