This month's question from the Molecular Biology Forums (online at molecularbiology.forums.biotechniques.com) comes from the “Cell Culture” section. Entries have been edited for concision and clarity. Mentions of specific products and manufacturers have been retained from the original posts but do not represent endorsements by, or the opinions of, BioTechniques.Molecular Biology Techniques Q&A
What is the best way to safely use viral expression systems? (Thread 25894)
Q I'm trying to express a construct in NIH 3T3 cells that alters a particular metabolic pathway to analyze the resulting cellular changes. I need to express the construct in all of the cells, which is not possible using lipofectamine transfection. I know viral transduction is highly efficient, but I've never worked with viruses before. Since most publications on viral transduction relate to gene therapy, I have been unable to find a basic summary or guide. Here are a few questions:
The available options are adeno-, lenti-, or retrovirus systems. My cells are actively dividing, so there's no need to worry about quiescent non-infected cells. I think transient expression will be fine since I only need expression for about two days. If possible, it would be nice to use the same system for transient expression and then to screen for stable expression. For these reasons, I am thinking of using an adenoviral system. What else do I need to consider when selecting a system?
What kits do you use and what has been your experience?
I would like to do this work in the cell culture hood in my lab. If I use an ecotropic virus that only infects mouse cells, will that sufficiently address safety concerns?
A Another option for NIH 3T3 cells is a targeted integration cell line using the Flp-In system from Invitrogen. We used this in our lab and it worked. You may also try using any stable transfection vector and then selecting clones. That would be a lot less work than setting up a viral system.
A To use a viral expression system requires a safety facility; a cell culture hood is not enough. In most cases, you can do a regular transfection, select the cells with antibiotics for two weeks without selecting clones, and then use those cells for your experiment.
Q Why isn't a regular cell culture hood sufficient for working with viruses? What is required to set up a safety facility?
A colleague told me he worked with retroviruses using a routine cell culture biosafety hood. Since I work for a startup company, we do not have a proper biohazard disposal facility. What precautions should I take?
A The safety rating of an organism depends not only on how dangerous it is alone, but also on the precautions you take, the danger to the environment in the event of viral release, where you are working, and other variables. To determine the necessary precautions, you will need to contact your local genetic modification safety officer to assess your experimental design to see if it is safe for the use of these viruses. I used an oncoretrovirus system based on a moloney-murine leukemia virus with the pMX expression vector, which contains an IRES site. The system consists of a virus backbone into which you clone your gene of interest. You can use the virus to stably or transiently transfect cells. The viruses are usually disabled so that they cannot replicate outside the lab environment. Our virus lacked both its gag-pol and env genes. The loss of gag-pol disables replication of the virus and the loss of the env gene allows variation in envelope proteins to match the target cell. Some envelope proteins will transfect murine cells, some human cells, and some all cell types. This system worked in 293T cells, but not in stem cells. We were able to work with this vector in a class II hood.
Unless you are transfecting stem cells, I would not recommend that you use lentivirus, SIV, or HIV, since this will increase the safety requirements. Lentivirus and SIV will transfect non-dividing cells since they can cross the nuclear envelope. Oncoretroviruses are safer since they only transfect dividing cells where the nuclear envelope has broken drown. Adenoviruses do not usually integrate into the genome since they replicate in the cytoplasm.
I got good transfection rates and multiplicity of infection (MOI) using the pMX/MSCV vector with a standard gag-pol gene and the VSVG envelope. I no longer work with the viruses, so I'm not certain where you can get them now. I suggest you scan the literature to see who has used them recently and then contact them to ask if they will give you some.
Q I am transfecting the packing cell line with VSV-g, gag-pol, and the gene of interest, then collecting the viral supernatant and infecting target cells. Aside from facility safety, what other precautions should I take?
A That really depends on what you are doing and which system you are working with. Are you using a xenotropic or murine specific env gene? Is the gene inserted in the virus oncogenic or apoptotic in humans?
Generally, your hood should be a class II HEPA filtered hood or better. You must treat the waste with bleach before disposal.
A All of the above, along with several other considerations, affect the safety of the procedure. When using genetically modified organisms, the effect on the worker and the environment should be considered. In general, if you are doing the following, your experiment should be considered a low risk:
Use a disabled virus vector missing essential genes that must be co-transfected with the backbone vector, such as gag-pol, Si (signal sequence), and env. This means that unless a rare triple recombination event occurs with the backbone vector, the virus cannot replicate within one of your cells or spread outside of an initial infection event.
Use an envelope specific for non-human cells so the virus should not infect you. VSVG is xenotropic and can infect human cells. I used it previously to infect stem cells. If you are worried about safety, try a murine specific vector for your mouse cells. This may be less toxic to the cells as well. VSVG is toxic, but I did not observe much cell death, even with high MOI.
Use a class II hood. This protects the cells rather than the operator, although these viruses generally do not infect via an aerosol route, so it should be fine.
Use non-hazardous genes of interest. If the gene inserted in the virus is not oncogenic, the damage will be limited in the event of infection.
Use oncoretroviruses. They are generally considered to be safer than lentiviruses, SIV, or HIV since they only infect dividing cells.
Be sure to follow good laboratory practices. Use gloves, be careful with syringes, etc.
Do not pour live virus down the sink or leave it standing outside the hood. Treat the waste accordingly before disposal.
Many people use retroviruses safely. As long as your work has been cleared by your local genetic modification safety officer or equivalent and you follow all the guidelines that came with that approval, you should be safe. I strongly recommend that you talk to your safety officer and see what other steps you can take to minimize chances of cross-infection.