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Setting up the optimal conditions for both transient transfection of synthetic siRNAs and lentiviral transduction of shRNAs into DC

Laboratory protocol

We performed experiments to set up the optimal conditions for both transient transfection of synthetic siRNAs and lentiviral transduction of shRNAs into DC.

To optimize transfection efficiency, while minimizing toxicity, non targeting FITC-conjugated siRNA were transfected in two different experimental settings:

a) fresh monocytes and
b) immature DCs (iDCs) obtained from monocytes in the presence of GM-CSF/IL-4.

In both experimental settings a considerably high transfection efficiency (up to 75%) was achieved in the absence of significant cell death. Moreover, the percentage of siRNA-FITC+ cells did not decline with time (up to 5 days post-transfection) suggesting high siRNA stability inside the DCs, in agreement with previously published data.

To optimize lentiviral delivery, experiments using a self-inactivating lentiviral vector (LV) expressing enhanced GFP (eGFP) from an internal CMV promoter were performed. DCs cells were transduced at different multiplicity of infection. FACS analysis indicated a good transfection efficiency (up to 80%). Evaluation of the effect of lentiviral infection on DC phenotype/activation, revealed that LV infection per se neither modified DC phenotype/activation state nor significantly interfered with their capacity to mature upon appropriate stimulation.

These approaches are currently being used to silence the transcription factor IRF4, and finely dissect its recently discovered function during human myeloid DC differentiation.

They will also be applied to the selective silencing of ILT3 (see below), based on the working hypothesis that disrupting molecular pathways leading to tolerance may result in the generation of a DC more successful in stimulating the proper immune response against tumours.

Protocol Steps:
Setting up the optimal conditions for both transient transfection of synthetic siRNAs and lentiviral transduction of shRNAs into DC Graph


created over 10 years ago (2 March 2009)    last modified over 7 years ago (28 September 2011)   [ RDF Rdf ]   [ RelFinder Relfinder ]