iLenti™ siRNA Expression System
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Product Details
Description
Recombinant lentiviral vectors have been shown to be a powerful tool for stable gene transfer to both dividing and non-dividing cells in vitro and in vivo. Through years of experience with lentiviral vectors, scientists at ABM have developed our own proprietary Lentiviral expression vectors and pLenti-combo packaging mix that allow for rapid production of recombinant lentiviral vectors with titers up to 107IU/ml. iLenti™ RNAi Expression System allows for the efficient expression of any target siRNA through transfection or lentiviral infection of target cells. The system is based on an unique convergent promoter design for greater efficiency of target gene knockdown without the need of a hair-pin loop structure design commonly required for a single promoter vector.
Advantages of ABM's iLenti RNAi Fusion Expression Vectors
- stable plasmid; rare plasmid DNA re-arrangement
- very high yielding plasmid
- no special competent cells required
- convergent promoter design
- higher efficiency of target gene knockdown
- ABM is THE source for your lentiviral expression needs!
User Manual 
Infection Protocol
Vectors
Related Products
Notes
NOT FOR RESALE without prior written consent of ABM. This product is distributed for laboratory research only.
Caution: Not for diagnostic use.
FAQs
- Is your lentiviral system Tat dependent or 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?
- 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).
- What generation is our iLenti-system?
- What is our lentivirus based on?
- What is the function of 3’ acceptor sites in lentiviral vectors?
- How do you ensure that your lentivirus is replication-defective?
- How efficient is the lentivirus transduction?
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 neomycin 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.
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.
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.