Q: What is required to make lentivirus?
A: To make lentivirus, you need:
- 293T cells, such as our 293LTV cell line
- A lentiviral expression vector to clone in a gene of interest
- A lentiviral packaging system which includes vectors containing lentivirus structural proteins.
Q: Why are your ViraSafe™ Lentiviral Expression Systems safer than other systems?
A: ViraSafe™ Lentiviral Expression Systems introduce added safety measures in a couple of ways:
1. ViraSafe™ Lentiviral Expression Systems are specifically engineered to minimize the chance of making replication-competent lentivirus (RCL). The ViraSafe™ systems use 3rd generation lentiviral packaging technology which provides for co-transfection of 4 separate plasmids, but sequence homology is reduced an additional 80-85%, making the chance of RCL even lower.
2. In addition to the standard VSV-G pseudotyped virus, which can infect virtually any cell regardless of species, you can now make an ecotropic lentivirus by choosing a ViraSafe™ Ecotropic Expression or Packaging System. Viruses made with an ecotropic system are packaged with a different envelope protein that will only readily infect mouse and rat cells.
Q: What is included with the Lentiviral Expression Systems?
A: We offer Lentiviral Expression Systems to produce either ecotropic (infects mouse and rat cells) or pantropic (infects most cell types) virus. Our Complete Expression Systems include one of 11 different expression vectors, a control vector, and three packaging vectors:
- pRSV-Rev packaging vector
- pCMV-VSVG envelope vector (pantropic) or pCMV-Eco envelope vector (ecotropic)
- pCgpV packging vector that expresses lentiviral Gag and Pol
The three packaging vectors are also available as a Lentiviral Packaging System, for researchers who may already have a 3rd generation expression construct. Expression vectors are also available individually.
Q: What is the difference between ecotropic and pantropic virus?
A: Ecotropic pseudotyped virus can only infect mouse or rat cells while pantropic pseudotyped virus can infect cells of any species. Ecotropic virus is safer to work with because it will not infect human cells; however it is less stable than pantropic virus and cannot handle ultracentrifugation procedures used for concentration and purification.
Q: How can I increase the plasmid prep yield of my pSMPUW expression vector?
A: The pSMPUW vectors are low copy plasmids and typically give a low yield. Here are some suggestions to get the maximal yield:
1. Bacterial culture of pSMPUW vectors should be done in medium containing 10ug/mL Kanamycin.
2. For maximal plasmid yield and quality, we recommend Stbl3 competent cells (Invitrogen) and treatment with alkaline proteinase (Promega #A1441 or Sigma #P8038) for 4-5 min using 10 units of proteinase per mL of bacterial lysate before adding neutralization solution. You can use Top10 or DH5a competent cells as long as you do a restriction enzyme digestion of the isolated plasmids to make sure the plasmid size is still correct and there is no recombination.
3. For each maxiprep, we use the bacterial pellet from 500 mL culture (24 hrs at 37ºC).
Q: What E. coli strain do you use to amplify pSMPUW expression vectors?
A: We recommend using Invitrogen Stbl3 competent cells for plasmid amplification, which will minimize recombination that can happen through the viral LTR sequences. If cells other than Stbl3 were used, such as DH5a or Top10, a restriction digest can be performed to confirm that the plasmid has maintained the original size.
Q: Should the insert contain a polyA site?
A: Inserts cloned into lentiviral expression vectors should not contain polyadenylation signals. The viral 3’ LTR contains a polyA site, therefore the presence of polyadenylation signals upstream may result in premature cleavage of the viral genome during transcription. This would affect viral genome packaging and result in low titer.
Q: Are your pSMPUW Expression Vectors compatible with 2nd generation packaging vectors?
A: Our lentiviral packaging and expression vectors are 3rd generation and have been modified to be safer to use (i.e. less infectious to humans) by supplying the necessary packaging elements as three separate plasmids, instead of the two that are used in 2nd generation systems. It is fine to use a 2nd generation packaging system with the 3rd generation pSMPUW lentiviral expression plasmid; however the titer may be lower than if you use our Lentiviral Packaging System.
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; 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: Can viral stocks be stored?
A: VSVG pseudotyped virus can be stored at -80ºC for approximately 6 months. Each freeze/thaw cycle drops the activity by about 20-30%, so it is recommended to aliquot before freezing. Lentiviral stocks should be thawed slowly on ice. Ecotropic lentivirus is not as stable and should not be frozen.
Q: What is the expected titer for lentivirus?
A: A typical titer for lentivirus 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 increase the titer of my lentivirus?
A: Here are some suggestions for higher-titer lentivirus production:
1. Because lentivirus 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 viruses are not very stable, and can't handle ultracentrifugation or freeze/thaw cycles. If you are making ecotropic virus, it is best to infect target cells immediately upon harvesting the supernatant.
5. We recommend using 293T cells that are healthy and at a low passage number for packaging virus.