Gene transfer methods using mammalian artificial chromosomes

我们的研究目标是开发利用哺乳动物人工染色体作为基因传递载体的基本技术，并与哥伦比亚大学的P.Hieter教授和加州大学的K.Mitani教授合作进行这项研究。冈崎的团队对HAC的构建提出了挑战，在HAC中，包含大量人类基因组区域的YAC克隆被用作起始材料。YAC克隆的臂被人端粒序列、loxP序列和哺乳动物选择标记修饰。为了通过Cre/loxP重组系统将Alphid序列插入到修饰的YAC中，构建了携带100kb Alphid序列和loxP序列的BAC。为了产生HAC，没有酵母基因组DNA的YAC纯化方法是至关重要的，我们通过与Hieter教授的讨论获得了有用的信息。为了将该HACs作为基因载体用于其他细胞系，我们利用微细胞介导的染色体转移(MMCT)方法将HT1080细胞系中形成的HACs导入小鼠A9细胞。我们证实，转移到A9细胞中的HAC保持稳定。Mitani的团队与我们的团队合作，成功地构建了含有人端粒序列和辅助依赖型腺病毒载体中的Alphid序列的Ad/HAC载体。用脂质体转染法将该DNA导入HT1080细胞，获得转化子。我们目前正在分析转化子中HAc的形成。HAc作为病毒颗粒感染细胞后，能否有效地由Ad/HAc载体形成HAc是今后的重要研究课题。

Our research objective is the development of basic technologies to utilize mammalian artificial chromosomes for gene delivery vectors and we collaborated with Prof. P.Hieter at Columbia University and Prof. K.Mitani at California University to proceed the research. Okazaki's group challenged the construction of HACs in which a YAC clone containing a large human genomic area was used as a starting material. The arms of the YAC clone were modified with human telomere sequences, loxP sequence and mammalian selection marker. To insert alphoid sequence into the modified YAC by Cre/loxP recombination system, a BAC carrying 100kb alphoid sequence and loxP sequence was constructed. In order to generate HACs, the method of the YAC purification free from yeast genomic DNA was crucial and we obtained useful information through discussion with Prof. Hieter. In order to use this HACs as gene delivery vectors in the other cell lines, we transferred HACs, which had been formed in the HT1080 cell line, into mouse A9 cells by microcell-mediated chromosome transfer (MMCT) method. We confirmed that HACs transferred into the A9 cells were stably maintained. Mitani's group in collaboration with our group succeeded in the construction of Ad/HAC vector containing human telomere sequences and alphoid sequence in a helper-dependent adenovirus vector. When this DNA was introduced into HT1080 cells by lipofection, transformants were obtained. We are currently analyzing the HAC formation in the transformants. It is important future subject whether HAC is formed efficiently from Ad/HAC vectors when infected to cells as virus particles.