Rapid creation of BAC-based human artificial chromosome vectors by transposition with synthetic alpha-satellite arrays.
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.
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Related Subject Headings
- Humans
- Genomics
- Genetic Engineering
- Gene Transfer Techniques
- Developmental Biology
- DNA-Binding Proteins
- DNA, Satellite
- DNA Transposable Elements
- Chromosomes, Artificial, Human
- Chromosomes, Artificial, Bacterial
Citation
Published In
DOI
EISSN
ISSN
Publication Date
Volume
Issue
Start / End Page
Related Subject Headings
- Humans
- Genomics
- Genetic Engineering
- Gene Transfer Techniques
- Developmental Biology
- DNA-Binding Proteins
- DNA, Satellite
- DNA Transposable Elements
- Chromosomes, Artificial, Human
- Chromosomes, Artificial, Bacterial