Frequently Asked Questions

Q: What is the required biosafety level for using recombinant lentivirus?
A: A biosafety level 2 facility is required for work involving recombinant lentiviruses according to guidelines issued by the NIH and Center for Disease Control. The VSV-G pseudotyped lentivirus is capable of infecting human cells, though these recombinant lentiviruses are replication-deficient. For more information on biosafety levels please visit http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm.

Q: Why are recombinant lentiviral vectors manufactured by Cellomics Technology the safest lentiviral vectors available in commercially available markets?
A: Great efforts have been made to improve the biosafety of lentiviral vectors, different safety features are built into lentiviral vectors to prevent the development of replication-competent lentiviruses (RCL), the biggest concern for lentiviral vectors. Even the earliest studies with HIV lentiviral vectors did not generate RCL in vitro or in vivo (Amado and Chen, 1999, Lentiviral Vectors—The Promise of Gene Therapy Within Reach? Science. vol. 285 (5428): 674-76), to minimize the possibility of RCL generation, Cellomics Technology routinely checks the RCL in lentiviral vectors produced to make sure our products are RCL-free.

Q: What is virus titer? How is lentiviral stock titer determined in Cellomics Technology?
A: In a strict sense, the definition of lentiviral vector titer is the number of lentivirus particles required to infect a cell, present in a volume. Titration of lentiviral preparation is important for consistent and reliable experiment results.

Two classes of titration methods have been described for the evaluation of lentiviral vector titers, including non-functional and functional titration methods. Functional assay includes the determination of transducing units following transducing cells with limiting dilution of lentiviral preparation, assessment of the number of colony-forming units following antibiotic selection, as well as FACS when fluorescent reporters are expressed. The latter includes determination of the quantity of p24 antigen by ELISA, reverse transcriptase activity, and the genomic RNA concentration in vector preparation. The non-functional methods provide information on the number of physical lentivirus particles in lentiviral preparation. Functional assay generates valuable data on the quality of these lentiviral vector particles.

Scientists of Cellomics Technology perform a functional assay to determine whether the lentiviral vector titer if it is feasible. Non-functional assays will be performed for some constructs if none of the functional assays are feasible.

Q: What's the optimal concentration of viruses that I should use for infection?
A: It depends on the purpose of the experiment. A higher titer of lentivirus should be used for in vivo experiments compared to in vitro experiments. Though the lentiviral vector is highly efficient in transduction, the transduction efficiency of the lentivirus depends heavily on the cell type to be transduced. A pilot experiment is highly recommended to determine how efficient the lentivirus is on your target cells.

Q: How much culture media should I use during infection?
A: For your reference, we recommend the following amount of virus-containing media for infection:

• 10-cm plate: 8-10 ml per plate
• 6-well plate: 1 ml per well
• 12-well plate: 0.5 ml per well
• 24-well plate: 0.2 ml per well
• This roughly reflects the surface area of each well or plate.

Q: What are the recommended storage conditions of recombinant lentiviruses?
A: For long-term storage, the virus should be kept at -80 °C. At -80 °C, the virus could be stable for 6 months to a year. Multiple freeze-thaw cycles should be avoided to prevent deterioration of lentiviruses.

Q: Does the freeze-thaw cycle infect the titer of lentiviruses?
A: Yes, multiple freeze-thaw cycles may reduce the functional titer of the virus stock by up to 2-4 folds. However, the first freeze-thaw cycle does not usually lower the lentiviral titer. Cellomics Technology recommends our customers not to use lentivirus that has gone through more than two freeze-thaw cycles for titer reasons.

Q: What is the capacity of a lentiviral vector as an expression system?
A: The cloning capacity for the transgene is approximately 3-4 kb for most vector formats.