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| 1. |
Birdsey GM, Dryden NH, Amsellem V, Gebhardt F, Sahnan K, Haskard DO, Dejana E, Mason JC, Randi AM.
(2008)
Transcription factor Erg regulates angiogenesis and endothelial apoptosis through VE-cadherin.
Blood
111
,
33498-33506
.
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Notes:
These authors showed that the ETS transcription factor Erg interacts with the VE-cadherin promoter region and regulates endothelial apoptosis through this interaction. They demonstrated that inhibition of Erg by siRNA resulted in decreased VE-cadherin mRNA and protein levels, and showed that Erg interacts with the VE-cadherin promoter using a CHIP assay. To show the functional relevance of this interaction, HeLa cells were transfected with a pGL4 Vector containing the VE-cadherin promoter region and an expression vector containing Erg2 cDNA. In this reporter assay, Erg overexpression resulted in ~1.8 fold transactivation of VE-cadherin promoter activity, as measured using the Dual-Luciferase® Reporter Assay System. Inhibition of Erg in human umbilical vein endothelial cells also resulted in a loss of viability and an increase in activation of caspase 3 and caspase 7. The authors showed that apoptosis could be significantly decreased by overexpression of VE-cadherin, indicating that Erg regulates survival partially via its interaction with VE-cadherin. The Caspase-Glo® 3/7 Assay was used to measure caspase activity in these experiments.
(0003872) |
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Products: Caspase-Glo® 3/7 Assay | Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack |
| 2. |
Garcia-Pedrero, J.M, Kiskinis, E., Parker, M.G., and Belandia, B.
(2007)
The SWI/SNF chromatin remodeling subunit BAF57 is a critical regulator of estrogen receptor function in breast cancer cells.
J. Biol. Chem.
281
,
22656–22664
.
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Notes:
To examine the role that BAF57, a transcriptional modulator of the estrogen receptor (ER), may have in breast cancer, BT549 cells were transfected with a reporter vector (pGL3-Basic with two estrogen response elements and the human pS2 promoter), the control pRL-CMV Vector and combinations of the following expression vectors: mERα or hERβ, BAF57, SRC1e, SRC1a and RAC3. After 16 hours, the cells were treated with 17β-estradiol. Twenty-four hours later, the cells were harvested and the luciferase activities assayed using the Dual-Luciferase® Reporter Assay System. GST-BAF57 (full-length, N- or C-domain) fusion protein was bound to Sepharose beads and incubated with 17β-estradiol or vehicle and wildtype or one of various mERα interaction domain mutants, which have been expressed and labeled with 35S methionine using a TNT® rabbit reticulocyte lysate system. The beads were washed and analyzed for bound protein. ZR-75-1 cells were transfected with BAF57 siRNA then treated with 17β-estradiol 24 hours later. Cell proliferation was measured using the CellTiter® 96 AQueous One Solution Cell Proliferation Assay.
(0003599) |
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Products: CellTiter 96® AQueous One Solution Cell Proliferation Assay | Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | pGL3-Basic Vector | pRL-CMV Vector |
| 3. |
Coldwell, M.J. and Morley, S.J.
(2007)
Specific isoforms of translation initiation factor 4GI show differences in translational activity.
Mol. Cell. Biol.
26
,
8448–8460
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Notes:
The authors explored the role of five different eukaryotic initiation factor (eIF) 4GI protein isoforms, which are encoded by alternatively spliced mRNAs, by using short interfering RNAs (siRNAs) to silence the eIF4GI gene. Three eIF4GI siRNA target sequences were evaluated for their ability to reduce eIF4GI mRNA levels in HeLa cells. To quantify the extent of gene silencing, a control plasmid that encodes an eIF4GI/Renilla luciferase fusion mRNA was created using the psiCHECK™-2 Vector. Cotransfection of HeLa cells with the eIF4GI siRNAs and psiCHECK™-2 control plasmid resulted in degradation of the eIF4GI/Renilla luciferase mRNA, leading to reduced Renilla luciferase activity and lower light output. The psiCHECK™-2 Vector encodes the firefly luciferase gene, which allowed normalization of Renilla luciferase expression. Firefly and Renilla luciferase activities were measured using the Dual-Luciferase® Reporter Assay System. Quantitative PCR (qPCR) was used to quantify the silencing of endogenous eIF4GI mRNA splice variants. Prior to qPCR, total RNA was isolated from siRNA-expressing HeLa cells, then reverse transcribed using the ImProm-II™ Reverse Transcription System. qPCR was The pGEM®-T Easy Vector was used in the creation of plasmids encoding siRNA-resistant eIF4GI isoforms, which were transfected into siRNA-expressing HeLa cells to restore eIF4GI function.
(0003778) |
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Products: Dual-Luciferase® Reporter Assay System | ImProm-II™ Reverse Transcription System | pGEM®-T Easy Vector System I | psiCHECK™-2 Vector |
| 4. |
Wang, B., Love, T.M., Call, M.E., Doench, J.G. and Novina, C.D.
(2006)
Recapitulation of short RNA-directed translational gene silencing in vitro.
Mol. Cell
22
,
553-560
.
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Notes:
microRNAs (miRNAs) are a class of endogenous short RNAs that repress gene expression. To assess miRNA-directed translational silencing, in vitro reactions were performed. Translational gene silencing by miRNAs in vitro requires target mRNAs to possess a 7-methyl G cap and a poly(A) tail. In vitro transcription was performed with 5μg of linearized plasmids containing zero, two, four, or six miCXCR4 binding sites, one siCXCR4 binding site or Renilla luciferase (pRL-TK; control mRNA) using the RiboMax™ T7 Large-Scale RNA Production kit. The mRNAs were modified by 7-methyl-G capping and polyadenylation. Translation was performed using nuclease-treated rabbit reticulocyte lysate containing 0.025pmole of mRNA with miCXCR4 or siCXCR4 binding sites, 0.025pmole Renilla control RNA, and different ratios of CXCR4 siRNA. Reaction products were separated by SDS-PAGE and visualized and quantitated by PhosphorImager analysis.
(0003412) |
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Products: pRL-TK Vector | Rabbit Reticulocyte Lysate System, Nuclease Treated | RiboMAX™ Large Scale RNA Production System—SP6 | RiboMAX™ Large Scale RNA Production System—T7 |
| 5. |
Koon, H-W., Zhao, D., Zhan, Y., Rhee, S.H., Moyer, M.P. and Pothoulakis, C.
(2006)
Substance P stimulates cyclooxygenase-2 and prostaglandin E2 expression through JAK-STAT activation in human colonic epithelial cells.
J. Immunol.
176
,
5050-9
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Notes:
The COX-2 promoter was cloned by PCR into the pGL3 Vector. NCM460-NK-1R cells were transiently transfected with the cloned promoter and the pRL-TK vector as an internal control or siRNA targeted against various JAK/STAT genes. The Dual-Luciferase® Assay was used to measure promoter activity. The wildtype COX-2 promoter was mutagenized using the GeneEditor™ in vitro Site-Directed Mutagenesis System.
(0003384) |
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Products: Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | GeneEditor™ in vitro Site-Directed Mutagenesis System | pGL3-Basic Vector | pGL3-Control Vector | pGL3-Enhancer Vector | pGL3-Promoter Vector |
| 6. |
Koon, H.W., Zhao, D., Zhan, Y., Rhee, S.H., Moyer, M.P. and Pothoulakis, C.
(2006)
Substance P stimulates cyclooxygenase-2 and prostaglandin E2 expression through JAK-STAT activation in human colonic epithelial cells.
J. Immunol.
176
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5050–5059
.
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Notes:
To examine the effect of Substance P (SP) on COX-2 expression, the COX-2 promoter region spanning –2069 to –66 bp was cloned by PCR and subcloned into the pGL3-Basic Vector (pGL3-Cox-2). Nontransformed human colonic epithelial NCM460 cells overexpressing neurokinin-1 receptor (NK-1R; NCM460-NK-1R) were seeded in 12-well plates and transiently transfected with pGL3-Cox-2 with either a transfection control pRL-TK Vector or siRNA or both. The siRNA molecules used were for AK1, JAK2 (Upstate Biotechnology), STAT3, STAT5, STAT6 or a control siRNA. The transfected cells were serum starved for 24 hours, treated with SP for 4 hours and then lysed. The cell extracts were measured for firefly and Renilla luciferase activities using the Dual-Luciferase® Reporter Assay System. Relative luciferase activity was a ratio of COX-2 promoter firefly activity to Renilla activity; results were expressed as percentage of control group without SP stimulation. To mutate the STAT binding elements, the pGL3-Cox-2 construct was modified using the GeneEditor™ in vitro Site-Directed Mutagenesis System. The resulting mutant constructs were tested in the same system as the wildtype COX-2 promoter.
(0003520) |
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Products: Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | GeneEditor™ in vitro Site-Directed Mutagenesis System | pGL3-Basic Vector | pRL-TK Vector |
| 7. |
Lee, S.J., Jang, B.C., Lee, S.W., Yang, Y.I., Suh, S.I., Park, Y.M., Oh, S., Shin, J.G., Yao, S., Chen, L. and Choi, I.H.
(2006)
Interferon regulatory factor-1 is prerequisite to the constitutive expression and IFN-gamma-induced upregulation of B7-H1 (CD274).
FEBS Lett.
580
,
755-762
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Notes:
Many cancer cells upregulate the co-signaling molecule B7-H1, confering resistance to anti-tumor immunity. The ability of interferon regulatory factor-1 (IRF-1) to upregulate B7-H1 expression was characterized by cloning fragments of the B7-H1 promoter upstream of the firefly luciferase reporter gene in the pGL3-Basic Vector and monitoring luciferase expression using the Dual Luciferase® Reporter Assay System. Firefly luciferase measurements were normalized using Renilla luciferase (pRL-CMV Vector). Putative IRF-1 binding sites in the B7-H1 promoter were identified using the Gel Shift Assay System. RT-PCR was used to examine B7-H1 mRNA levels in interferon-γ-treated or untreated A549 cells exposed to various concentrations of IRF-1 siRNA. cDNA synthesis was performed with the ImProm-II™ Reverse Transcription System.
(0003451) |
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Products: Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | Gel Shift Assay Core System | Gel Shift Assay System | ImProm-II™ Reverse Transcriptase | ImProm-II™ Reverse Transcription System | pGL3-Basic Vector | pRL-CMV Vector |
| 8. |
Chew, J.L., Loh, Y.H., Zhang, W., Chen, X., Tam, W.L., Yeap, L.S., Li, P., Ang, Y.S., Lim, B., Robson, P. and Ng, H.H.
(2005)
Reciprocal transcriptional regulation of Pou5f1 and Sox2 via the Oct4/Sox2 complex in embryonic stem cells.
Mol. Cell. Biol.
25
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6031–46
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Notes:
The authors studied the effects of Embryonic Stem Cell (ESC)-specific regulation on the Pou5f1 promoter in human and mouse cells. To examine the effect of knockdown of Oct4 and Sox2 (two genes involved in ESC regulation) on the Pou5f1 promoter, a 3kb fragment of the human POU5F1 promoter was cloned into pGL3-Basic Vector and 100ng cotransfected with 100ng shRNA plasmids into mouse E14 ESCs. Five nanograms of pRL-SV40 Vector served as a transfection control. For the enhancer assay, a 461bp fragment of genomic DNA containing the SRR2 enhancer of Sox2 was amplified and cloned into the pGL3-Promoter Vector. The same amounts of plasmid, shRNA and transfection control were transfected into E14 ESCs as in the Pou5f1 promoter assay. To investigate gene knockdown in 293T cells, 5ng of the two open reading frame (ORF) constructs (the Luc-Sox2 and the Luc-Pou5f1 ORFs cloned into the psiCHECK™-2 Vector) were cotransfected with 100ng shRNA plasmid. The outcome was examined 48–60 hours post-transfection using the Dual-Luciferase® Reporter Assay System.
(0003291) |
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Products: Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | pGL3-Basic Vector | pGL3-Promoter Vector | pRL-SV40 Vector | psiCHECK™-2 Vector |
| 9. |
Rose, S.D., Kim, D.H., Amarzguioui, M., Heidel, J.D., Collingwood, M.A., Davis, M.E., Rossi, J.J. and Behlke, M.A.
(2005)
Functional polarity is introduced by Dicer processing of short substrate RNAs.
Nucleic Acids Res.
33
,
4140–56
.
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Notes:
Having observed that blunt 27mers had increased potency in RNAi compared to 21mers or 27mers with 3' or 5' overhangs, these authors investigated what differences may account for these changes in gene silencing activity using the same target sequence in enhanced green fluorescent protein (EGFP). For one experiment, a PCR-generated fragment of the EGFP coding region spanning sites EGFPS1 and EGFPS2 was cloned into psiCHECK™-2 Vector in both sense and antisense orientations. Also, a PCR-generated fragment of the human heterogeneous nuclear ribonucleoprotein H (hnRNPH) coding region spanning sites H1 and H3 was similarly cloned in sense and antisense orientations. HEK293 cells were transfected with 150ng EGFP sense and antisense vectors plus EGFPS2 or control duplex RNAs. HCT116 cells were transfected with 100ng sense and antisense hnRNPH vectors with H3 or control duplex RNAs. The Dual-Luciferase® assay was used to evaluate luciferase expression 24 hours post-transfection. In a separate EGFP RNAi experiment, the Steady-Glo® Luciferase Assay System was used to monitor firefly luciferase activity to normalize transfection of HEK 293 cells.
A further RNAi experiment targeted the firefly luciferase gene in the pGL3-Control Vector cotransfected with 20, 2 or 0.4 nM siRNA duplexes into HeLa cells. After 48 hours, the cells were lysed and 10µl tested using the Luciferase Assay System. To test the level of expression of human La antigen targeted for gene silencing, total RNA was harvested from HeLa cells using the SV 96 Total RNA Isolation System, reverse transcribed and used in real-time PCR.
(0003289) |
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Products: Luciferase Assay System | pGL3-Control Vector | psiCHECK™-2 Vector | Steady-Glo® Luciferase Assay System | SV 96 Total RNA Isolation System |
| 10. |
Lin, X., Ruan, X., Anderson, M.G., McDowell, J.A., Kroeger, P.E., Fesik, S.W. and Shen, Y.
(2005)
siRNA-mediated off-target gene silencing triggered by a 7 nt complementation.
Nucleic Acids Res.
33
,
4527-4535
.
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Notes:
To identify novel regulators of the HIF-1 pathway, the authors performed a knockdown experiment using a synthetic siRNA library against a number of kinases. Two of their ‘top hits’ directly down-regulated the hif-1a mRNA through a 7nt complementation. They built an siRNA library using an HIF-1 reporter assay. For siRNA library screening, the HIF-1 reporter and a control reporter (pRL-TK Vector, Promega) were transfected into H1299 cells. The Dual-Glo™ Luciferase Assay System was used to analyze cells transfected with an siRNA against HIF-1a as a positive control and an siRNA irrelevant to the HIF-1 pathway as a negative control.
(0003538) |
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| 11. |
Landthaler, M., Yalcin, A., and Tuschl, T.
(2004)
The Human DiGeorge Syndrome Critical Region Gene 8 and Its D. melanogaster Homolog Are Required for miRNA Biogenesis
Curr. Biol.
12
,
2162-2167
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Notes:
In this study, the psiCHECK-2 vector was used to assist in selection of siRNA sequences that work optimally against their selected target. Dicer and DGCR8 coding sequences were individually cloned into the psiCHECK-2 multiple cloning site. The resulting vector and a synthetic siRNA duplex targeting the gene coding sequence were transfected into 293 cells and Renilla luciferase activity was measured using the Dual Luciferase® Assay System. Firefly luciferase activity was used to normalize the data. siRNAs that caused greater than 80% reduction in Renilla luciferase signal were selected for further use.
(0003220) |
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Products: Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | psiCHECK™-2 Vector |
| 12. |
Dunoyer P., Lecellier C.H., Parizotto E.A., Himber C. and Voinnet O.
(2004)
Probing the microRNA and small interfering RNA pathways with virus-encoded suppressors of RNA silencing.
Plant Cell.
16(5)
,
1235-1250
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Notes:
The authors cloned five distinct silencing suppressor proteins from five different plant viruses in order to examine the pathways involving both small interfering RNA and micro RNA in Arabidopsis thaliana. These viral factors [P1- HcPro of Turnip mosaic virus (TuMV), P38 protein of Turnip crinkle virus (TCV), P19 protein of Tomato bushy stunt virus(TBSV), P25 protein of Potato virus X, and the P15 protein of Peanut clump virus (PCV)] were inserted into a mammalian expression vector and tested for protein production using the TNT® Quick Coupled Transcription/Translation System. The suppression effects of these plant viral proteins were also tested in HeLa cells. The CMV promoter was cloned from pRL-CMV into the pGL3-Basic Vector and both plasmids were transfected at 500ng each plus 1µg of each of the five suppressor-expressing vectors. After one day, 300ng siRNA targeting the firefly luciferase gene was added. Twenty-four hours later, the Dual Luciferase® Reporter Assay System was used to determine the ratio of firefly:Renilla luciferase expression and see if the viral suppressor protein had an effect.
(0003085) |
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Products: Dual-Luciferase® Reporter Assay System | pGL3-Basic Vector | pRL-CMV Vector |
| 13. |
Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T.
(2001)
Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.
Nature
411
,
494-498
.
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Notes:
In this landmark paper describing RNA interference in mammalian cells, the firefly and Renilla luciferase genes were targeted for degradation. NIH/3T3, HEK293, HeLa S3, COS-7, and S2 cells were transfected with 1μg pGL2-Control or pGL3-Control Vector, 0.1μg pRL-TK Vector, and 0.21μg siRNA duplex targeting either firefly of Renilla luciferase. The Dual-Luciferase® Assay was used 20 hours post-transfection to monitor luciferase expression. It was found that transfection with 21bp dsRNA can cause the specific degradation of a targeted sequence. This was the first demonstration of the RNAi effect in mammalian cells.
(0003027) |
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Products: Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | pGL2-Control Vector | pGL3-Control Vector | pRL-TK Vector |