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siRNA mediated knockdown of ERbeta in vivo in magnocellular neurosecretory cells of the rat paraventricular nucleus
Author(s) -
Doherty Faye C,
Sladek Celia D
Publication year - 2009
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.23.1_supplement.968.1
Subject(s) - gene knockdown , downregulation and upregulation , medicine , endocrinology , chemistry , small interfering rna , estrogen receptor , receptor , transfection , biology , gene , biochemistry , cancer , breast cancer
Estrogen receptor β (ERβ) is a transcription factor that is downregulated to undetectable levels in the nuclei of magnocellular neurosecretory cells (MNCs) in response to hyperosmolality or hypovolemia in rats. To determine if downregulation of ERβ allows upregulation of other genes in the MNCs during dehydration, we used siRNA to locally and acutely reduce ERβ expression in the paraventricular nucleus (PVN) of rats. This avoids the systemic and developmental effects of knockout models. We used an siRNA construct specific to ERβ and modified to enhance stability and nuclease resistance (siSTABLE, Dharmacon) as well as a corresponding non‐targeting construct. siRNA (6.48ng in 0.5ul PBS) was stereotaxically injected into PVN, using the ERβ‐specific construct on one side and the non‐targeting one on the other. On the side of the PVN injected with the ERβ‐specific construct, the number of ERβ‐positive nuclei was reduced to 50±13% (p=0.013; n=2) compared to the control side. This reduced expression is not due to dehydration since hematocrit in these rats (35±1% n=2) was lower than in dehydrated animals (41±1%, n=10; p=0.012), but not lower than in unmanipulated controls (37.8±0.7%, n=9; p=0.137). The effect of the siRNA is highly localized; misplaced injections in the caudal PVN did not effect ERβ expression in the MNCs. This supports the feasibility of using siRNA to reduce ERβ in discrete neuronal populations.