Navigation Links
pSilencer 4.1-CMV: Versatile Vectors for Expression of siRNA, miRNA, and mRNA


Ambion's pSilencer 4.1-CMV plasmid vectors are designed for long-term gene silencing experiments in a broad range of cell lines. The vectors carry an RNA polymerase II-type CMV promoter (human cytomegalovirus immediate-early promoter) and an optimized SV40 polyadenylation signal to drive high-level expression of hairpin siRNA. For stable transfections requiring long-term antibiotic selection, an SV40 promoter expresses one of three antibiotic resistance genes (hygromycin, neomycin, or puromycin) for long-term antibiotic selection. Here, we show that pSilencer 4.1-CMV is a versatile vector that can be used to express siRNA as well as microRNA (miRNA) or messenger RNA (mRNA).


Efficient Gene Silencing

To demonstrate the utility of the pSilencer CMV vectors in RNA interference experiments, a pSilencer 4.1-CMV vector was engineered to express a hairpin siRNA targeting GAPDH. This construct was then transfected into HeLa cells. As shown in Figure 1, the level of GAPDH protein was reduced by ~80% two days after transfection. This suggests that the hairpin siRNA was efficiently expressed and correctly processed in the cell to generate a mature, functional siRNA. Long-term antibiotic selection of the transfected cells produced cell lines exhibiting reduced levels of GAPDH mRNA and protein for over 4 months (see Drive Long-Term Silencing with CMV Vectors).


Expression of Mature miRNA

Expression of hairpin siRNAs for gene silencing is only one application of the pSilencer 4.1-CMV vectors; these vectors can also be used to express miRNA. miRNAs are endogenous, single-stranded small RNAs that are believed to play critical roles in eukaryotic development by controlling post-transcriptional gene expression. Like siRNAs, miRNAs are expressed as longer hairpin molecules. These primary transcripts (pri-miRNAs) are then cleaved in the nucleus by the Drosha protein to release 60-70 nt pre-miRNA hairpins. Pre-miRNAs are exported to the cytoplasm by an Exportin 5-dependent mechanism where they are processed by Dicer to generate mature 21-24 nt miRNAs [1, 2].

To evaluate whether pSilencer 4.1-CMV vectors could express pri-miRNA hairpins that could be processed by the cellular machinery to mature miRNA, genomic DNA fragments containing either the predicted mir-16 or mir-124 pri-miRNA hairpins were cloned in pSilencer 4.1-CMV hygro vectors. These plasmid constructs were then transfected into HeLa cells where they were expected to drive expression of the 180 nt mir-16 pri-miRNA transcript or the 150 nt mir-124 pri-miRNA (Figure 2A).

Figure 1. siRNA-mediated Knockdown of GAPDH Using pSilencer 4.1-CMV puro. HeLa cells were not transfected (NT) or transfected with pSilencer 4.1-CMV puro vectors engineered to express an siRNA targeting GAPDH mRNA (CMV-Gap) or a scrambled sequence (CMV-Scr). (A) Immunostaining of HeLa cells two days after transfection showing reduction of GAPDH levels in cells transfected with CMV-Gap. Green: GAPDH protein. Blue: DAPI stained nuclei. (B) Relative levels of GAPDH expression compared with HeLa cells not transfected (NT).

Figure 2. Expression of miRNA Using pSilencer 4.1-CMV hygro. pSilencer 4.1-CMV was engineered to express mir-16 or mir-124 pri-miRNA. (A) Schematic representation of the mir-16 and mir-124 transcripts. Each transcript included the predicted hairpin at its center and flanking genomic sequences of various sizes. (B) Two days after transfection into HeLa cells, total RNA was isolated with the mirVana miRNA Isolation Kit and 1 g of total RNA was analyzed on a denaturing agarose gel. A Northern blot was used to detect let-7 miRNA, 5.8S and 5S rRNA while miR-16 and miR-124 miRNA were detected by solution hybridization with the mirVana miRNA Detection Kit. All probes were labeled and purified with the mirVana Probe & Marker Kit. NT = not transfected.


Two days after transfection, total RNA was isolated from the cell cultures using the mirVana miRNA Isolation Kit, which provides quantitative yield of small RNAs, including miRNA, siRNA, tRNA, and 5S/5.8S rRNAs. Analysis of miRNA expression by solution hybridization assay with the mirVana miRNA Detection Kit showed a significant overexpression of miR-16 in cells transfected with the pSilencer 4.1-CMV miR-16 vector (Figure 2B). In contrast, no variation in the levels of let-7 miRNA or 5.8S and 5S rRNAs was observed. The exogenous expression of mature miRNA was further confirmed by solution hybridization using a probe specific for miR-124. Expression of this brain-specific miRNA was detected only in cells transfected with the CMV miR-124 vector (Figure 2B). Like Chen et al. [3], we found that native genomic sequences flanking each side of the predicted hairpin are required for correct processing of pri-miRNA into mature miRNA. Expression of shorter pri-miRNA transcripts lacking the flanking sequences resulted in accumulation of unprocessed pre-miRNA hairpins as determined by Northern blot analysis (data not shown).


High Level of Protein Expression

In addition to providing high-level expression of mature siRNA or miRNA, the CMV promoter is excellent for stable or transient expression of protein in a wide range of mammalian cells. To test the ability of the pSilencer 4.1-CMV vectors to drive high levels of mRNA and protein expression, vectors containing either a firefly luciferase cDNA or a chloramphenicol acetyltransferase (CAT) cDNA were prepared and transfected into HeLa cells. The close correlation between luciferase or CAT gene transcript levels and the enzymatic activity of their corresponding proteins have made these systems powerful genetic reporters for investigating transcriptional and post-transcriptional regulation in mammalian cells. As shown in Figure 3, high levels of reporter protein were readily detected in the transfected cells 24 or 48 hours after transfection. The presence of a hygromycin, neomycin, or puromycin antibiotic resistance genes on each pSilencer 4.1-CMV vector further allows for selection of stable cell lines constitutively expressing these reporters.

Figure 3. Expression of mRNA Using pSilencer 4.1-CMV puro. HeLa cells were transfected in triplicate with pSilencer 4.1-CMV vectors carrying either a firefly luciferase cDNA or a CAT cDNA. (A) Relative luciferase activity 24 hours after transfection. (B) Relative CAT activity 48 hours after transfection. NT = not transfected. RU = relative units.


The pSilencer 4.1-CMV vectors are provided linearized and ready for ligation. They are part of Ambion's extensive line of plasmid and viral expression systems. For more information or to determine which vector best suits your needs, please visit www.ambion.com/prod/psilencer.


back to top


Ordering Information
Cat# Product Name Size 1552 mirVana miRNA Detection Kit 100 rxns 1554 mirVana Probe & Marker Kit 30 rxns + 10 marker rxns 1560 mirVana miRNA Isolation Kit up to 40 purifications 5775 pSilencer 4.1-CMV puro 20 rxns 5777 pSilencer 4.1-CMV hygro 20 rxns 5779 pSilencer 4.1-CMV neo 20 rxns
'"/>

Source:


Page: All 1 2 3 4 5 6

Related biology technology :

1. Selecting siRNA Sequences to Incorporate into the pSilencer Vectors
2. Your Data: Use of pSilencer 1.0-U6 siRNA Expression Vector in the Analysis of Synaptotagmin Location and Function
3. A Versatile Power Supply for All Electrophoresis Applications
4. Versatile Vectors for Ponasterone A- Inducible Control of Gene Expression in Mammalian Cells
5. Versatile Reporter Vectors for Monitoring Viral Transduction
6. Versatile Transfection Reagent Offers Low Toxicity and Consistent Performance
7. Generate Adenovirus Vectors in E. coli by Homologous Recombination with the AdEasy Adenoviral Vector System
8. Epitope-Tagging Vectors for Functional Analysis in Yeast
9. New Mammalian Expression Vectors Employ Stable, High-Level Fluorescence Humanized Renilla GFP Reporter
10. Efficiently Insert Unique Restriction Sites into Plasmid Vectors
11. High-Efficiency Site-Directed Mutagenesis of Large Plasmid Vectors
Post Your Comments:
*Name:
*Comment:
*Email:
TAG: pSilencer CMV Versatile Vectors for Expression siRNA miRNA and mRNA

(Date:1/22/2015)... | RBJ today announces the firm brokered a long-term lease ... leading biopharmaceutical company, at Two Ledgemont Center in ... , president, and Brian Cohen , senior vice president, ... at 95 Hayden Ave. Photo - http://photos.prnewswire.com/prnh/20150122/170765 ...
(Date:1/22/2015)... Rootstown, OH (PRWEB) January 22, 2015 Crystal ... announced today that it has received AOAC-PTM Certifications for the ... O111, O121, and O145; collectively referred to as STEC or ... at 1 colony forming unit (cfu) per 325 g of ...
(Date:1/22/2015)... 2015 Controlled Substance Compliance Services (CSCS), ... companies check the legal requirements around using controlled substances ... , As their international operations expand and become ... solutions built as a result of the first phase ...
(Date:1/22/2015)... , Jan. 22, 2015   Cypher Genomics, ... Sequenom, Inc. (NASDAQ: SQNM ), the ... agreement for next generation noninvasive prenatal tests (NIPT). ... interpretation technology, called Mantis™, to advance analysis of ...
Breaking Biology Technology:Transwestern?RBJ Advises Shire in 202,000 SF Lease, Creating Boston's Largest Suburban Biotech Campus 2Crystal Diagnostics Awarded AOAC-PTM Accreditation for the Rapid Detection of “Big 6” E.coli Food Pathogens 2Global Compliance Service for Controlled Substances to Expand to China 2Cypher Genomics and Sequenom Announce Development Agreement 2Cypher Genomics and Sequenom Announce Development Agreement 3Cypher Genomics and Sequenom Announce Development Agreement 4
... 28 Boston,Scientific Corporation (NYSE: BSX ) ... that Boston Scientific has completed its acquisition of ... April 16.,Under the terms of the transaction, all ... per share in cash, for a total of,approximately ...
... N.J., May 28 National Stem Cell,Holding, Inc. (OTC: ... steps to,change its name to Proteonomix, Inc. The change ... Michael Cohen, President/CEO, stated: "Our new name reflects the ... development stage and,commencing the commercialization of our technologies. These ...
... Provides Additional Global Corporate Finance, Management and ... Manufacturing ... (TSX: NVN) announced today the appointment of Gordon H.,Busenbark to ... chief financial officer of Xytis Pharmaceuticals, a privately,held development-stage specialty ...
Cached Biology Technology:Boston Scientific Completes Acquisition of CryoCor 2Boston Scientific Completes Acquisition of CryoCor 3National Stem Cell Holding, Inc. Announces the Change of Its Name to Proteonomix, Inc. 2National Stem Cell Holding, Inc. Announces the Change of Its Name to Proteonomix, Inc. 3Nventa Appoints Gordon H. Busenbark to Board of Directors 2
(Date:12/24/2014)... launch in December 2014, the 1U™ app has ... to remember their usernames and passwords through replacing the antiquated ... assist people who have struggled to remember usernames and passwords, ... focuses on redefining identity, announced today that it is offering ...
(Date:12/19/2014)... Markets ( http://www.researchandmarkets.com/research/86ncd6/micro_market ) has announced the addition ... Systems Market" report to their offering. ... security market is estimated to grow at a CAGR of ... a larger share in this market, Canada ...
(Date:12/17/2014)... , Dec. 15, 2014  HITLAB SM announced ... audit to confirm its adherence to current U.S. Food ... to conduct regulated smart device and smart phone application ... research quality. "HITLAB is determined to improve ...
Breaking Biology News(10 mins):1U Offers Best Solution to the Username / Password Dilemma: For FREE! 21U Offers Best Solution to the Username / Password Dilemma: For FREE! 3Micro Market Monitor: North America Perimeter Security Systems Market (Intrusion Detection Sensor, Video Surveillance, Communication/Alarm and Notification, Access Control System) 2Micro Market Monitor: North America Perimeter Security Systems Market (Intrusion Detection Sensor, Video Surveillance, Communication/Alarm and Notification, Access Control System) 3HITLAB Completes GCP Audit for FDA Smart Device & App Compliance 2
... with storing nuclear waste and the possibility of it ... no longer be obstacles on the road to cleaner ... University of Notre Dame, led by Thomas E. Albrecht-Schmitt, ... professor of chemistry and biochemistry, showcases Notre Dame Thorium ...
... problems in the world today can be traced back to the ... requiring energy, space and resources to survive, the stress on the ... is enormous. Richard Cardullo , a professor of ... free public lecture on campus in which he will discuss whether ...
... Childrens Research Institute and the University of Melbourne have ... allergy. It is hoped the test would be ... minimise over-diagnosis of peanut allergy in the community. ... standard for diagnosing peanut allergy, and while an oral ...
Cached Biology News:New paper by Notre Dame researchers describes method for cleaning up nuclear waste 2Is Earth overpopulated? 2Test to improve peanut allergy diagnosis 2
...
GLP expression profiling service on the Affymetrix platform. Starting material can be blood, tissue or RNA. 5 day turnaround. Standard and custom post analyses of microarray data available....
... enables early selection of the best ... quantitative data for sensitivity, dynamic range, ... Furthermore quantitative antibody fingerprints can be ... your antibodies., The full-service package includes ...
... allows for rapid, convenient, and efficient Denatured ... process utilizes a spin column to make ... concentration of proteins which were electro-eluted in ... ion exchangers, offering high binding capacity fo ...
Biology Products: