Gene-silencing in human DC by siRNA approaches

We are currently performing gene silencing experiments in human monocyte-derived DCs (MDDCs) with the dual aim of generating relevant knowledge and data on

(i) the transcriptional regulation of DC differentiation, and
(ii) the safe and rationale in vitro manipulation of gene expression in DCs.

One of the two different approaches that have been set up in parallel is:

Transient transfection of synthetic siRNA: to this aim, commercially available siRNA Smart Pool from Dharmacon targeted against the STAT3 transcription factor have been successfully transfected in human MDDCs (Lipofectamine 2000 as transfection reagent). The efficiency of silencing, as detected by western blot or FACS analysis of the protein was of 50-80% (depending on the donor), and peaked at 72 h post-transfection. Transfection efficiency was reproducibly higher than 80% and did not induce any type I IFN production. A total of 9 different donors have been transfected under these optimized conditions and a detailed analysis of the phenotypic and functional consequences of STAT3 silencing have been performed upon

(a) Toll-like receptor (TLR) mediated DC activation (e.g LPS or pI:C) and
(b) activation in a tolerogenic microenvironment (e.g. combined stimulation TLR-L + IL-10).

In particular, we analysed the immunophenotype, the antigen up-take activity, the secretion of cytokines/chemokines, and the ability of silenced MDDCs to stimulate T cell allogenic proliferation in classical MLR assays.

No consistent and reproducible differences in the immunophenotype and antigene uptake capability of both iMDDCs and TLR-matured MDDCs have been observed. Modest inhibitory effects have instead been observed in the production IL-10 and IL-6, whose biological activity is controlled through STAT3. We noticed, however, that the transfection itself caused a non specific activation of MDDCs (of variable extent from donor to donor) that rendered difficult the interpretation of the results (especially MLR).

Global microarrays were used to define differentially modulated genes in STAT3 silenced MDDCs versus control cells.

• molecule type
• gene,
• signal transducer and activator of transcription 3,
• small interfering RNA

• organism type
• Homo sapiens

• cell type
• dendritic cell

• experimental design type
• genetic modification design: knock-out experiment,
• in vitro design experiment

created over 15 years ago (2 March 2009)    last modified over 13 years ago (28 September 2011)   [ RDF ]   [ RelFinder ]