2026-06-27T10:32:01Zhttps://docta.ucm.es/rest/oai/request
oai:docta.ucm.es:20.500.14352/930872025-02-27T00:48:28Zcom_20.500.14352_14col_20.500.14352_15
Docta Complutense author Kotzamanis, George author Cheung, Wing author Abdulrazzak, Hassan author Pérez Luz, Sara author Howe, Steven author Cooke, Howard author Huxley, Clare 2024-01-15T12:40:36Z 2024-01-15T12:40:36Z 2005 Kotzamanis, George, et al. «Construction of Human Artificial Chromosome Vectors by Recombineering». Gene, vol. 351, mayo de 2005, pp. 29-38. https://doi.org/10.1016/j.gene.2005.01.017. 0378-1119 10.1016/j.gene.2005.01.017 https://hdl.handle.net/20.500.14352/93087 https://doi.org/10.1016/j.gene.2005.01.017 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. eng Attribution-NonCommercial-NoDerivatives 4.0 International Construction of human artificial chromosome vectors by recombineering journal article 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URL https://docta.ucm.es/bitstreams/5cc16221-39aa-4692-9ece-e7e946a8519d/download File MD5 5b5654b8c2dc17bcf72c7ffd21336753 2440127 application/pdf Human_artificial_chromosome_vectors.pdf