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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 the recombinant lentiviruses according to guideline issued by the NIH and Center for Disease Control. The VSV-G pseudotyped lentivirus 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 recombinant lentiviral vectors manufactured by Cellomics Technology are the safest lentiviral vector available in commercially available market?
A: Great efforts have been made to improve the biosafety of lentiviral vectors, different safety features are built in 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 check 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 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 to consistent and reliable experiment result.
Two classes of titration methods have been described for evaluation of lentiviral vector titers, including non-functional and functional titration methods. Functional assay includes 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 the information of 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 functional assay to determine the lentiviral vector titer if it is feasible. Non-functional assays will be performed for some constructs if none of functional assays is feasible.

Q: What's the optimal concentration of viruses that I should use for infection?
A: It depends on the purpose of the experiment. Higher titer of lentivirus should be used for in vivo experiment compared to in vitro experiment. Though lentiviral vector is highly efficient in transduction, the transduction efficiency of lentivirus depends heavily on the cell type to be transduced. Pilot experiment is highly recommended to determine how efficient the lentivirus 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 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, first one freeze-thaw cycle does not lower the lentiviral titer, usually. Cellomics Technology recommends our customer not to use lentivirus that has gone through more than two freeze-thaw cycles for titer reason.

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