| Budget Amount *help |
¥16,680,000 (Direct Cost: ¥15,300,000、Indirect Cost: ¥1,380,000)
Fiscal Year 2007: ¥5,980,000 (Direct Cost: ¥4,600,000、Indirect Cost: ¥1,380,000)
Fiscal Year 2006: ¥4,900,000 (Direct Cost: ¥4,900,000)
Fiscal Year 2005: ¥5,800,000 (Direct Cost: ¥5,800,000)
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| Research Abstract |
Gene targeting is a method to manipulate a specific genomic loci by using homologous recombination that is one of an intrinsic DNA repair system. Since only the target gene can be disrupted precisely, this is a faithful method as compared with RNAi.
In this project, to improve the gene targeting efficiency, we constructed in vivo homologous recombination (HR) assay system and screened the genes related to HR in insect cells. As a result, we isolated silkworm AGO2 gene as a suppressor of HR at an extra-chromosomal DSB site. Furthermore, our results suggested that the Ago2 protein, at least in part, plays an important role to discriminate whether DSB is on the chromosome or not, through chromatin.
Consequently, we isolated and characterized chromatin regulators from silkworm cells to utilize the improvement of gene targeting efficiency. In silkworm chromatin, histone H3K9 was possible to be mono-, di-, or tri-methylated by. Su(Var)3-9. Furthermore, methylated H3K9 was recognized by HP1a and HP1b. Compared with other organisms, recruitment of the HP1a to the 5'-flanking region, however, is less effective to induce the transcription repression via hetrochromatin formation.
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