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Nat Protoc. 2011 Jun;6(6):806-16. doi: 10.1038/nprot.2011.327. Epub 2011 May 19.

A simple, versatile and efficient method to genetically modify human monocyte-derived dendritic cells with HIV-1-derived lentiviral vectors.

Author information

1
Department of Human Virology, Ecole Normale Supérieure de Lyon, France.

Abstract

Lentiviral vectors derived from the human immunodeficiency type 1 virus (HIV-1 LV) are among the finest tools available today for the genetic modification of human monocyte-derived dendritic cells (MDDCs). However, this process is largely inefficient because MDDCs show a strong resistance to HIV-1 transduction. Here we describe a step-by-step protocol from the production of LVs to cell transduction that allows the efficient genetic modification of MDDCs. This protocol can be completed in 23 d from the initial phase of LV production to the final analysis of the results of MDDC transduction. The method relies on the simultaneous addition of HIV-1 LVs along with noninfectious virion-like particles carrying Vpx, a nonstructural protein encoded by the simian immunodeficiency virus (Vpx-VLPs). When thus provided in target cells, Vpx exerts a strong positive effect on incoming LVs by counteracting the restriction present in MDDCs; accordingly, 100% of cells can be transduced with low viral inputs. Vpx-VLPs will improve the efficiency of LV-mediated transduction of MDDCs with vectors for both ectopic gene expression and depletion studies.

PMID:
21637200
DOI:
10.1038/nprot.2011.327
[Indexed for MEDLINE]

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