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siRNA‐mediated knockdown of endogenously expressed bestrophin in smooth muscles.
Author(s) -
Larsen Per,
Matchkov Vladimir,
Nilsson Holger,
Aalkjaer Christian,
Pedersen Finn Skou
Publication year - 2007
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.21.5.a653-a
Subject(s) - downregulation and upregulation , gene knockdown , transfection , microbiology and biotechnology , vascular smooth muscle , chemistry , western blot , gene expression , messenger rna , biology , gene , smooth muscle , endocrinology , biochemistry
We have recently characterized in smooth muscle cells a unique cGMP‐dependent Ca 2+ ‐activated Cl − current (I Cl(cGMP‐Ca) ) that co‐exists with a “classical” Ca 2+ ‐activated Cl − current. We hypothesized that bestrophin‐4 (a product of the VMD2‐like 3 gene) could be responsible for the I Cl(cGMP‐Ca) based on similarities between membrane current produced by bestrophin heterologous expression and this endogenous current. This was supported by a similar distribution pattern of the I Cl(cGMP‐Ca) and the bestrophin expression. To test this hypothesis siRNA‐mediated downregulation of gene expression was used. Cultured aortic smooth muscle cells (A7r5) were transfected with siRNA directed against bestrophin‐4 and cultured for 3 days. The efficiency of transfection was demonstrated by specific perinuclear fluorescence of Cy3‐labelled siRNA. The downregulation of targeted protein expression was controlled by qPCR and Western blot. The downregulation of bestrophin‐4 expression (by 88% in mRNA) with siRNA was a associated with significant reduction (by 83%) of the I Cl(cGMP‐Ca) while the “classical” Ca 2+ ‐activated Cl − current was not affected. Our studies provide evidence that bestrophin‐4 is responsible for the I Cl(cGMP‐Ca) in smooth muscle cells. This study presents a novel efficient technique for specific downregulation of gene expression in blood vessels, much needed in studies of vascular function.