Q: What is required to make retrovirus?
A: Retrovirus can be packaged using 293T or 293RTV cells. We also offer Platinum Packaging Cell Lines that can be used to package either amphotropic, ecotropic, or pantropic retrovirus. Here are the options for making retrovirus:
1. Transfect a Platinum retrovirus packaging cell line, such as Plat-A (amphotropic) or Plat-E (ecotropic), which stably expresses both MMLV Gag-Pol and an amphotropic or ecotropic envelope protein, with a retroviral expression vector. These cells require transfection of only an expression vector to produce retrovirus. Unlike VSVG pseudotyped virus, amphotropic and ecotropic viruses are not very stable, and can't handle ultracentrifugation.
2. Cotransfect the Plat-GP Retroviral Packaging Cell Line, which stably expresses MMLV Gag-Pol, with pCMV-VSVG and an expression vector. This cell line produces VSVG pseudotyped retrovirus that can infect any cell type.
3. Cotransfect 293T or 293RTV cells with 3 vectors:
a. pCMV-Gag-Pol vector
b. Envelope vector, either pCMV-VSVG, pCMV-Ampho, or pCMV-Eco
c. A Retroviral Expression Vector
Q: What is the difference between ecotropic, amphotropic, and pantropic virus?
A: Ecotropic pseudotyped virus can only infect mouse or rat cells, amphotropic can infect most mammalian cells, and pantropic (VSVG pseudotyped) virus can infect cells of any species. Ecotropic virus is safer to work with because it will not infect human cells. Both ecotropic and amphotropic are less stable than pantropic virus and are unable to withstand ultracentrifugation or a freeze/thaw cycle.
Q: How much total DNA should I use for transfection?
A: The total amount of DNA used for transfection will be determined by the transfection protocol that you are using. We recommend following the manufacturer’s protocol for the reagent you are using, which should specify the total DNA amount based on the plate size.
Q: Should antibiotics be present in the media during the transfection?
A: Any antibiotics that are used to maintain stable cell lines should be avoided when packaging the virus because they may be toxic to target cells if carried over in the media during transduction.
Q: When should retrovirus be harvested?
A: We recommend harvesting the viral supernatant 48 hours after transfection. To harvest, collect the virus containing media and pass through a 0.45 µm filter unit.
Q: Do you have a protocol for harvesting retrovirus?
A: We recommend harvesting the viral supernatant 48 hours after transfection. At this time, transfection efficiency should be at least 80% to get a good titer. If the transfection efficiency is lower than this, our suggestion is to optimize transfection conditions. Amphotropic and ecotropic retroviruses are not very stable and therefore we don’t recommend centrifuging the viral supernatant. To harvest, you will just collect the virus containing media and pass through a 0.45um filter unit. It is also recommended to use the viral supernatant the same day it is harvested because one freeze/thaw cycle can reduce the titer by up to 100-fold.
Q: What is a typical titer for retrovirus?
A: A typical titer for retrovirus under optimized conditions is 1x10^6 TU/ml, however there are many variables that can influence this value, such as the packaging cells, transfection efficiency, insert size, and the incubation time until harvest. We can’t guarantee a titer value because there are so many factors that contribute to this value that are dependent upon the researcher.
Q: How can I ensure good, efficient packaging of my retrovirus?
A: Here are some suggestions:
1. Because retrovirus contains LTR repeat sequences, recombination can happen after transformation, resulting in a plasmid of reduced size. We recommend using Invitrogen Stbl3 competent cells for plasmid amplification, which will minimize recombination. If you did not use these cell lines to amplify your plasmids, you should confirm that the plasmids have maintained the correct size by running a restriction digest.
2. The insert should not exceed the cloning capacity of the expression vector. Viral packaging becomes less efficient with large inserts, so you can expect a low titer if you clone an insert size that approaches the maximum.
3. Transfection conditions should be optimized to achieve at least 80% transfection efficiency. Less than 80% may result in low titer.
4. Unlike VSVG pseudotyped virus, ecotropic and amphotropic viruses are not very stable, and can't handle ultracentrifugation or freeze/thaw cycles.
5. We recommend using 293 cells that are healthy and at a low passage number for packaging virus.
6. Amphotrophic and ecotropic retroviruses are extremely unstable and are unable to handle ultracentrifugation. It is also recommended to use the viral supernatant the same day they are harvested because one freeze/thaw cycle can reduce the titer by up to 100-fold.
7. If you are unable to achieve the high titer you require, you can concentrate the virus by one of the following methods:
a. A concentrator unit with a 100,000 kDa molecular weight cut off membrane. Up to 2 mL of supernatant can be concentrated down to 200 µL, which should increase the concentration by 10-fold.
b. An ultracentrifugation step if the virus is VSV-G pseudotyped.
Q: Can retrovirus be frozen?
A: VSVG-pseudotyped retrovirus can be frozen, but it will degrade even at -80ºC. On average, each freeze/thaw cycle will result in a 2-4 fold decrease in titer. Amphotropic and ecotropic viruses are not able to handle a freeze/thaw cycle and should be used fresh.