Navigation Links
siRNA Expression Vectors with Selectable Markers

For Long Term Studies in Mammalian Cells


Six new pSilencer siRNA expression vectors are now available, each with an antibiotic resistance gene to facilitate selection in mammalian cultured cells. These new vectors are ideal for the long-term study of gene specific knockdown. Gene specific silencing in mammilian cells can be readily accomplished by inducing the RNA interference pathway with siRNAs (see Gene Specific Silencing by RNAi). Advantages of using siRNA expression vectors over siRNAs prepared by chemical synthesis, in vitro transcription or RNase III digestion of long dsRNA include the ability to perform long term gene silencing studies and eliminating the need to work directly with RNA.


How siRNA Expression Vectors Work
Vectors that express siRNAs within mammalian cells typically use an RNA polymerase III promoter to drive expression of a short hairpin RNA that mimics the structure of an siRNA (See Why RNA Polymerase III Promoters? in sidebar). The insert that encodes this hairpin is designed to have two inverted repeats separated by a short spacer sequence (Figure 1). One inverted repeat is complementary to the mRNA to which the siRNA is targeted. A string of thymidines added to the 3' end serves as a pol III transcription termination site. (See Design of Vector Encoded siRNAs in sidebar). Once inside the cell, the vector constitutively expresses the hairpin RNA, which induces silencing of the target gene.


Click here for larger image

Figure 1. Schematic of a Typical siRNA Expression Vector . A. siRNA encoding insert. B. Vector. C. Hairpin siRNA.


Selectable Markers are Ideal for Long Term Studies
The use of mammalian siRNA expression vectors with antibiotic resistence genes provides many benefits. Selectable markers can help compensate for poor plasmid transfection efficiencies seen with some cell lines. In these cases, only a fraction of the transfected cells express the siRNA, and reduction in target gene expression with even a potent siRNA can be difficult to detect. Transient use of an antibiotic can be used to enrich the population of cells that have taken up the marker-containing plasmid. This means that useful results can be obtained from experiments where transfer of the siRNA expression plasmid into the cells is inefficient.

In addition, use of selectable markers permits long term gene silencing studies of cells that take up the siRNA expression vector. Changes in phenotype due to reduced gene expression that may not be readily apparent only a few days after transfection can be followed over several weeks .


New!
siRNA Expression Vectors wi th Puromycin, Neomycin, and Hygromycin Selectable Markers
Six pSilencer siRNA Expression Vectors with antibiotic selectable markers are now available from Ambion (click here for comparison chart). These vectors feature the same promoters, ampicillin resistance gene, and E. coli origins of replication as the pSilencer 2.0-U6 and pSilencer 3.0-H1 siRNA Expression Vectors, but have the added benefit of an antibiotic resistence gene.

pSilencer 2.1-U6 puro and pSilencer 3.1-H1 puro contain the resistance gene pac, which confers resistance to puromycin.

pSilencer 2.1-U6 hygro and pSilencer 3.1-H1 hygro contain the resistance gene hph, which inactivates hygromycin.

pSilencer 2.1-U6 neo and pSilencer 3.1-H1 neo contain the neomycin resistance gene, which confers resistance to the antibiotic G418.

All six siRNA expression plasmids allow for the selection of cells that have taken up the plasmid and are expressing the resistance gene.

To demonstrate the use of Ambion's new selectable marker containing pSilencer siRNA expression vectors, HeLa cells expressing GFP were transfected with pSilencer 2.1-U6 hygro encoding an siRNA to GFP and selected with the antibiotic hygromycin (Figure 2). Three weeks after transfection, GFP levels were analyzed by fluorescence microscopy. GFP levels were found to be reduced 94% as compared to cells transfected with pSilencer 2.1-U6 hygro without an insert. This reduction in gene expression was maintained for at least four weeks.

The selectable marker-containing pSilencer vectors are supplied with (1) linearized and purified vector ready for ligation; (2) a DNA insert encoding a GFP-specific siRNA; (3) a circular, negative control pSilencer vector that expresses a scrambled control siRNA; and (4) 1X DNA Annealing Solution.

Figure 2. Long Term Silencing of GFP with pSilencer 2.1-U6 hygro. HeLa cells expressing cycle 3 GFP were transfected with pSilencer 2.1-U6 hygro containing an insert encoding an siRNA targeting cycle 3 GFP under the control of the human U6 promoter (Panel A) or pSilencer 2.1-U6 hygro without an siRNA-encoding insert (Panel B). Following transfection, the cells were selected with hygromycin. Three weeks following selection, the cells were analyzed for GFP expression by fluorescence microscopy. Green: GFP. Blue: DAPI stained nuclei. GFP levels were remarkably reduced (94%) in cells transfected with the GFP siRNA-encoding pSilencer 2.1-U6 hygro siRNA Expression Vector as compared to those transfected with an "empty" siRNA expression vector.


back to top


Ordering Information
Cat# Product Name Size 5760 pSilencer 2.1-U6 hygro 20 rxns 5762 pSilencer 2.1-U6 puro 20 rxns 5764 pSilencer 2.1-U6 neo 20 rxns 5766 pSilencer 3.1-H1 hygro 20 rxns 5768 pSilencer 3.1-H1 puro 20 rxns 5770 pSilencer 3.1-H1 neo 20 rxns
'"/>

Source:


Page: All 1 2 3 4 5

Related biology technology :

1. Custom and library siRNA for efficient gene silencing
2. Custom and library siRNA for efficient gene silencing
3. Cancer siRNA Oligo Set Version 1.0
4. Library siRNA
5. Custom siRNA Oligo Synthesis Service
6. Efficient RNAi-mediated gene silencing in neuronal cells using QIAGEN siRNA and TransMessenger Transfection Reagent*
7. Quantification of siRNA Silencing Efficiency Using the LightCycler System
8. Housekeeping Genes: Universal Positive Controls in siRNA Knockdown Experiments
9. Confirming gene silencing mechanism by pGFP/GFP22 siRNA co-transfection
10. siRNA transfection optimization with the Agilent 2100 bioanalyzer
11. siRNA Design Guidelines
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:6/27/2016)... , June 27, 2016  Liquid Biotech ... announced the funding of a Sponsored Research Agreement ... circulating tumor cells (CTCs) from cancer patients.  The ... in CTC levels correlate with clinical outcomes in ... These data will then be employed to support ...
(Date:6/24/2016)... ... 24, 2016 , ... Researchers at the Universita Politecnica delle Marche in Ancona ... or pleural mesothelioma. Their findings are the subject of a new article on the ... are signposts in the blood, lung fluid or tissue of mesothelioma patients that can ...
(Date:6/23/2016)... SILVER SPRING, Md. , June 23, 2016 ... evidence collected from the crime scene to track the criminal ... sick, and the U.S. Food and Drug Administration (FDA) uses ... Sound far-fetched? It,s not. ... whole genome sequencing to support investigations of foodborne illnesses. Put ...
(Date:6/23/2016)... June 23, 2016   EpiBiome , a precision ... million in debt financing from Silicon Valley Bank (SVB). ... and to advance its drug development efforts, as well ... "SVB has been an incredible strategic partner ... a traditional bank would provide," said Dr. Aeron ...
Breaking Biology Technology:
(Date:4/28/2016)... First quarter 2016:   , Revenues amounted ... quarter of 2015 The gross margin was 49% (27) ... the operating margin was 40% (-13) Earnings per share ... operations was SEK 249.9 M (21.2) , Outlook   ... M. The operating margin for 2016 is estimated to ...
(Date:4/19/2016)... , UAE, April 20, 2016 ... implemented as a compact web-based "all-in-one" system solution for ... biometric fingerprint reader or the door interface with integration ... modern access control systems. The minimal dimensions of the ... readers into the building installations offer considerable freedom of ...
(Date:4/14/2016)... April 14, 2016 BioCatch ... Detection, today announced the appointment of Eyal Goldwerger ... role. Goldwerger,s leadership appointment comes at a ... of the deployment of its platform at several of ... technology, which discerns unique cognitive and physiological factors, is ...
Breaking Biology News(10 mins):