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Infectious delivery of the 132 kb CDKN2A/CDKN2B genomic DNA region results in correctly spliced gene expression and growth suppression in glioma cells

Gene Therapy volume 11, pages 1195–1204 (2004)Cite this article

Abstract

The expression of genes from genomic loci can be relatively complex, utilizing exonic, intronic and flanking sequences to regulate tissue and developmental specificity. Infectious bacterial artificial chromosomes (iBACs) have been shown to deliver and express large genomic loci (up to 135 kb) into primary cells for functional analyses. The delivery of large genomic DNA inserts allows the expression of complex loci and of multiple splice variants. Herein, we demonstrate for the first time that an iBAC will deliver and correctly express in human glioma cells the entire CDKN2A/CDKN2B genomic region, which encodes for at least three important cell-cycle regulatory proteins (p16INK4a, p14ARF and p15INK4b). Two of these proteins are expressed from overlapping genes, utilizing alternative splicing and promoter usage. The delivered locus expresses each gene at physiological levels and cellular responses (apoptosis versus growth arrest) occur dependent on cellular p53 status, as expected. The work further demonstrates the potential of the iBAC system for the delivery of genomic loci whose expression is mediated by complex splicing and promoter usage both for gene therapy applications and functional genomics studies.

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Acknowledgements

We thank Dr David Louis (Massachusetts General Hospital, Charlestown, MA, USA) for providing samples from the Massachusetts General Hospital Brain Tumor Bank, for initially suggesting this experiment and for helpful discussion and advice. This work was supported by grants from The Wellcome Trust, the National Cancer Institute (CA692460) and the Berkowitz-Knott Fund for Brain Tumor Research to EAC. RW-M was a Wellcome Trust International Prize Travelling Research Fellow, and thanks the Trust for their continued support.

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  1. R Wade-Martins

    Present address: The Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK

Authors and Affiliations

  1. Molecular Neuro-Oncology Laboratories, Neurosurgery Service, Massachusetts General Hospital-East and Harvard Medical School, Charlestown, MA, USA

    R Inoue, K A Moghaddam, M Ranasinghe, Y Saeki, E A Chiocca & R Wade-Martins

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Inoue, R., Moghaddam, K., Ranasinghe, M. et al. Infectious delivery of the 132 kb CDKN2A/CDKN2B genomic DNA region results in correctly spliced gene expression and growth suppression in glioma cells. Gene Ther 11, 1195–1204 (2004). https://doi.org/10.1038/sj.gt.3302284

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