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  • Nonviral Transfer Technology
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Stable integration of large (>100 kb) PAC constructs in HaCaT keratinocytes using an integrin-targeting peptide delivery system

Gene Therapy volume 7, pages 1600–1605 (2000)Cite this article

Abstract

Transfer of large DNA constructs in gene therapy studies is being recognised for its importance in maintaining the natural genomic environment of the gene of interest and providing tissue-specific regulation and control. However, methods used to deliver such constructs have been poorly studied. We used a receptor-mediated, integrin-targeting transfection system enhanced by liposomes, to deliver a 110 kb PAC (P1-based artificial chromosome) to HaCaT keratinocytes. The PAC contained the collagen VII locus, an EGFP (enhanced green fluorescent protein) reporter gene and the puromycin resistance gene (pac) to allow selection of stably transfected cells. Analysis of puromycin resistant and EGFP-expressing colonies by Western blot showed that collagen VII production increased dramatically after transfection, indicating successful transfer of a large fully functional genomic locus. Fluorescent in situ hybridisation (FISH) and Southern blot analysis revealed that the PAC had integrated as at least one copy per cell. EGFP expression has persisted for 35 weeks, suggesting stable transgene expression. We conclude that the integrin-targeting peptide method of gene delivery is an effective means of stably delivering large DNA constructs to human keratinocytes and could be of benefit for genomic gene therapy approaches.

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Acknowledgements

We thank Prof Norbert Fusenig for the gift of HaCaT cells, Dr M McBurney for the gift of a plasmid containing the mouse pgk-1 gene and Dr E Fuchs for the gift of a plasmid containing the keratin 14 promoter. We also thank Rob White for advice on PAC DNA preparation. This work was supported by the European Community (Biomed 2 program, contract BMH4-CT97–2559) and the Dystrophic Epidermolysis Bullosa Research Association (DEBRA) UK.

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Authors and Affiliations

  1. The Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK

    S H Compton, M Rice, E R Levy & A Hovnanian

  2. Department of Dermatology, University of Münster, Münster, Germany

    S Mecklenbeck & L Bruckner-Tuderman

  3. The Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK

    J E Mejía & Z Larin

  4. Molecular Immunology Unit, Institute of Child Health, University College London Medical School, London, UK

    S L Hart

  5. Laboratory of Epithelial Cell Differentiation, Ecole Normale Supérieure, Paris, France

    R Cervini & Y Barrandon

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  1. S H Compton
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Compton, S., Mecklenbeck, S., Mejía, J. et al. Stable integration of large (>100 kb) PAC constructs in HaCaT keratinocytes using an integrin-targeting peptide delivery system. Gene Ther 7, 1600–1605 (2000). https://doi.org/10.1038/sj.gt.3301280

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  • DOI: https://doi.org/10.1038/sj.gt.3301280

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