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<p>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.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年2月21日

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  • Teruhiko Suzuki,
  • Mana Yamakawa,
  • Shiho Sasaki,
  • Akane Okada,
  • Kanako Kazuki,
  • Satoshi Abe,
  • Seia Nara,
  • Masayuki Su’etsugu,
  • Takahiko Hara,
  • Yasuhiro Kazuki

摘要

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.