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Construction of Modular Lentiviral Vectors for Effective Gene Expression and Knockdown

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Lentiviral Vectors and Exosomes as Gene and Protein Delivery Tools

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1448))

Abstract

Elucidating gene function is heavily reliant on the ability to modulate gene expression in biological model systems. Although transient expression systems can provide useful information about the biological outcome resulting from short-term gene overexpression or silencing, methods providing stable integration of desired expression constructs (cDNA or RNA interference) are often preferred for functional studies. To this end, lentiviral vectors offer the ability to deliver long-term and regulated gene expression to mammalian cells, including the expression of gene targeting small hairpin RNAs (shRNAmirs). Unfortunately, constructing vectors containing the desired combination of cDNAs, markers, and shRNAmirs can be cumbersome and time-consuming if using traditional sequence based restriction enzyme and ligation-dependent methods. Here we describe the use of a recombination based Gateway cloning strategy to rapidly and efficiently produce recombinant lentiviral vectors for the expression of one or more cDNAs with or without simultaneous shRNAmir expression. Additionally, we describe a luciferase-based approach to rapidly triage shRNAs for knockdown efficacy and specificity without the need to create stable shRNAmir expressing cells.

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Acknowledgements

This work was supported by the Canadian Institutes of Health Research [operating grant MOP-97925 to DD, Graduate scholarship to AdB]; the Canadian Cancer Society [CCSRI# 2011-700876] and the Canadian Cancer Society [CRS #19087]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Authors and Affiliations

  1. Department of Biology, McGill University, Montréal, QC, H3G 0B1, Canada

    Angeline de Bruyns, Ben Geiling & David Dankort

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  1. Angeline de Bruyns
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  2. Ben Geiling
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  3. David Dankort
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Correspondence to David Dankort .

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  1. Istituto Superiore di Sanità, National AIDS Center, Rome, Italy

    Maurizio Federico

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de Bruyns, A., Geiling, B., Dankort, D. (2016). Construction of Modular Lentiviral Vectors for Effective Gene Expression and Knockdown. In: Federico, M. (eds) Lentiviral Vectors and Exosomes as Gene and Protein Delivery Tools. Methods in Molecular Biology, vol 1448. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3753-0_1

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  • DOI: https://doi.org/10.1007/978-1-4939-3753-0_1

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3751-6

  • Online ISBN: 978-1-4939-3753-0

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