iLenti ™ siRNA
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Service Description Cat. No. Price
iLenti™ siRNA
Cloning
1 iLenti™ Construct, 500ng C043 $85.00
1 iLenti™-GFP Construct, 500ng C043-G $85.00
4 iLenti™ Constructs, 4 x 500ng C044 $275.00
4 iLenti™-GFP Constructs, 4 x 500ng C044-G $275.00
Negative Control Scramble Vector, 500ng LV015 $85.00
Negative Control Scramble Vector with GFP, 500ng LV015-G $85.00
iLenti siRNA Lentivirus
Production
1 iLenti Construct, Packaged Lentivirus(107 IU/ml; 2 x 200ul) C043V $475.00
1 iLenti-GFP Construct, Packaged Lentivirus(107 IU/ml; 2 x 200ul) C043V-G $475.00
Pooled Lentiviruses, 4 iLenti Constructs Pooled (107 IU/ml; 2 x 200ul) C044V $595.00
Pooled Lentiviruses, 4 iLenti-GFP Constructs Pooled (107 IU/ml; 2 x 200ul) C044V-G $595.00
Scrambled siRNA Lentivirus LVP015 $285.00
Scrambled siRNA GFP Lentivirus LVP015-G $285.00
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Detailed Information
Description
Custom-made, ready-to-use iLenti™ and iLenti™-GFP siRNA for transfection or infection.

  • A single construct for $85
  • 4 constructs for only $275!
  • Guaranteed knockdown for any gene
  • Every construct with a GFP reporter
  • Less than the material cost, why do it yourself?

Simply provide us with your gene's accession number for any target gene or siRNA target sequence and we will create the lentivirus siRNA construct for you within 2 weeks!

Once we get your gene name, we will search our siRNA database of over 20,000 publications covering over 7,000 human and mouse genes. Then we will design four constructs with high knockdown efficiency against the target. If your gene of interest is not yet published, we will design four target sequences using our internal siRNA software. With four siRNA constructs, at least one of the constructs should give over 70% gene knockdown efficiency in appropriate target cells with over 70% transfection efficiency.

We also have a scramble construct in our proprietary vector as the ideal control for your experiments.
Advantages of iLenti™ siRNA:
  1. siRNA in lentiviral vectors for either transfection or viral infection.
  2. iLenti™ vectors are very stable, no special competent cells are required.
  3. iLenti™ uses more potent 27-29 bp oligos.
  4. iLenti™ uses convergent promoters to avoid hairpin loop structures, making sequencing and plasmid growth much easier.
Custom siRNA Lentivirus (iLenti™) Services Agreement    
User Manual    
Infection Protocol    
Vectors
Notes
NOT FOR RESALE without prior written consent of ABM. This product is distributed for laboratory research only. Caution: Not for diagnostic use.
Product Documents
FAQs
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Is your lentiviral system Tat dependent or independent?
Tat-Independent.
About the negative control (scramble), what is it? How do you know it is not targeting other genes? Also does it have any marker for me to say that the cells have got the control siRNA, but it is not bringing down my gene?
It is specifically designed that it should bring down any gene based on sequence search. Using antibiotics as your selection marker can show whether the negative control was successfully incorporated by the cell.
How come I cannot see EGFP in my cells after infection with the iLenti-EGFP product? I have seen fluorescent activity with 293T cells but no fluorescent activity in my target cells (not 293T cells).
It is easy to see EGFP in 293 cells. Most of the other cell lines take about 48-73h to see the EGFP, and even then, the fluorescence activity may be low. The promoter strength for the EGFP gene varies with different cell line.

Refer to the paper "Transgenes Delivered by Lentiviral Vector are Suppressed in Human Embryonic Stem Cells in a Promoter-Dependent Manner" by Xia et al. in Stem Cells and Development 16:167-176 (2007).

EGFP expression is dependent on promoter strength, which may differ between different cell types. The promoter for the EGFP gene is from CMV. Even though the promoter is ubiquitous, the expression of EGFP may be suppressed in some target cells. That does not necessarily mean that the vector is not in the cells. The vector may be there and the siRNA may be expressed because the promoters for the siRNA are different.

You can try Western blotting against the target gene product along with a standard curve to evaluate whether siRNA is being expressed and functional in the target cells.
What generation is our iLenti-system?
It is third generation lentivirus.
What is our lentivirus based on?
Our system is based on inactivated HIV. All our packaging plasmids cannot be integrated into the host and they are transiently expressed only.
What is the function of 3’ acceptor sites in lentiviral vectors?
It's for biosafety reason. The 5' splice donor and 3' acceptors sites enhance the biosafety of the vector by facilitating removal of the packaging sequence and RRE such that expression of the gene of interest in the transduced host cell is no longer Rev-dependent (Dull et al., 1998). It will inhibit viral production in stable cell lines.
How do you ensure that your lentivirus is replication-defective?
We cannot send a protocol for you to view but I can summarize what we do to ensure the virus' replication deficiency.

Lentiviral vector stocks are tested for the presence of replication-competent lentivirus (RCL) by monitoring p24 antigen expression in the culture medium of transduced 293T cells for 30 days. Serial passaging of the transduced p24 cells over this period allows for the amplification of RCL. Since the vectors are replication-defective, no amplification of p24 signal would normally be expected. However, if there were an increase in P24 signal over time, it would be indicative of RCL contamination. The p24 antigen is assayed with a commercial kit from http://www.zeptometrix.com/ . This assay has been performed on multiple vector lots without a positive result.
How efficient is the lentivirus transduction?
This depends on the cell line or type as each one is different. In general, the lentivirus is very efficient in most cell lines with 100% for 293 and other commonly used cells. The lowest efficiency observed is 10% and customers have to do evolution for a specific cell line using EGFP lentivirus. For customers that just need to generate a stable cell line, 1% trandsuction efficiency is enough as every transduced cell is permanently integrated with the vector. Therefore, 10ml of 10^6 cfu/ml would contain more than enough transduced cells.
Does the final siRNA produced by this system have overhangs or not?
Once expressed in the host cell, Dicer will process the expressed dsRNA duplex into siRNA with 3' overhangs.
I cannot open link for information on lentiviral particle production for generation of stable cell lines. Please send link.
Here is the link for iLenti lentivirus production for generation of stable cell lines:

http://www.abmgood.com/RNAex/pdfs/iLenti-siRNA%20Expression%20System.pdf
I want to linearize the siRNA vector for electroporation purposes. Can you suggest a cut site outside the expression cassette that I can use?
HpaI or PacI will work to linearize the vector. Both sites are located outside of the LTRs.
What volume are the siRNA constructs provided in?
10 µl
I am having difficulties linearizing my vector with PacI, can you suggest any reasons why this isn't working?
In our experience, PacI is an inefficient cutter and when carrying out in-house digests with this enzyme, we will normally do a 3 hour digestion. We suggest doing a 20-fold overdigestion at 3 hours to linearize the vector if you choose to use use this site.

Can I use the BbsI sites shown on the map to cut the siRNA insert out of the vector?
The BbsI sites shown on the vector map of our siRNA constructs are used for cloning the siRNA inserts into the vector backbone. Once the cloning step is carried out these sites are destroyed and cannot be used to cut out the insert from the vector for further downstream applications.
What primers can I use to sequence my vectors?
One forward sequencing reaction using the U6 primer should be sufficient:

U6 forward sequencing primer
5'-TACGTCCAAGGTCGGGCAGGAAGA-3'

You can use the H1 Primer to sequence in the reverse direction if required:

H1 sequencing primer: 5'-CAACCCGCTCCAAGGAATCG-3'