DNA replication timing (RT) refers to the temporal order in which segments of chromosomes are replicated during the cell cycle, which can change during differentiation of embryonic stem cells (ESCs) into defined cell types. Because many diseases such as cancer exhibit a disrupted RT program compared to that of healthy cells, it is possible for the mechanism controlling RT to offer information about the origins of these diseases. It is expected that DNA sequence alone is sufficient to control the mechanism of changes in DNA RT. Introducing artificial chromosomes that carry segments of human DNA into human ESCs will demonstrate whether these segments dictate their original temporal replication. Furthermore, one can observe if the segments then change their RT appropriately during differentiation of ESCs to different cell types. If this hypothesis is correct, proper regulation of RT will be recapitulated in this artificial system. Furthermore, it will be possible to narrow down the smallest sequences necessary to regulate RT changes through various DNA sequence deletions. Through genetic engineering and nucleofection technology, I have already created a stable ESC line with human bacteria artificial chromosomes (BACs) and confirmed their long-term retention (>60 days), showing that my autonomously replication BAC system works. / / Key words: replication timing, bacteria artificial chromosomes, embryonic stem cells, development /
