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Journal of Virology, January 2009, p. 283-294, Vol. 83, No. 1
0022-538X/09/$08.00+0     doi:10.1128/JVI.01865-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Insertional Gene Activation by Lentiviral and Gammaretroviral Vectors{triangledown} ,{dagger}

Marieke Bokhoven,1 Sam L. Stephen,1 Sean Knight,1 Evelien F. Gevers,2 Iain C. Robinson,2 Yasuhiro Takeuchi,1 and Mary K. Collins1*

Infection and Immunity, University College London, Windeyer Building, 46 Cleveland Street, London W1T 2AH,1 Division of Molecular Neuroendocrinology, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom2

Received 4 September 2008/ Accepted 15 October 2008

Gammaretroviral and lentiviral vectors are promising tools for gene therapy, but they can be oncogenic. The development of safer vectors depends on a quantitative assay for insertional mutagenesis. Here we report a rapid, inexpensive, and reproducible assay which uses a murine cell line to measure the frequency of interleukin-3 (IL-3)-independent mutants. Lentiviral and gammaretroviral vectors cause insertional mutagenesis at similar frequencies; however, they use different mechanisms. Human immunodeficiency virus (HIV)-based vectors generate mutants by insertion only into the growth hormone receptor (Ghr) locus. The HIV enhancer/promoter is active in the absence of the HIV Tat protein in this locus, and an HIV/Ghr spliced transcript expresses GHR and cells respond to GH. Deletion of the enhancer/promoter in a self-inactivating HIV-based vector prevents this mechanism of insertional mutagenesis. In contrast, gammaretroviral vectors insert into other loci, including IL-3 and genes identified as common insertion sites in the Retroviral Tagged Cancer Gene Database (RTCGD).

* Corresponding author. Mailing address: Infection and Immunity, University College London, Windeyer Building, 46 Cleveland Street, London W1T 2AH, United Kingdom. Phone and fax: 44-2076799301. E-mail: mary.collins{at}ucl.ac.uk

{triangledown} Published ahead of print on 22 October 2008.

{dagger} Supplemental material for this article may be found at http://jvi.asm.org/.

Journal of Virology, January 2009, p. 283-294, Vol. 83, No. 1
0022-538X/09/$08.00+0     doi:10.1128/JVI.01865-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



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