Published online 26 January 2005
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Rapid creation of BAC-based human artificial chromosome vectors by transposition with synthetic alpha-satellite arrays
1 Institute for Genome Sciences and Policy, Duke University CIEMAS Room 2379, 101 Science Drive, Durham, NC 27708, USA 2 Athersys, Inc. 3201 Carnegie Avenue, Cleveland, OH 44115, USA
*To whom correspondence should be addressed. Tel: +1 919 668 4477; Fax: +1 919 668 0795; Email: joydeep.basu{at}duke.edu
Received October 20, 2004. Revised January 5, 2005. Accepted January 5, 2005.
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.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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