Construction of human artificial chromosome vectors by recombineering George Kotzamanis a , Wing Cheung a , Hassan Abdulrazzak a,b , Sara Perez-Luz a , Steven Howe a,1 , Howard Cooke c , Clare Huxley a, T a Division of Biomedical Sciences, Imperial College London, Exhibition Road, South Kensington, London SW7 2AZ, UK b MRC Clinical Sciences Centre, Imperial College London, Hammersmith Hospital Campus, London W12 ONN, UK c MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK Received 4 October 2004; received in revised form 9 January 2005; accepted 24 January 2005 Received by V. Larionov Available online 15 April 2005 Abstract Human artificial chromosomes (HACs) can be formed de novo by transfection of large fragments of cloned alphoid DNA into human HT1080 cells in tissue culture. In order to generate HACs carrying a gene of interest, one can either co-transfect the alphoid DNA and the gene of interest, or one can clone both into a single vector prior to transfection. Here we describe linking ~70 kb of alphoid DNA onto a 156- kb BAC carrying the human HPRT gene using Red homologous recombination in the EL350 Escherichia coli host [Lee et al., Genomics 73 (2001) 56–65]. A selectable marker and EGFP marker were then added by loxP/Cre recombination using the arabinose inducible cre gene in the EL350 bacteria. The final construct generates minichromosomes in HT1080 cells and the HPRT gene is expressed. The retrofitting vector can be used to add the ~70 kb of alphoid DNA to any BAC carrying a gene of interest to generate a HAC vector. The method can also be used to link any unrelated BAC or PAC insert onto another BAC clone. The EL350 bacteria are an excellent host for building up complex vectors by a combination of homologous and loxP/Cre recombination. D 2005 Elsevier B.V. All rights reserved. Keywords: Homologous recombination; HAC; MAC; HPRT; BAC; Centromere; Minichromosome 1. Introduction Human artificial chromosomes (HACs) can be formed de novo when alphoid DNA is transfected into HT1080 cells (Harrington et al., 1997; Ikeno et al., 1998). The resulting minichromosomes are stably maintained at low copy number in the human cells by a functioning centromere. Such HAC vectors have been used for the expression of therapeutic genes in human cells (Grimes et al., 2001; Mejı ´a et al., 2001a,b; Ikeno et al., 2002). HACs have various advantages as gene expression vectors with potential for use in gene therapy. They are stably maintained alongside the host genome without selection and should therefore confer long-term gene expression without selection even in dividing cells, after only a single administration. Stable maintenance is achieved without integration so there is little danger from insertional mutagenesis that has been found to be a serious problem with integrating retrovirus vectors. They also contain no viral DNA or proteins so they should not cause severe immunogenic responses that have been found to be a serious problem with adenoviral vectors. Finally, they are by nature large and are thus suitable for carrying intact mammalian genes surrounded by all their long-range controlling elements that should confer physiological levels of fully controlled gene expression. Bacterial artificial chromosomes (BACs) provide a con- venient vector for construction and production of HAC vector 0378-1119/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2005.01.017 Abbreviations: HAC, human artificial chromosome; BAC, bacterial artificial chromosome; PAC, P1 artificial chromosome; HPRT, hypoxan- thine phosphoribosyltransferase; EGFP, enhanced green fluorescent protein. T Corresponding author. Tel.: +44 20 7594 3028; fax: +44 20 7594 3015. E-mail address: c.huxley@imperial.ac.uk (C. Huxley). 1 Current address: Molecular Immunology Unit, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK. Gene 351 (2005) 29 – 38 www.elsevier.com/locate/gene