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Effect of nuclear localization and hydrodynamic delivery-induced cell division on φC31 integrase activity

Gene Therapy volume 17, pages 217–226 (2010)Cite this article

Abstract

Phage φC31 integrase is a recombinase that can be expressed in mammalian cells to integrate plasmids carrying an attB sequence into the genome at specific pseudo attP locations. We show by immunofluoresence that wild-type φC31 integrase is cytoplasmic and that addition of the SV40 nuclear localization signal (NLS) localized φC31 integrase to the nucleus. Unexpectedly, the NLS depressed integration efficiency in HeLa cells and provided no benefit when used to integrate the human Factor IX (hFIX) gene into mouse liver. As breakdown of the nuclear membrane during mitosis could allow cytoplasmic integrase access to the chromosomes, we analyzed whether cell division was required for integration into liver cells in vivo. Hepatocytes were labeled with iododeoxyuridine to mark cells that underwent DNA replication during the week after hydrodynamic injection. Hydrodynamic delivery led to DNA replication in one-third of hepatocytes. Approximately three out of four cells having φC31 integrase-mediated stable hFIX expression did not undergo replication, indicating that cell division was not required for integrase function in liver. Therefore, although the bulk of φC31 integrase protein seems to be cytoplasmic in mammalian cells, integration can still occur in the nucleus, even without cell division.

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Acknowledgements

Many thanks to Julien Sage and members of his laboratory at Stanford for assistance with the liver-staining experiments. Thanks to Anne Brunet and members of her laboratory for assistance with their microscope. We appreciate the contributions of Christopher L Chavez to the conclusions made in this paper. PHS Grant Number CA09302, awarded by the National Cancer Institute, DHHS, supported LEW. RTH was supported by a medical research training fellowship from the Howard Hughes Medical Institute. This work was supported by NIH grant HL068112 to MPC.

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  1. Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA

    L E Woodard, R T Hillman, A Keravala, S Lee & M P Calos

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  1. L E Woodard
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  2. R T Hillman
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  3. A Keravala
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  4. S Lee
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Correspondence to M P Calos.

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Woodard, L., Hillman, R., Keravala, A. et al. Effect of nuclear localization and hydrodynamic delivery-induced cell division on φC31 integrase activity. Gene Ther 17, 217–226 (2010). https://doi.org/10.1038/gt.2009.136

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