New services available from the Virus Tech Core Facility
We have developed a lenti/retroviral titration method measuring transducing units, or number of proviral copies in the infected cells.
If your transgene has no reporter, the only available option you had to titer your viruses was by analysing the number of RNA copies (physical titration). This method usually overestimates the actual number functional viral particles in a 100-150 fold (Geraerts et al., 2006 and based in our own analyses), when compared to functional titration or expression of reporter genes in the infected cells upon FACS analysis.
We can now offer a new titration method based in the integration of the provirus, by analysing the number of copies of provirus integrated in the transduced cells and normalizing to a house keeping gene. In our hands the values obtained by this new method compared to the FACS functional methods were comparable, thus presenting a viable titration method when no reporter is present in the trangene.
Purification and concentration of viruses
We used precipitation/concentration and ultracentrifugation to ensure an almost 99,6% of purificed viruses in our products.
We offer several services of virus media concentration depending on the necessities of our customers:
- 100 KDa Centricon Plus-70 Centrifugal Filter: This is our standard method to concentrate supernatants. Pros: the concentrated product is ready to use, fast and reliable. Cons: Limited virus media volumen per prep.
- PEG precipitation: An alternative method of virus precipitation. Pros: Higher volumes of viral supernatant per prep, PEG reduces the inmunoresponse when viral vectors are applied in vivo, viruses titer lost upon thawing and freezing cycles is dramatically reduced (25% of titer lost reported against a 3% in our experiments). Cons: PEG precipitates other impurities, so ultracentrifugation is needed as a further purification step.