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Method Article
Nicolas Manel
INSERM U932, Institut Curie, France
Corresponding Author
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
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.
Keywords
Human monocyte-derived dendritic cells, shRNA, RNAi, Vpx
Figure 1
The authors declare no competing financial interests
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Associated Publications
A cryptic sensor for HIV-1 activates antiviral innate immunity in dendritic cells
by Nicolas Manel, Brandon Hogstad, Yaming Wang, +3
Nature (13 December, 2010)
Method Article
Nicolas Manel
INSERM U932, Institut Curie, France
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 14 Dec, 2010
No community comments so far
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
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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Associated Publications
A cryptic sensor for HIV-1 activates antiviral innate immunity in dendritic cells
by Nicolas Manel, Brandon Hogstad, Yaming Wang, +3
Nature (13 December, 2010)