3rd Generation Packaging System Mix
For the production of lentiviral particles, three components are generally required: 1) a lentiviral vector containing your inserts of interest (cDNA, shRNA, or miRNA), 2) one or two packaging vectors which contain all necessary viral structure proteins, 3) an envelope vector expressing Vesicular Stomatitis Virus (VSV) glycoprotein (G). The 3rd generation packaging system offers maximal biosafety as the lentiviral Rev gene is supplied as an independent vector from other structure genes, further eliminating the possibility of reverse recombination of vectors into replication competent viral particles. The 3rd generation lentiviral packaging mix will only support lentiviral expression vectors with a chimeric 5' LTR in which the HIV promoter is replaced with CMV or RSV, thus making it TAT-independent. The 3rd generation lentiviral vectors will not support the production of 2nd generation lentiviral particle productions. All of the lentiviral vectors marketed by ABM are TAT-independent.
|Unit quantity||200 µl|
|Caution||Not for diagnostic use.|
NOT FOR RESALE without prior written consent of ABM. This product is distributed for laboratory research only.
- Enhanced Lentivirus Safety Features: Replication Incompetency
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- Lentiviral System FAQ
|What is the usual survival rate of cells after initial infection? I transduced two types of cells and after 72 hour incubation, one cell line had 50% dead, the other had just about 85-90% dead. Any pointers?|
Each cell line is different; like two different people, one with a drug sensitivity and one without, there is no means of comparison between cell lines. There are variations in the membrane transporters for the selection marker as well as cell transduction efficiency, so each cell line must be treated differently and their optimal killing curves must be determined independently.
|Are there any safety issues to consider if using lentiviral vectors in the same laboratory where replication competent adenovirus work is being carried out? For instance, can elements of one become incorporated into another under certain conditions (e.g . giving rise to a replication competent retrovirus)?|
If the adenovirus does not code for the viral packaging genes of the lentivirus, there is no safety issue. To remove all safety concerns, we recommend not to clone the viral packaging genes of the lentivirus into the adenoviral genome.
|Which cell line is better for high-titer lentivirus production - 293T or 293FT cells?|
Our 293T was clonally selected to be fast-growing and transfected easily and thus also supports high-level expression of viral proteins. We routinely use them to produce very high titer (10^10) lentivirus. Both cell lines are capable for proficient lentivirus production.
|What is the ratio of the three packaging plasmids when packaging via 293 cells?|
The three plasmids are present in equal molarity and in equal ratio.
|Is it possible to grow the plasmids up when they are mixed?|
No, it is not possible to grow up the plasmids from the product.
|Which system should I use to package my TAT dependent vector?|
TAT dependent vectors will need to be packaged using a 2nd generation system (abm Cat LV003). 3rd generation packaging systems (abm Cat. LV053) will not be compatible with this type of vector.
|What is the titer for viruses made by Cat# LV053 with 293T cells?|
For both our 2nd or 3rd generation packaging mixes, the crude viral supernatant (i.e. not concentrated nor purified) titer will be around 10^6IU/ml.
|Is RRE present?|
Yes, it is present after Pol in pLenti-P3A.
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- Song, J et al. "miR-370 and miR-373 regulate the pathogenesis of osteoarthritis by modulating one-carbon metabolism via SHMT-2 and MECP-2, respectively" Aging Cell 5:826-837 (2015). DOI: 10.1111/acel.12363. PubMed: 26103880.
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- Kang, et al. "PCGEM1 stimulates proliferation of osteoarthritic synoviocytes by acting as a sponge for miR-770" Journal of Orthopaedic Research 34.3:412 (2016). DOI: 10.1002/jor.23046. Application: Lentiviral infection.
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