RNAi in human monocyte-derived dendritic cells using shRNA vectors
Human monocyte-derived dendritic cells (MDDC) constitute a widely used experimental model to study human dendritic cells. However, loss-of-function experiments in MDDC have been limited by the poor efficiency of traditional RNAi techniques. We have developed a RNAi method in MDDC using shRNA lentiviral vectors. Importantly, this method of MDDC transduction does not appear to interfere with the process of DC differentiation. Furthermore, this method does not provoke MDDC maturation. This process is highly efficient and >90% transduction is routinely obtained. This protocol is focused on the use of shRNAs in the widely available LKO.1 backbone, with puroR or GFP as a marker. It is adaptable to other lentiviral shRNA vectors, to gain-of-function approaches (overexpression) and for differentiation into macrophages instead of dendritic cells.
Figure 1
Posted 14 Dec, 2010
RNAi in human monocyte-derived dendritic cells using shRNA vectors
Posted 14 Dec, 2010
Human monocyte-derived dendritic cells (MDDC) constitute a widely used experimental model to study human dendritic cells. However, loss-of-function experiments in MDDC have been limited by the poor efficiency of traditional RNAi techniques. We have developed a RNAi method in MDDC using shRNA lentiviral vectors. Importantly, this method of MDDC transduction does not appear to interfere with the process of DC differentiation. Furthermore, this method does not provoke MDDC maturation. This process is highly efficient and >90% transduction is routinely obtained. This protocol is focused on the use of shRNAs in the widely available LKO.1 backbone, with puroR or GFP as a marker. It is adaptable to other lentiviral shRNA vectors, to gain-of-function approaches (overexpression) and for differentiation into macrophages instead of dendritic cells.
Figure 1
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