Rapid creation of BAC-based human artificial chromosome vectors by transposition with synthetic alpha-satellite arrays

被引:51
作者
Basu, J
Stromberg, G
Compitello, G
Willard, HF
van Bokkelen, G
机构
[1] Duke Univ, Inst Genome Sci & Policy, CIEMAS, Durham, NC 27708 USA
[2] Athersys Inc, Cleveland, OH 44115 USA
关键词
D O I
10.1093/nar/gki207
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:587 / 596
页数:10
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