使用表面固定有酵母人工染色体的生物活性珠子对酵母进行转化。
Transformation of yeast using bioactive beads with surface-immobilized yeast artificial chromosomes.
作者信息
Kawakami Shigeki, Harashima Satoshi, Kobayashi Akio, Fukui Kiichi
机构信息
Venture Business Laboratory Center for Advanced Science and Innovation, Osaka University, Japan.
出版信息
Methods Mol Biol. 2006;349:61-5. doi: 10.1385/1-59745-158-4:61.
Yeast artificial chromosomes (YACs) are useful cloning vectors with the capacity to carry large DNA inserts. The largest barrier using such large DNA molecules in transformation experiments has been their physical instability in a solution. We developed a new method for transforming yeast with chromosome-sized DNA. The method uses bioactive beads composed of calcium alginate to immobilize yeast chromosomal DNAs. Chromosomal DNA immobilized on bioactive beads is physically stable when compared with naked chromosomal DNAs. The bead-mediated transformation performed well, not only with respect to the transformation frequency, but also in successful transformation using split chromosomal DNA that exceeded 450 kb in size. In this chapter we introduce a new method for transforming yeast using bioactive beads. In conjunction with genomic YAC libraries and the yeast chromosome-splitting method, this technique will pave the way to stable and effective transfer of YACs into yeast cells.
酵母人工染色体（YACs）是有用的克隆载体，能够携带大片段DNA插入物。在转化实验中使用如此大的DNA分子的最大障碍是它们在溶液中的物理不稳定性。我们开发了一种用染色体大小的DNA转化酵母的新方法。该方法使用由海藻酸钙组成的生物活性珠子来固定酵母染色体DNA。与裸露的染色体DNA相比，固定在生物活性珠子上的染色体DNA在物理上是稳定的。珠子介导的转化不仅在转化频率方面表现良好，而且在使用大小超过450 kb的分裂染色体DNA进行成功转化方面也表现出色。在本章中，我们介绍一种使用生物活性珠子转化酵母的新方法。结合基因组YAC文库和酵母染色体分裂方法，这项技术将为YACs稳定有效地转移到酵母细胞中铺平道路。
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