Efficient in vivo knock-down of estrogen receptor alpha: application of recombinant adenovirus vectors for delivery of short hairpin RNA

BACKGROUND: Adenovirus (Ad) mediated gene transfer is a well-established tool to transiently express constructs in livers of mice in vivo. In the present study, we determined the specificity and efficiency of Ad vectors expressing short hairpin (sh) RNA constructs to knock-down the estrogen receptor α (ERα). RESULTS: Two different shRNA constructs derived from the murine ERα coding sequence were designed (shERα). In vitro, transfection of three mouse cell lines with pSUPER-shERα constructs resulted in up to 80% reduction of endogenous ERα activity. A single mismatch in the target sequence eliminated the reduction of ERα activity, demonstrating the specificity of shERα. The subsequently generated Ad.shERα vectors were equally effective in vitro. In vivo, intravenous administration of Ad.shERα resulted in 70% reduced hepatic mouse ERα mRNA levels. Co-injection of Ad.shERα with an Ad vector containing a luciferase (luc) gene driven by an estrogen responsive element (ERE) containing promoter resulted in a significant (90% on day five) down-regulation of hepatic luciferase activity, as determined by non-invasive optical imaging. Down-regulation was sustained up to day seven post-injection. CONCLUSION: Ad mediated transfer of shERα expression constructs results in efficient and specific knockdown of endogenous ERα transcription both in vitro and in vivo

A comparative study between catalase gene therapy and the cardioprotector monohydroxyethylrutoside (MonoHER) in protecting against doxorubicin-induced cardiotoxicity in vitro

50) limited the possibility to increase catalase activity more than 3.5-fold, which was not enough to protect infected NeRCaMs against doxorubicin-induced cardiotoxicity and (2) confirms the efficacy of monoHER as a cardioprotector. Thus, the use of monoHER proves more suitable for the prevention of doxorubicin-induced cardiotoxicity than catalase gene transfer employing adenovirus vectors

Efficient knock-down of estrogen receptor alpha: application of recombinant adenovirus vectors for delivery of short hairpin RNA-2

<p><b>Copyright information:</b></p><p>Taken from "Efficient knock-down of estrogen receptor alpha: application of recombinant adenovirus vectors for delivery of short hairpin RNA"</p><p>BMC Biotechnology 2006;6():11-11.</p><p>Published online 28 Feb 2006</p><p>PMCID:PMC1403768.</p><p>Copyright © 2006 Krom et al; licensee BioMed Central Ltd.</p>days after Ad. administration and subjected to taqman analysis. The cyclophillin gene was used as internal standard. Data represented as mean ± SD

Efficient knock-down of estrogen receptor alpha: application of recombinant adenovirus vectors for delivery of short hairpin RNA-0

<p><b>Copyright information:</b></p><p>Taken from "Efficient knock-down of estrogen receptor alpha: application of recombinant adenovirus vectors for delivery of short hairpin RNA"</p><p>BMC Biotechnology 2006;6():11-11.</p><p>Published online 28 Feb 2006</p><p>PMCID:PMC1403768.</p><p>Copyright © 2006 Krom et al; licensee BioMed Central Ltd.</p>hERα_1103, or pSUPER- shERα_tandem. Subsequently, the cells were treated 24 hours with 10M 17-β-estradiol. Luciferase activity was measured 48 hours after transfection and after correction for LacZ expression, represented as the mean (n = 3) ± SD relative to the transfection with pSUPER-empty. (A) Endogenous mouse ERα mediated transcription in EOMA, H5V and MXT cells after introducing pSUPER +/- shERα. (B) The 19-nt target-recognition sequence of ERα_1395 contains one mismatch with human ERα and five mismatches with the mouse ERβ sequence. (C) ERα mediated transcription in EOMA cells after over expression of either mouse ERα- or human ERα-expression vectors in presence of pSUPER empty or pSUPER shERα_1395 (D) Western blot analysis of H5V cells co-transfected with pCMV-mERα and pSUPER-empty or pSUPER-shERα_1395. The lysates were analysed by immunoblotting (insert-photo) with anti-mouse ERα and anti-p38. The intensity of the bands was quantified and normalized to cells transfected with pSUPER-empty. The relative ERα protein levels are presented (bar-diagram) as mean (n = 3) +/- SD

Efficient knock-down of estrogen receptor alpha: application of recombinant adenovirus vectors for delivery of short hairpin RNA-3

<p><b>Copyright information:</b></p><p>Taken from "Efficient knock-down of estrogen receptor alpha: application of recombinant adenovirus vectors for delivery of short hairpin RNA"</p><p>BMC Biotechnology 2006;6():11-11.</p><p>Published online 28 Feb 2006</p><p>PMCID:PMC1403768.</p><p>Copyright © 2006 Krom et al; licensee BioMed Central Ltd.</p>cipients were treated for 6 hours with increasing amounts of estrogen (0–50 μg/kg, s.c). Then, the mice were sacrificed, and the livers were processed for luciferase assays. Luciferase activity is expressed as relative luciferase units (RLU) per mg total liver protein. (B) Male C57Bl/6 mice (n = 5) were injected with Ad.ERE-Luc (5 × 10pfu) plus Ad.Empty or Ad.shERα_1103 (3 × 10pfu). Three or seven days post-infection, the mice were injected with 5 μg/kg estrogen. The (inset) photo shows the result of optical imaging of the bioluminescence at day three, the bar-diagram is a quantitative representation of hepatic luciferase activity at day three or day seven. (C) Male C57Bl/6 mice (n = 5) were co-injected with Ad.ERELuc (5 × 10pfu) + Ad.Empty or Ad.shERα_1103 (3 × 10pfu). Five days later, the mice received 0 or 5 μg/kg estrogen. After 6 hours, the animals were sacrificed, and hepatic luciferase activity was determined. Luciferase activity is expressed as relative luciferase units (RLU) per mg total liver protein. Data represented as mean ± SD

Efficient knock-down of estrogen receptor alpha: application of recombinant adenovirus vectors for delivery of short hairpin RNA-1

<p><b>Copyright information:</b></p><p>Taken from "Efficient knock-down of estrogen receptor alpha: application of recombinant adenovirus vectors for delivery of short hairpin RNA"</p><p>BMC Biotechnology 2006;6():11-11.</p><p>Published online 28 Feb 2006</p><p>PMCID:PMC1403768.</p><p>Copyright © 2006 Krom et al; licensee BioMed Central Ltd.</p>tudy. (B) EOMA and MXT cells were co-transfected with pERE-Luc and pCMV-LacZ and than infected either with Ad.Empty, Ad.shERα_1395, or Ad.shERα_1103. 10M Estrogen was administrated for 24 hours. Luciferase activity was measured 48 hours after infection. Data represented as mean ± SD relative to infection with Ad.Empty