Our RNA interference lentiviral vectors contain siRNAs. We employ a dual-convergent promoter system where the sense and antisense strands of the siRNA are expressed by two different promoters rather than in a hairpin loop - this is to avoid any possible recombination events that can occur.

abm guarantees that at least one out of the four siRNA Lentivector constructs purchased in a set will result in over 70% knockdown of gene expression within target cells showing >80% transfection efficiency. If this is not the case, we will provide a one-time replacement of four new constructs with alternative siRNA sequences. To qualify for this replacement, the vectors must be transfected at ≥5 nM and assayed 48 hours post-transfection. Customers must provide adequate data to show >80% transfection efficiency with a positive control, plus additional qPCR data to evaluate the level of gene expression. The replacement set will not covered by the same guarantee, and if these constructs are also considered to be ineffective then, it is most likely the gene is not susceptible to siRNA knockdown.

abm limits its obligation and liability for the success of this technology to providing one replacement of any siRNA lentivector product only. Before sending your inquiry, please make sure you have extensively optimized your experiments, including (where applicable) increasing your MOI, increasing the duration of infection (up to 72 hours), and carrying out clone screening before assaying for knockdown. Please send all replacement inquiries and experimental data to technical@abmgood.com.

*Due to the unpredictable nature of customized knockdown, this guarantee does not apply to siRNAs designed 1) against species other than human, mouse, or rat, 2) against regions other than the coding region (CDS), or 3) with customer-provided siRNA target sequences. All sales are final for custom designs.

NOT FOR RESALE without prior written consent of abm. This product is distributed for laboratory research only. Caution: Not for diagnostic use.

Tat-Independent.

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.

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.

iLenti is third generation lentivirus.

Our system is based on inactivated HIV. All our packaging plasmids cannot be integrated into the host and they are transiently expressed only.

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.

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.

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% transduction efficiency is enough as every transduced cell is permanently integrated with the vector. Therefore, 10ml of 10^6 IU/ml would contain more than enough transduced cells.

Once expressed in the host cell, Dicer will process the expressed dsRNA duplex into siRNA with 3' overhangs.

HpaI or PacI will work to linearize the vector. Both sites are located outside of the LTRs.

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.

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.

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'