Application of a Bacterial Artificial Chromosome Modification System for a Human Artificial Chromosome Vector

Exactly controlled conditional gene expressing systems are crucial for genomic functionalresearch, animal transgenesis and gene therapy. Bacterial artificial chromosomes (BACs)are optimal for harboring long fragments of genomic DNA or large cDNA up to 300 kb insize. Therefore, BACs are available to produce transgenic cells and animals for thefunctional studies of genes. However, BAC can insert DNA randomly into the host genome,possibly causing unpredicted expression. We previously developed a human artificialchromosome (HAC) vector from human chromosome 21 using chromosome engineering. The HACvector has several important characteristics desired for an ideal gene delivery vector,including stable episomal maintenance, and the ability to carry large genomic DNAcontaining its own regulatory element, thus allowing physiological regulation of thetransgene in a manner similar to that of the native chromosome. In this study, we developa system fusing BAC library and HAC technology together to allow tight control of geneexpression. This system enables BAC to be cloned into the defined locus on the HAC vectorby the Cre/loxP system. In addition, the genome in the BAC is possible to be engineeredfreely by the BAC recombineering technology. This system is a highly efficient tool forthe rapid generation of stringently controlled gene expression system on the HACvector.