Construction of neocentromere-based human minichromosomes for gene delivery and centromere studies

Human neocentromeres are fully functional centromeres that arise naturally in non-centromeric regions devoid of alpha-satellite DNA. We have successfully produced a series of minichromosomes by telomere-associated truncation of a marker chromosome mardel(10) containing a neocentromere. The resulting minichromosomes are either linear or circular in nature, and range in size from approximately 650 kb to 2 Mb. These minichromosomes exhibit full centromeric activity, bind to essential centromere proteins, and are mitotically stable over many generations. They provide a useful system for dissecting the functional domains of complex eukaryotic centromeres and as vectors for therapeutic gene delivery.