Rapid creation of BAC-based human artificial chromosome vectors by transposition with synthetic alpha-satellite arrays.
Rapid creation of BAC-based human artificial chromosome vectors by transposition with synthetic alpha-satellite arrays.
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DOI：
10.1093/nar/gki207
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发表时间：
2005
影响因子：
14.9
通讯作者：
Van Bokkelen G
中科院分区：
文献类型：
--
作者：
Basu J;Stromberg G;Compitello G;Willard HF;Van Bokkelen G
高效构建基于BAC的人类人工染色体（HACs）需要优化每个关键功能单元以及开发用于快速和可靠操作高分子量BAC载体的技术。在此，我们基于天然D17 Z1阵列典型的16单体重复长度创建了合成的17号染色体衍生的α-卫星阵列，其中与D17 Z1 α-卫星（5/16单体）相比，共有CENP-B盒元件完全不存在（0/16单体）或密度增加（16/16单体）。使用这些载体，我们表明，CENP-B盒元件的存在是一个有效的从头着丝粒形成的要求，增加CENP-B盒元件的密度可能会提高从头着丝粒形成的效率。此外，我们还开发了一种新的高通量方法，该方法允许通过转座子介导的合成α-卫星阵列和其他关键功能单元的修饰将任何基因组BAC靶标快速转化为HAC载体。总之，这些方法提供了显着推进基于BAC的HAC用于基因组的功能注释和用于基因转移的应用的潜力。
Efficient construction of BAC-based human artificial chromosomes (HACs) requires optimization of each key functional unit as well as development of techniques for the rapid and reliable manipulation of high-molecular weight BAC vectors. Here, we have created synthetic chromosome 17-derived alpha-satellite arrays, based on the 16-monomer repeat length typical of natural D17Z1 arrays, in which the consensus CENP-B box elements are either completely absent (0/16 monomers) or increased in density (16/16 monomers) compared to D17Z1 alpha-satellite (5/16 monomers). Using these vectors, we show that the presence of CENP-B box elements is a requirement for efficient de novo centromere formation and that increasing the density of CENP-B box elements may enhance the efficiency of de novo centromere formation. Furthermore, we have developed a novel, high-throughput methodology that permits the rapid conversion of any genomic BAC target into a HAC vector by transposon-mediated modification with synthetic alpha-satellite arrays and other key functional units. Taken together, these approaches offer the potential to significantly advance the utility of BAC-based HACs for functional annotation of the genome and for applications in gene transfer.