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Gene targeting is enhanced in human cells overexpressing hRAD51

Gene Therapy volume 6, pages 1282–1290 (1999)Cite this article

Abstract

The ideal therapy for single gene disorders would be repair of the mutated disease genes. Homologous recombination is one of several cellular mechanisms for the repair of DNA damage. Recombination between exogenous DNA and homologous chromosomal loci (gene targeting) can be used to repair an endogenous gene, but the low efficiency of this process is a serious barrier to its therapeutic potential. Recent progress in the isolation and characterisation of mammalian genes and proteins involved in DNA recombination has raised the possibility that the cellular biochemistry of recombination can be manipulated to improve the efficiency of gene targeting. As an initial test of this approach, we have overexpressed the gene encoding hRAD51, a protein with homologous DNA pairing and strand exchange activities, in human cells and measured its effect on gene targeting. We report a two- to three-fold increase in gene targeting, and enhanced resistance to ionising radiation in hRAD51-overexpressing cells with no obvious detrimental effects. These observations provide valuable genetic evidence for the involvement of hRAD51 in both gene targeting and DNA repair in human cells. Our data also establish overexpression of recombination genes as a viable approach to improving gene targeting efficiencies.

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Acknowledgements

We are grateful to Dr Dennis Hellgren for pCMV51, to Dr Elizabeth Fisher for the λDASH II library, to Dr Fiona Benson and Dr Stephen C. West for the hRAD51 antibody and to Mr Peter England for clones of 6–16 genomic DNA.

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Author notes

  1. This paper is dedicated to the memory of Eladio Viñuela

Authors and Affiliations

  1. Gene Targeting Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, DuCane Road, London, W12 0NN, UK

    R J Yáñez & A C G Porter

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  2. A C G Porter
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Yáñez, R., Porter, A. Gene targeting is enhanced in human cells overexpressing hRAD51. Gene Ther 6, 1282–1290 (1999). https://doi.org/10.1038/sj.gt.3300945

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