Human artificial chromosomes containing chromosome 17 alphoid DNA maintain an active centromere in murine cells but are not stable Anas M. Alazami, Jose ´ E. Mejı ´a, 1 and Zoia Larin Monaco * Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headley Way, Oxford OX3 9DS, United Kingdom Received 15 September 2003; accepted 14 November 2003 Abstract Human artificial chromosomes (HACs) are autonomous molecules that can function and segregate as normal chromosomes in human cells. De novo HACs have successfully been used as gene expression vectors to complement genetic deficiencies in human cultured cells. HACs now offer the possibility of studying the regulation and expression of large genes in a variety of cell types from different tissues and correcting gene deficiencies caused by human inherited diseases. Complementary gene expression studies in mice, especially in mouse models of human genetic diseases, are also important in determining if large human transgenes can be expressed appropriately from artificial chromosomes. Toward this aim we are establishing artificial chromosomes in murine cells as novel gene expression vectors. Initially we transferred HAC vectors into murine cells, but were unable to generate de novo HACs at a reasonable frequency. We then transferred HACs previously established in human HT1080 cells to three different murine cell types by microcell fusion, followed by positive selection. We observed that the HACs in murine cells bound centromere protein C (CENP-C), a marker of active centromeres, and were detected under selection but rapidly lost when selection was removed. These results suggest that the HACs maintain at least a partially functional centromere complex in murine cells, but other factors are required for stability and segregation. Artificial chromosomes containing mouse centromeric sequences may be required for better stability and maintenance in murine cells. D 2004 Elsevier Inc. All rights reserved. Introduction HACs have been generated in the human fibrosarcoma cell line (HT1080) either by modifying natural chromo- somes via telomere-directed truncation, producing succes- sively smaller minichromosomes [1,2], or by introducing defined sequences such as human telomeres, alphoid DNA, and specific genomic fragments into human cells to generate de novo artificial chromosomes [3–7]. Both approaches have determined that alphoid DNA forms a functional centromere in a human artificial chromosome (HAC). In addition, the presence of a centromere protein B sequence (referred to as a CENP-B box) in alphoid DNA is a requirement for the formation of a functional centromere in a de novo HAC [8]. Work from two groups has shown that HACs can function as gene expression vectors to correct a genetic defect. HAC constructs containing chromosome 17 alphoid (17a) DNA and the hypoxanthine guanine phosphoribosyl- transferase (HPRT) genomic locus generated de novo arti- ficial chromosomes in HPRT-deficient HT1080 cells, which complemented the deficiency [7,9]. HACs were relatively stable for several months without selection and contained one or more copies of the HPRT gene. In further studies, HAC vectors containing 17a DNA generated de novo HACs at a much higher frequency in HT1080 cells in comparison to similar vectors with Y alphoid DNA, sug- gesting that different alphoid DNA templates may have the capability of generating HACs at different frequencies [10,11]. Minichromosomes have been established in mice follow- ing transfer of engineered human Y minichromosomes containing mouse centromeric DNA [12,13], human chro- mosome fragments [14–16], or stable small accessory chromosomes [17]. Although reports have indicated varia- 0888-7543/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ygeno.2003.11.011 * Corresponding author. Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headley Way, Oxford UK OX3 9DS. E-mail address: zoia.larin-monaco@molecular-medicine.oxford.ac.uk (Z.Larin Monaco). 1 Present address: INSERM U563, Pavillon Lefebvre, Purpan Hospital, Place du Dr Baylac, 31059 Toulouse cedex 03, France. www.elsevier.com/locate/ygeno Genomics 83 (2004) 844 – 851