Reconstitution of DNA fragments on HAC/MAC via the fragment-assembly system

Human/mouse artificial chromosomes (HACs/MACs) serve as gene vectors for transferring of megabase DNA sequences and multiple gene cassettes into mammalian cells. However, their full potential is hindered by the lack of effective gene-loading methods. In this study, we developed a fragment assembly (FA) system that enables reconstitution of introduced sequences in HACs/MACs by simultaneously loading multiple gene-loading vectors (GLVs) while removing the GLV vector backbone sequences. As a proof of principle, we simultaneously loaded three GLVs, which encode the promoter, open reading frame of a gene, and a poly A signal sequence, onto a MAC carrying a gene-loading pad for the FA system. We successfully obtained clones bearing a reconstituted gene expression cassette on the MAC. Further introduction of three GLVs in two gene-loading steps enabled the reconstitution of another gene expression cassette in the MAC. The gene-loaded MAC was transferable to other cells, and the introduced cells expressed the reconstituted genes. These data demonstrate that the FA system is useful for reconstituting multiple sequences in HACs/MACs.