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Product Description

The siRNA expression vector with antibiotics labeling can continuously suppress the target gene expression, lasting for several weeks or even longer

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GenePharma SuperSilencing shRNAi Vector

The siRNA expression vector with antibiotics labeling can continuously suppress the target gene expression, lasting for several weeks or even longer.
When a validated siRNA sequence (for example, obtained by screening in siRNA synthesis method) needs to maintain gene silencing for a longer period of time, we recommend the use of shRNA vector system. GenePharma is committed to providing the most advanced and convenient shRNA related tools. The company recently introduces a new generation of shRNA expression plasmid, a highly efficient ready to use vector, which can produce shRNAs sustainedly in cell, so as to achieve lasting suppression of target gene expression.

GenePharma SuperSilencing shRNA Expression Vector System

Characteristics of shRNA Expression Vector System.

  1. Produces short-hairpin RNA (shRNA) to achieve RNA interference.
  2. The method is simple, economical, and only needs to synthesize DNA oligos.
  3. The use of cloning vector is convenient, and the vector has selectable markers.
  4. shRNA plasmid is stable and easy to operate.

GenePharma SuperSilencing shRNA Expression Vector characteristics:

  1. The cloning vector has two cloning restriction sites BamH I and Bbs I. Bbs I is a special restriction endonuclease; it can produce asymmetrical complementary sticky ends to ensure the correct direction of inserted fragments, and to prevent the self-circularization of vectors.
  1. Multiple screening markers can help to establish stable transfected cell lines.

Neo:  Neomycin resistance gene.
Hygro: Hygromycin B resistance gene.
GFP: Neo: GFP report gene and the Kan/G418 resistance gene.

GenePharma can construct eight kinds of RNAi vectors.
pGPU6, pGPH1, pGPU6/Neo, pGPH1/Neo, pGPU6/Hygro, pGPH1/Hygro, pGPU6/GFP/Neo, pGPH1/GFP/Neo.
pGPU6, pGPH1 is the RNAi vector without screening marker.
pGPU6/Neo, pGPH1/Neo is the RNAi vector with Neomycin resistance screening marker. pGPU6/Hygro, pGPH1/Hygro is the RNAi vector with Hygromycin resistance screening marker.
pGPU6/GFP/Neo, pGPH1/GFP/Neo is the RNAi vector with Neomycin resistance and GFP report gene expression.

 

GenePharma SuperSilencing shRNA Expression Vector Pack
Each pack contains 4 shRNA vectors designed for a target gene; we guarantee that at least one of them can cause more than 70% inhibition efficiency in mRNA level.

Pack composition

Number

shRNA vector for a target gene

4

positive control shRNA vector

1

negative control shRNA vector

1

Attention:Each plasmid in this pack is 50ug.

 




 

Product

Price

Times

SuperSilencing shRNA Expression Vector Pack

 

2 weeks

GenePharma SuperSilencing shRNA vector system
Each set includes linear or cyclic shRNA expression vectors, the corresponding negative control vector and a GFP positive control vector. We also provide you with the overall solution.

Cat. No.

Product

Promoter

Selection marker

Quantity

Price

E-01

pGPU6

U6

6*50μg

$650

E-02

pGPH1

H1

6*50μg

$650

E-03

pGPU6/Neo

U6

Neo

6*50μg

$650

E-04

pGPH1/Neo

H1

Neo

6*50μg

$650

E-05

pGPU6/Hygro

U6

Hygro

6*50μg

$650

E-06

pGPH1/Hygro

H1

Hygro

6*50μg

$650

E-07

pGPU6/GFP/Neo

U6

Neo

6*50μg

$650

E-08

pGPH1/GFP/Neo

H1

Neo

6*50μg

$650

GenePharma ready-to-use SuperSilencing shRNA Expression Vector Cloning and Construction Service
We can help you to insert the DNA fragment encoding target shRNA into the plasmid and do sequence validation. You just need to tell us the target gene sequence or Gene ID and the name of the inserted vector, and we will help you to construct the expression vector. Furthermore, we will provide you with sufficient purified expression plasmid encoding your target shRNA. 

Cat. No.

Product

Promoter

Selectable Marker

Quantity

Price

Times

C02001

pGPU6

U6

50μg

$149

2 weeks

C02002

pGPH1

H1

50μg

$149

2 weeks

C02003

pGPU6/Neo

U6

Neo

50μg

$149

2 weeks

C02004

pGPH1/Neo

H1

Neo

50μg

$149

2 weeks

C02005

pGPU6/Hygro

U6

Hygro

50μg

$149

2 weeks

C02006

pGPH1/Hygro

H1

Hygro

50μg

$149

2 weeks

C02007

pGPU6/GFP/Neo

U6

Neo

50μg

$149

2 weeks

C02008

pGPH1/GFP/Neo

H1

Neo

50μg

$149

2 weeks

,,
 
GenePharma’s eight different RNAi Vector map:

1.GPU6,  2. pGPH1, 3. pGPU6/Neo, 4.pGPH1/Neo, 5. pGPU6/Hygro, 6. pGPH1/Hygro, 7. pGPU6/GFP/Neo,  8. pGPH1/GFP/Neo.





GenePharma pGPU6/GFP/Neo shRNA vectorProduct Description and Background
siRNA and RNA Interference
 
Small Interfering RNAs (siRNAs) are short, double-stranded RNA molecules that can target mRNAs with complementary sequence for degradation via a cellular process termed RNA interference (RNAi) (Elbashir 2001). Researchers in many disciplines employ RNAi to analyze gene function in mammalian cells. The siRNA used in early studies was typically prepared in vitro and transfected into cells. More recent publications feature plasmids that express functional siRNA when transfected into mammalian cells (Sui 2002, Lee 2002, Paul 2002, Paddison 2002, Brummelkamp 2002). Using siRNA expression vectors has the advantage that the expression of target genes can be reduced for weeks or even months (Brummelkamp 2002), eclipsing the 6–10 days typically observed with in vitro prepared siRNA used for transient transfection (Byrom 2002).
 
pGPU6/GFP/Neo siRNA Expression Vector
pGPU6/GFP/Neo plasmid is supplied ligation-ready
The pGPU6/GFP/Neo siRNA Expression vector is linearized with both Bam HI and Bbs I to facilitate directional cloning. They are purified to remove the digested insert so that it cannot re-ligate with the vector. This greatly increases the percentage of clones bearing the hairpin siRNA-coding insert after ligation, reducing the time and effort required to screen clones. Both pGPU6/GFP/Neo and pGPH1/GFP/Neo are linearized with the same restriction enzymes, so that a given hairpin siRNA insert can be subcloned into either vector using the 5' overhangs left by restriction enzyme digestion. A basic pGPU6/GFP/Neo vector map is shown in Figure 1 on page 3; more detailed sequence information about the pGPU6/GFP/Neo vector is available from the Technology Support of GenePharma, Inc..
 
Feature
Nucleotide position
CMV IE promoter
1-589
GFP ORF
613-1410
SV40 polyA
1499-1549
T3 promoter binding site
1602-1621
HU6 promoter
1720-1992
T7 promoter binding site
2005-2026
F1 origin
2033-2488
SV40 promoter
2829-3011
Kanamycin/Neomycin resistance gene
3013-3807
pUC origin of replication
4392-5035
Kit Components and Storage
Each pGPU6/GFP/Neo siRNA Expression Vector Kit includes 4 components:
• Linearized pGPU6/GFP/Neo siRNA Expression Vector ready for ligation
• Circular, negative control pGPU6/GFP/Neo vector that expresses a hairpin siRNA with limited homology to any known sequences in the human, mouse, and rat genomes
• human GAPDH-specific, hairpin siRNA insert that can be used as a positive control for ligation
• 1XDNA Annealing Solution to prepare annealed DNA oligonucleotides for ligation into the pGPU6/GFP/Neo vector
 
 
E-07
F-07
Component
20 μl
---
pGPU6/GFP/Neo (circular) (50 ng/ul)
---
20μl
pGPU6/GFP/Neo (linearized) (50 ng/ul)
10 μl
10 μl
pGPU6/GFP/Neo Negative control (0.5 μg/μl)
10 μl
10 μl
GAPDH Control Insert (20 nM)
0.5 ml
0.5 ml
10×shDNA Annealing Solution
 
Store the pGPU6/GFP/Neo siRNA Expression Vector Kit at –20°C (if desired the 1XDNA Annealing Solution can be stored at room temp). Properly stored kits are guaranteed for 6 months from the date received.
 
Other Required Material
Ligation and transformation
• Two complementary oligonucleotides targeting the gene of interest for RNAi (design and ordering is discussed in section II starting on page 8)
• DNA ligase, ligase reaction buffer, and competent E. coli cells are needed to subclone the siRNA inserts.
• Kanamycin containing plates and liquid media will also be needed to propagate the plasmids.
Plasmid purification
For efficient transfection into mammalian cells it is crucial that preparations of pGPU6/GFP/Neo be very pure.
Mammalian cell transfection reagents
The optimal mammalian cell transfection conditions including transfection agent and plasmid amount must be determined empirically.
Cell culture facility and supplies
In addition to routine cell culture media, culture media containing G418 (a neomycin analog) will be needed for selection of pGPU6/GFP/Neo-transfected cells.
Optimizing Antibiotic Selection Conditions
G418 titration (kill curve)
a.       Plate 20,000 cells into each well of a 24 well dish containing 1 ml of culture medium.
b.       After 24 hr, add 500 μl culture medium containing 25–4000 μg/ml G418.
c.       Culture the cells for 10–14 days, replacing the antibiotic-containing medium every 3 days.
d.       Examine the dishes for viable cells every 2 days.
Identify the lowest G418 concentration that begins to give massive cell death in approximately 7–9 days, and kills all cells within 2 weeks. Use this G418 concentration to select cells containing the pGPU6/GFP/Neo plasmid after transfection.
Optimal plating density
a.       Plate several different amounts of cells into separate wells of a 24 well dish containing 1 ml of culture medium.
b.       After 24 hr, add 500 μl culture medium containing G418; use the concentration identified in the previous experiment.
c.       Culture the cells for 5–14 days, replacing the antibiotic-containing medium every 3 days.
Identify the cell plating density that allows the cells to reach 80% confluency before massive cell death begins; and use it to plate cells transfected with your pGPU6/GFP/Neo clone.
loning Hairpin siRNA Inserts into pGPU6/GFP/Neo
Prepare a 1 μg/μl solution of each oligonucleotide
a.       Dissolve the hairpin siRNA template oligonucleotides in approximately 100 μl of nuclease-free water.
b.       Dilute 1 μl of each oligonucleotide 1:100 to 1:1000 in TE (10 mM Tris, 1 mM EDTA) and determine the absorbance at 260 nm. Calculate the concentration (in μg/ml) of the hairpin siRNA oligonucleotides by multiplying the A260 by the dilution factor and then by the extinction coefficient (~33 μg/ml).
Dilute the oligonucleotides to approximately 100 μM.
Anneal the siRNA template oligonucleotides
a.       Assemble the 50 μl annealing mixture as follows:
Amount
Component
5 μl
10XshDNA Annealing Solution
5 μl
sense siRNA template oligonucleotide (100 uM)
5 μl
antisense siRNA template oligonucleotide (100 uM)
35 μl
ddH2O
b.       Heat the mixture to 95°C for 3 min, then turn off the heater and cool to room temperature slowly.
c.       The annealed siRNA template insert can either be ligated into a pGPU6/GFP/Neo vector or stored at –20°C for future ligation.
Transfecting pGPU6/GFP/Neo into Mammalian Cells
Transfect cells and culture 24 hr without selection
We recommend using GenePharma’s RNAi-Mate transfection reagent (Cat.No. C-01) to deliver pGPU6/GFP/Neo plasmids into mammalian cells with high efficiency and minimal toxicity. Follow the instructions for using RNAi-Mate provided with the product. RNAi-Mate is a proprietary formulation of polyamines that can be used in the presence or absence of serum in the culture medium. It is suitable for the transfection of a wide variety of cell types.
Transfect the purified plasmid into the desired cell line, plate transfected cells at the plating density identified in step II.C.2 on page 10, and culture for 24 hr without selection.
It is important to include two non-transfected control cultures. One is subjected to neomycin or G418 selection to control for the fraction of cells that survive selection; it will help determine the effectiveness of the transfection and selection. The second control is grown without neomycin or G418 selection as a positive control for cell viability.
Selecting Antibiotic-Resistant Transfected Cells
Once they are prepared, pGPU6/GFP/Neo siRNA expression vectors can be used in transient siRNA expression assays, or to create cell populations or a clonal cell line that stably expresses your siRNA. Note that with normal (nontransformed) and primary cell lines, it may be difficult to obtain clones that stably express siRNA. For these types of cells, we recommend choosing the antibiotic selection strategies outlined in sections 1 and 2 below.
Short term antibiotic selection for enrichment of cells that transiently express the siRNA
In experiments where the transfection efficiency is low, a rapid antibiotic selection can be used to kill cells that were not transfected with the pGPU6/GFP/Neo siRNA expression vector. This enrichment for transfected cells can be useful for reducing background when analyzing gene knockdown.
a.       Culture the cells for 1–3 days in the antibiotic-containing medium (added in step B.2) to enrich the culture for cells that were successfully transfected.
Analyze the population for an expected phenotype and/or the expression of the target gene.
Selecting a population of cells that stably express the siRNA
Creating a population of cells stably expressing the siRNA involves treating cells with neomycin or G418 for several days to eliminate cells that were not transfected. The surviving cell population can then be maintained and assessed for reduction of target gene expression.
a.       Culture the cells in medium containing neomycin or G418 (added in step B.2) until all of the cells in the non-transfected control culture are killed. At this point, the selection is complete and the cells can be grown without antibiotic until they repopulate the culture vessel.
b.       Analyze expression of the target gene at any time after the cells in the non-transfected control culture have been killed.
Pool and passage antibiotic-resistant cell cultures as needed. It is a good idea to periodically grow the cells with a minimal level of antibiotic selection, to prevent the accumulation of cells that no longer express antibiotic resistance. Often this “minimal level” is about half the antibiotic concentration used to kill off nontranfected cells, but this value varies widely among different cell types.
Selecting for clones that stably express the siRNA
For many researchers, the goal is to create a clonal cell line that expresses the siRNA template introduced with pGPU6/GFP/Neo. Cloning stably expressing cell lines is advantageous because strains that exhibit the desired amount of gene knockdown can be identified and maintained, and clones that are neomycin-resistant but which do not express the siRNA can be eliminated.
Typically the levels of siRNA expression and gene knockdown vary widely among cells. In fact pGPU6/GFP/Neo-transfected cells that survive antibiotic selection may not have a significant reduction in expression of the target gene. Instead, they may have found a way to mitigate the effects of a reduction in the target gene expression by compensating in another fashion or by shutting down expression of the siRNA. To avoid this, it can be useful to isolate clones that can be screened to identify the cells that cause the desired reduction in target gene expression.
 
Troubleshooting
2. Poor ligation efficiency
If the ligation reaction (section III.A.3 on page 11) is inefficient, then there will be relatively few plasmids to transform. Possible causes of poor ligation efficiency include:
a.    The concentration of the annealed siRNA template insert is lower than expected.
Evaluate ~5 μl of the insert DNA (from step III.A.3.c on page 9) using a 12% native polyacrylamide gel and compare its ethidium bromide staining to bands from a molecular weight marker or another standard of known concentration.
b.    The ligase or ligase reaction buffer have become inactive.
Test your ligation components using another vector and insert or replace your ligation components and retry the siRNA insert cloning.
c.    One or both of the hairpin siRNA template oligonucleotides have high levels of non-full-length products.
The size of oligonucleotides can be evaluated on an 12% native polyacrylamide gel.
d.    The oligonucleotide annealing reaction was ineffective.
A low concentration of one of the oligonucleotides or incomplete denaturation of individual oligonucleotides could have reduced the relative amount of dsDNAs.
Compare the annealed siRNA template insert to each of the single-stranded oligonucleotides using native 8–12% polyacrylamide gel electrophoresis. If the annealed siRNA template insert has bands corresponding to the single-stranded oligonucleotides, then adjusting the concentrations of the single-stranded DNA molecules and heat-denaturing at a higher temperature during siRNA insert preparation (step III.A.2.b on page 9) might improve the percentage of dsDNA products. Alternatively, in some cases, gel purifying the band corresponding to annealed insert may result in better ligation.
 
  1. Xu, B., G. Chen, et al. "shRNA-Mediated BAALC knockdown affects proliferation and apoptosis in human acute myeloid leukemia cells." Hematology 17(1): 35-40.

  2. Wang, T. S., R. H. Guo, et al. "Expression of livin in gastric cancer and induction of apoptosis in SGC-7901 cells by shRNA-mediated silencing of livin gene." Biomedicine & Pharmacotherapy 64(5): 333-338.

  3. Yang, Y., Y. Gao, et al. "Downregulation of survivin expression and enhanced chemosensitivity of MCF-7 cells to adriamycin by PDMAE/survivin shRNA complex nanoparticles." International Journal of Pharmaceutics 405(1): 188-195.

  4. Zhou, L., Y. Jiang, et al. (2008). "Silencing of N-Ras gene expression using shRNA decreases transformation efficiency and tumor growth in transformed cells induced by anti-BPDE." Toxicological sciences 105(2): 286-294.

  5. Zhou, X., Z. Zhang, et al. (2008). "Inhibition of cyclin D1 expression by cyclin D1 shRNAs in human oral squamous cell carcinoma cells is associated with increased cisplatin chemosensitivity." International Journal of Cancer 124(2): 483-489.

  6. Yao, X., G. Zhao, et al. "Overexpression of high-mobility group box 1 correlates with tumor progression and poor prognosis in human colorectal carcinoma." Journal of cancer research and clinical oncology 136(5): 677-684.

  7. Wang, X., D. Xing, et al. (2009). "BimL directly neutralizes Bcl-xL to promote Bax activation during UV-induced apoptosis." FEBS letters 583(12): 1873-1879.

  8. Zhang, C. M., X. Q. Zeng, et al. "Effects of NYGGF4 knockdown on insulin sensitivity and mitochondrial function in 3T3-L1 adipocytes." Journal of bioenergetics and biomembranes 42(5): 433-439.

  9. Guo, H., Y. Lv, et al. "Downregulation of p57 accelerates the growth and invasion of hepatocellular carcinoma." Carcinogenesis 32(12): 1897-1904.

  10. Zhao, L., C. Zhang, et al. "RNAi-mediated inhibition of PDGF-D leads to decreased cell growth, invasion and angiogenesis in the SGC-7901 gastric cancer xenograft model." Cancer biology & therapy 9(1): 42-48.

  11. Huang, Q. R., Q. Li, et al. "Involvement of anion exchanger-2 in apoptosis of endothelial cells induced by high glucose through an mPTP-ROS-Caspase-3 dependent pathway." Apoptosis 15(6): 693-704.

  12. Wang, X., W. Gong, et al. "p21-activated kinase 5 inhibits camptothecin-induced apoptosis in colorectal carcinoma cells." Tumor Biology 31(6): 575-582.

Ordering
The first problem we meet for the application of siRNA is to design the sequences. Although there are quite a lot principles for the designing of published sequences and quite a few companies supply on-line design service, up to now, no software can guarantee the efficiency of the oligos designed.
Shanghai GenePharma Co, Ltd. and our collaboration laboratories have been involved in RNAi research for some time. For each specific gene target, if 4 pairs of oligos were designed and synthesized, normally you will have at least one pair showing 70% down regulation.
Our price: 2OD, PAGE purified, $89/pair
*If one single order is less than 5 pairs extra shipping fee of $40 will be charged. Payment terms are 30 days. We accept cheque or wire transfer into our USD account.
Specifications of siRNA Oligos:
Quality Control:
All our siRNA oligos undergo vigorous process monitoring and strict quality control. Length and labeling are systematically controlled by PAGE or mass spectrometry analysis. Quantity is systematically validated by UV absorbance at 260 nm.
Purification: Fully deprotected and desalted.
Purified by PAGE or RP-HPLC upon request
Length: 19 to 23 mers
Bases: RNA (A, C, G or U)
DNA (A, C, G or T)
Backbone: Phosphodiester bond
Labels and modifications: Fluorescein, biotin and phosphate: 3’ or 5’end
Other labels available upon request
Format: Single-strand RNA oligos is delivered in dry form
Storage and stability
Although oligonucleotides are stable in solution at 4°C for up to 2 weeks, we recommend storage should be at -20°C. Repetitive freeze-thaw cycles should be avoided by storing as aliquots. For long-term storage, siRNA oligos should be dried.
Oligonucleotides with fluorescent labels should be protected from light. We guarantee our oligonucleotides for six months, when stored under the above conditions.
Shipment: Shipped by express delivery, dry in individual, transparent tubes.
Oligonucleotide Technical Data Sheet
Oligonucleotides are delivered with an Oligonucleotide Technical Data Sheet, which includes oligonucleotide name, sequence, concentration, size,  purification method.

Services available upon request

  1. Aliquoting
  2. Free design support
  3. Additional services may increase turn-around time

Ordering:Please fill in the following order form and send it to: order@genepharma.com or by fax to:00862151320295 our staff will confirm with you asap.