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Virally expressed siRNA mediates localized gene silencing in specific cardiovascular (CV) regulatory nuclei of mouse brain
Author(s) -
Burmeister Melissa A,
Lazartigues E.,
Tian X.,
Kutschke W.,
Sharma R. V.,
Kapusta D. R.,
Davisson R. L.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a364-a
Subject(s) - gene knockdown , gene silencing , green fluorescent protein , transduction (biophysics) , transgene , biology , viral vector , rna interference , microbiology and biotechnology , genetically modified mouse , small hairpin rna , reporter gene , small interfering rna , gene expression , gene , transfection , rna , genetics , biochemistry , recombinant dna
Dissecting the molecular underpinnings of central neuro‐cardiovascular control is challenging due to the involvement of many genes in multiple circuits. siRNA is an effective strategy for gene silencing; however, in vivo applications require efficient intracellular transduction in target tissues. Here we tested the hypothesis that adenoviral‐mediated delivery of siRNA would induce efficient, stable and localized knockdown in CV regions of mouse brain. Ad‐siGFP was designed to express both a U6‐driven siRNA hairpin targeted against green fluorescent protein (GFP) and LacZ as a reporter for viral expression. GFP transgenic mice underwent injection of Ad‐siGFP or a control virus (Ad‐Con) into the paraventricular nucleus (PVN) or ICV to target the subfornical organ (SFO). Mice were sacrificed at 3, 14 or 28 days, and alternating brain sections were processed for GFP visualization or X‐gal. GFP levels in PVN were reduced by 3 days in Ad‐siGFP mice (38±3%, n=4) compared to Ad‐con mice (0±6%, n=4, p<0.05), with further marked knockdown at 2 wks (Ad‐siGFP 60±2% vs Ad‐con 0±11%, n=5 each, p<0.05). Robust and region‐specific LacZ was observed in Ad‐siGFP mice at these times. GFP levels in PVN nearly had returned to baseline in Ad‐siGFP mice after 4 wks. A similar GFP/LacZ profile was seen in SFO. These results establish proof‐of‐principle that efficient, stable and localized gene silencing can be achieved in CV regions of mouse brain using viral‐delivered siRNA.