RT Journal Article
T1 Construction of human artificial chromosome vectors by recombineering
A1 Kotzamanis, George
A1 Cheung, Wing
A1 Abdulrazzak, Hassan
A1 Pérez Luz, Sara
A1 Howe, Steven
A1 Cooke, Howard
A1 Huxley, Clare
AB 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.
SN 0378-1119
YR 2005
FD 2005
LK https://hdl.handle.net/20.500.14352/93087
UL https://hdl.handle.net/20.500.14352/93087
LA eng
NO 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.
NO European Commission
DS Docta Complutense
RD 15 abr 2025