太鼎生物科技/太鼎食安科技

siRNA客製化病毒顆粒

方便又省時 快速有效

不用自已養病毒、定量病毒。高力價siRNA Lenti病毒顆粒，直接感染細胞

【siRNA客製化病毒顆粒, 產品問與答】

1. 【問與答】慢病毒表現系統是依賴Tat或不依賴Tat ?

Tat-Independent.

2. 【問與答】如何知道陰性對照組是什麼?如何知道此對照組, 不會針對其它基因發生作用, 是否有任何標記?

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.

3. 【問與答】為何在感染iLenti-EGFP後, 細胞沒有表現EGFP螢光蛋白, 如此現像在在293T細胞株是有表現EGFP, 但是其它的細胞株則沒有表現EGFP蛋白?.

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.

4. 【問與答】iLenti-system是第幾代?

It is third generation lentivirus.

5. 【問與答】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.

6. 【問與答】lentiviral vectors的3’ acceptor sites 功能是什麼?

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.

7. 【問與答】如何確保 lentivirus 為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.

8. 【問與答】lentivirus transduction效能如何?

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.

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