Gene Therapy (2002) 9, 724–726  2002 Nature Publishing Group All rights reserved 0969-7128/02 $25.00 www.nature.com/gt Construction of neocentromere-based human minichromosomes for gene delivery and centromere studies LH Wong, R Saffery and KHA Choo The Murdoch Childrens Research Institute, Royal Children’s Hospital, Melbourne, Victoria, Australia Human neocentromeres are fully functional centromeres that arise naturally in non-centromeric regions devoid of -satel- lite DNA. We have successfully produced a series of minich- romosomes 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 Keywords: neocentromere; human engineered chromosomes; CENP-A; gene delivery Introduction Centromeres are essential for the process of stable chro- mosome inheritance. They are the site of kinetochore for- mation and are responsible for attachment to, and move- ment along microtubules for the faithful separation of chromosomes during mitotic and meiotic cell divisions. In recent years, there have been major efforts in the study of simple and complex centromeres and in the appli- cation of centromere knowledge for the construction of human engineered chromosomes (HECs) to provide a tool to further understand chromosome and centromere properties, and for potential gene delivery and expression studies in gene therapy. Other authors in this volume have covered the use of conventional centrom- eres that contain repetitive DNA in HEC construction. We will focus specifically on the use of non-repetitive DNA-based centromeres (or neocentromeres) to achieve HEC construction, and discuss some of the unique appli- cations of the ensuing HECs. Neocentromere discovery and its wide distribution within the human genome In 1993, we described the first neocentromere lacking the -satellite DNA traditionally associated with human cen- tromere function. 1 This neocentromere was identified on a mitotically stable marker chromosome, designated mardel(10), that was devoid of normal centromeric -sat- ellite DNA but had acquired a fully functional kineto- chore at an interstitial 10q25 region with a DNA content Correspondence: KHA Choo, The Murdoch Childrens Research Institute, Royal Children’s Hospital, Flemington Road, Melbourne, Victoria 3052, Australia 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 com- plex eukaryotic centromeres and as vectors for therapeutic gene delivery. Gene Therapy (2002) 9, 724–726. DOI: 10.1038/sj/gt/3301756 that appears unaltered from the progenitor chromosome 10. 2–6 Recently, we have used a procedure based on chromatin immunoprecipitation and array (CIA) analysis to determine the distribution pattern of the histone-H3- related centromere binding protein CENP-A – a key component of the centromeric nucleosome that distingu- ishes functional centromeres from the surrounding chromatin. Using this procedure, which involves immun- oprecipitation of centromeric chromatin and the hybridization of the extracted DNA to contiguous gen- omic BAC arrays constructed across neocentromeric regions, we have identified a 330 kb and 460 kb CENP- A-binding domain on the 10q25 and a second 20p12 neo- centromere, respectively. 7,8 Up to the present time, over 51 cases of human neocen- tromeres originating from 18 different human chromo- somes have been described at different genomic locations, thus establishing neocentromeres as a relatively widespread phenomenon within the human genome. 9,10 Construction of neocentromere-based minichromosomes (NC-MiCs) We have employed the strategy of telomere-associated chromosome truncation to remove non-essential chromo- some arms to produce minichromosomes in situ. Human telomeric DNA consists of simple tandem repeats of a hexanucleotide TTAGGG sequence. By introducing small arrays of cloned telomeric DNA into cells, it is possible to truncate the distal portion of a chromosomal arm hence leading to the seeding of a new telomere at the site of integration. Targeted truncation is facilitated by the inclusion of homologous genomic sequences within the truncation construct. Through sequential rounds of tar- geted truncation using homologous sequences in close proximity to the neocentromere, both chromosomal arms