|Budget Amount *help
¥3,500,000 (Direct Cost : ¥3,500,000)
Fiscal Year 2005 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 2004 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 2003 : ¥1,800,000 (Direct Cost : ¥1,800,000)
1. The improvement of detection methods of DNA-PKcs mutation
For typing of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) mutation to obtain the mice used the following experiments ; the detection methods of DNA-PKcs mutation were improved by newly designing primers, making a fluoresence-labeled oligomer and the application of AFLP analysis using a DNA sequencer.
2. The effect of DNA-PKcs deficiency on mutant frequency and mutation spectrum in spontaneous and AOM-induced mutations
The detection of deletion mutations by Spi^- assay was performed. In spontaneous mutations, no significant difference in mutant frequencies (MF) was detected between homozygotes (scid/scid) and wild type mice (+/+) of DNA-PKcs mutation, but in azoxymethane (AOM)-induced mutation, MF of scid/scid were detected with a higher tendency than those of +/+ (P=0.12). By analysis of mutation spectra, deletion mutations more than 1 kbp (large deletion) in mutants obtained from scid/scid were tended to be more than those from +/+, and in AOM-induced mutation, those from scid/scid were significantly higher than those from +/+.
3. The effect of haploid insufficiency of DNA-PKcs in AOM-induced mutation and colon carcinogenesis
In the case of spontaneous mutation, MF obtained from heterozygotes (scid/+) had a higher tendency than those from +/+ in deletion mutations less than 100 bp (small deletion). In the case of AOM-induced mutation, MF from scid/scid, scid/+ and +/+ were 7.3±1.6, 25.9±12.5, 4.7±1.1 per 10^6 plaques. MFs from scid/+ were significantly higher than those from +/+ in small deletions and also large deletions. In DNA repair of small deletions and also large deletions, the possibility was suggested of the presence of haploid insufficiency. This was proceeding histological analysis in colon carcinogenesis.