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Howard Cooke

Publications and source records attributed to Howard Cooke.

4 recordsLinked to original sources
PubMed2005

Construction of human artificial chromosome vectors by recombineering.

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 approximately 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 approximately 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.

Cell Line, Tumor↗
PubMed2004

Localisation of centromeric proteins to a fraction of mouse minor satellite DNA on a mini-chromosome in human, mouse and chicken cells.

Centromeres are required for faithful segregation of chromosomes in cell division. It is not clear how centromere sites are specified on chromosomes in vertebrates. We have previously introduced a mini-chromosome, named ST1, into a variety of cell lines including human HT1080, mouse LA9 and chicken DT40. This mini-chromosome, segregating faithfully in these cells, contains mouse minor and major, and human Y alpha-satellite DNA repeats. In this study, after determining the organisation of the satellite repeats, we investigated the location of the centromere on the mini-chromosome by combined immunocytochemistry and fluorescence in situ hybridisation analysis. Centromeric proteins were consistently co-localised with the minor satellite repeats in all three cell lines. When chromatin fibres were highly stretched, centromeric proteins were only seen on a small portion of the minor satellite repeats. These results indicate that a fraction of the minor satellite repeats is competent in centromere function not only in mouse but also in human and chicken cells.

Animals↗
PubMedDate not supplied

Generation of a telomere-based episomal vector.

We have developed a telomere-based episome by large-scale amplification in Escherichia coli cells. This episome consists of a PAC vector in which a 6 Kb sequence, containing an array of telomeric repeats spaced by a synthetic sequence, is tandemly repeated by large-scale multimerization in E. coli. After transfection in human HT1080 cells, the construct, called clone 106, was able to persist in episomal form or integrated into some endogenous chromosomes. Integrations occurred exclusively at the telomeres. Episomes were still present in HT1080 cells after more than 100 days in the absence of selection. Integrations of clone 106 into the telomeric regions were retained only under selective conditions, and when the selection was removed the construct was progressively eliminated from the chromosome. The long-term maintenance of clone 106 into human cells as an episome and its ability to integrate transiently into the telomeres of the host chromosomes suggest that this PAC-based episome is potentially a good candidate vector for gene therapy applications.

Cell Line, Tumor↗