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Strategies to Insulate Lentiviral Vector-Expressed Transgenes

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Lentivirus Gene Engineering Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 614))

Abstract

Lentiviruses are capable of infecting many cells irrespective of their cycling status, stably inserting DNA copies of the viral RNA genomes into host chromosomes. This property has led to the development of lentiviral vectors for high-efficiency gene transfer to a wide variety of cell types, from slowly proliferating hematopoietic stem cells to terminally differentiated neurons. Regardless of their advantage over gammaretroviral vectors, which can only introduce transgenes into target cells that are actively dividing, lentiviral vectors are still susceptible to chromosomal position effects that result in transgene silencing or variegated expression. In this chapter, various genetic regulatory elements are described that can be incorporated within lentiviral vector backbones to minimize the influences of neighboring chromatin on single-copy transgene expression. The modifications include utilization of strong internal enhancer-promoter sequences, addition of scaffold/matrix attachment regions, and flanking the transcriptional unit with chromatin domain insulators. Protocols are provided to evaluate the performance as well as the relative biosafety of lentiviral vectors containing these elements.

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Acknowledgments

This work was supported in part by National Institutes of Health grants R01HL65519 and R01HL66305, and by an Elaine H. Snyder Cancer Research Award and a King Fahd Endowed Professorship (to R.G.H.) from The George Washington University Medical Center.

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Authors and Affiliations

  1. Department of Anatomy and Regenerative Biology, The George Washington University Medical Center, Washington, DC, USA

    Ali Ramezani & Robert G. Hawley

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  1. Ali Ramezani
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  2. Robert G. Hawley
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Correspondence to Robert G. Hawley .

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  1. Div. Patogenesi dei Retrovirus, Centro Nazionale AIDS, Viale Regina Elena 299, Roma, 00161, Italy

    Maurizio Federico

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Ramezani, A., Hawley, R.G. (2010). Strategies to Insulate Lentiviral Vector-Expressed Transgenes. In: Federico, M. (eds) Lentivirus Gene Engineering Protocols. Methods in Molecular Biology, vol 614. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-533-0_5

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  • DOI: https://doi.org/10.1007/978-1-60761-533-0_5

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