The MISSION TRC shRNAs are cloned into the pLKO.1 transfer vector that is compatible with standard 2 plasmid (packaging vector with rev gene and envelope vector) or 3 plasmid (packaging vector without rev gene, envelope vector, and rev expression vector) packaging systems.
No, we do not currently offer packaging cells. We recommend the commonly available engineered cell line HEK 293T. It is imperative that the cells you use be healthy, never grown to more than 80% confluence, and a maximum of 20 passage number. Following these recommendations will guarantee the best viral titers.
The standard offering provides each clone as 200 µL containing approximately 1 x 106 TU/mL (via p24 titering assay). Should you require high volumes or titers, we can accommodate your request.
High-Throughput Lentiviral Particle Production Results
We have optimized our viral production and this titer gives excellent integration efficiency in cultured cells. You may be more familiar with adenovirus, which titers at a range around 109. Lentivirus, however, has many advantages over adenovirus such as stable integration and ability to transduce primary and non-dividing cells, etc. If there are applications that require you to use higher titer virus, the highly stable VSV-G envelope allows concentration of lentiviral particle via ultracentrifugation.
We determine the TU/mL based upon the p24 assay from ZeptoMetrix, which is an ELISA against the p24 viral capsid protein. We use the conversion from Didier Trono to determine the relationship between pg/mL p24 and viral titer or MOI:
There are approximately 2000 molecules of p24 per physical particle (PP) of lentivirus:
(2 x 103) x (24 x 103 Da of p24 per PP), 48 x 106/Avogadro = (48 x 106) / (6 x 1023) = 8 x 10-17 g of p24 per PP, approximately 1 PP per 1 x 10-16 g of p24, 1 x 104 PP per pg of p24
A reasonably well packaged, VSV-G pseudotyped lentiviral vector will have an infectivity index in the range of 1 TU per 1000 physical particles (PP) to 1 TU per 100 PP (or less). Thus, the range is approximately 10 to 100 TU/pg of p24. It is through this conversion that TU/mL is obtained. Our current viral packaging protocol has given us highly reproducible titers.
Viral vector systems have been developed with enhanced safety features. It is recommended to use a third generation lentiviral vector system. It is a three plasmid system consisting of:
The packaging vector, which contains the minimal set of lentiviral genes required to generate the virion structural proteins and packaging functions.
The vesicular stomatitis virus G-protein (pCMV-VSV-G) envelope vector, which provides the heterologous envelope for pseudotyping, and
The shRNA transfer vector, which contains the sequence of interest as well as the cis acting sequences necessary for RNA production and packaging. The multi-plasmid approach results in no single plasmid containing all the genes necessary to produce packaged lentivirus.
Resulting particles are replication-incompetent and deletion in the U3 portion of the 3’ LTR eliminates the promoter-enhancer region, further negating the possibility of viral replication. The system has also removed virulence genes which are not necessary for shRNA packaging. These features combined have improved biosafety and handling. It is recommended to use a third generation lentiviral system for its enhanced biosafety features. Though there are no known incidents of third generation systems producing replication competent virus, it is important to monitor for replication competency when performing routine lentiviral packaging. NIH guidelines recommend replication-incompetent lentiviral particles be handled as Risk Group-Level 2 (RGL2). Additional precautions may be required based upon local, state, or country regulations.
One safety question regarding the large-scale manufacturability of viral vectors is the generation of replication-competent helper viruses during the packaging of the vector, which can occur by homologous recombination. Good laboratory practices are necessary to prevent any forms of contamination that may result in an opportunity for a replication-competent virus to form. Replication-competent virus testing is necessary. Because viral structural genes have been placed on different genetic units, multiple recombination events must occur before a replication competent helper virus is generated. Furthermore, areas of homology among the units expressing the helper virus proteins have been minimized. Also, heterologous promoters for the helper virus proteins are used. CDC-directed guidelines for BSL-2 + practices should dictate the handling of large amounts of virus and of concentrated virus.
Our production facility conducts standardized operation procedure of testing for the spontaneous production of a replication-competent lentivirus (RCV) at on a regular basis.
No, the viral particles cannot be propagated for biosafety reasons. The MISSION TRC lentiviral particles are replication incompetent. The particles are made using features of 2nd and 3rd generation lentiviral packaging systems. Genes for replication and structural proteins are absent in the packaged viral genome since these genes are supplied by other plasmids in the packaging cells. The viral genome contains only the region between the 5’ and 3’ LTRs of pLKO.1. In addition, the lentiviral vector contains a self-inactivating 3’ LTR that renders it unable to produce infectious virus once it integrates into the host chromosome.
The pLKO.1 vector is a lentiviral (HIV)-based plasmid. The vector is regarded as a biosafety level 2 material and safe to use due to its modified features (deletion of a number of accessory genes implicated in the virulence of HIV, minimal genome of the viral particles, non-replicating and self-inactivation features), making it incapable of producing virus once infected into the host cell. Please consult with your institution’s biosafety officer on specific requirements.
The library is lentiviral based. This allows for the production of viral particles in an appropriate packaging cell line. Upon infection with the resulting lentiviral particles, the shRNA sequence is integrated into the chromosome for stable expression of the hairpin RNA. The VSV-G envelope is pantropic and allows delivery to virtually any cell. In addition, lentivirus does not require a mitotic event for integration into the host cell genome.
No, this is not possible. The vector is used to make the particles in the packaging or producer cells. The viral genome contains only the RNA version of the region found between the 5' and 3' LTRs of pLKO.1 (promoter, hairpin sequence, puromycin resistance gene, etc.)
Given the size of the particles and the current VSV-G pseudotype, it is not expected that lentivirus will pass through the BBB (although we have no data to support this claim). Multiple researchers targeting areas of the brain have shown that direct cranial injection is optimal.
The NIH recommends preparing your lab for BSL-2 standards when using lentivirus. Please consult with your institution's biosafety officer on specific requirements.
The MISSION Lentiviral Packaging Mix can be used if you wish to produce more virus in your own lab since the viral particles we produce for purchase are replication-incompetent. The product number is SHP001. Our packaging mix is an optimized formulation of two plasmids consisting of a packaging vector containing the minimal set of lentiviral genes required to generate the virion structural proteins and packaging functions, and an envelope plasmid containing the vesicular stomatitis virus (VSV) G-protein. The exact contents and nature of the formulation are proprietary. The Lentiviral Packaging Mix is designed to be co-transfected along with a compatible lentiviral transfer vector into HEK293T cells in order to create high-titer pseudo-typed lentiviral particles used for downstream transduction applications. The procedure is available in the Product Information Sheet (286 Kb PDF) for SHP001.
It is commonly believed that lentivirus integrates randomLy within the genome. However, some literature suggests that lentivirus is known to insert into active genes.
For questions about the library, pricing and quotes or other concerns, please e-mail us at: MISSIONRNAi@milliporesigma.com. MISSION is a trademark of Merck KGaA, Darmstadt, Germany. Label License.
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