Viral vector production is a worthwhile skill that can be made even easier with a few tips and tricks. In general, transfection of multiple plasmids into a producer cell line results in infectious, non-replicative virus. However, it is important to ensure that your vector preparation is efficient, giving your experiments the best chance of success. Poor virus titres can mean that it’s not possible to deliver your sequence of interest to enough of your target cells, leading to less than ideal results.
There are many different protocols available on the internet and elsewhere describing how best to produce viral vectors. Different authors make varying claims about the best ways to ensure success as well as the pitfalls to avoid. But how many of these claims are legitimate and how many are simply myths? To find out, read on:
Virus Producing Cells Must Be Below a Certain Passage Number: MOSTLY FALSE
The most important tip about your virus producing cells is to take good care of them. If you passage irregularly or with poor technique, the cells will not produce good virus titres no matter the passage number. HEK 293 cells are sensitive to passage number and should be replaced regularly. However, HEK 293Ts (with the SV40 T antigen) are more resilient and, if diligently maintained, can be used for six months or even longer with no significant reduction in virus titre. Of course, if you are using cells prepared or frozen by someone else, you may be uncertain of the original quality. If all else fails, feel free to blame the name on the tube!
If You Perform the Procedure Correctly, a Certain Titre Is Guaranteed: FALSE
Virus titre depends on a number of factors and is by no means guaranteed. Lentivirus titres are very dependent on the transfer plasmid. Large plasmids containing complex transgenes can be particularly problematic. For AAV, different serotypes can also produce very different titres. The choice of transgene can also affect virus production, with some sequences poorly tolerated by the 293 producer cells.
Viral Vectors Can Be Stored at -80°C for Many Years: SOMETIMES TRUE
AAV has a long shelf life at -80 degrees and can anecdotally be kept for many years without significant loss of virus. Most people working with lentivirus know that the virus should not be freeze-thawed repeatedly. However, even if lentivirus stays frozen at -80 degrees it will still slowly degrade over time. Viral vectors older than 6 months should be re-titered before use. If you pull a vial of lentivirus out of the depths of the freezer and the production date begins with ‘200-‘, it is probably best to throw it away!
If My Virus Works Well in Cell Culture, It Will Work Just as Well In Vivo: FALSE
You can measure the titre of viruses expressing fluorescent protein or drug selection markers by infecting 293s cells, assuming that the transgene promoter isn’t tissue-specific. However, to infect other cultured cells or animal tissues, it may be necessary to use a higher multiplicity of infection (MOI), more concentrated virus and longer time points to check for viral expression.
On the other hand, some AAV serotypes are more efficient in vivo than in vitro. For example, AAV-8 works very well in the rodent nervous system, but titres measured in culture using 293s cells are not usually as robust. For this reason, AAV preparations are often titered using QPCR.
If I See Fluorescence After Transfection but Not After Transduction My Viral Vector Production Didn’t Work: FALSE
Keep in mind that plasmid transfection delivers hundreds of thousands of copies of your fluorescent protein DNA. In contrast, virus transduction may only result in a handful of virus particles successfully entering a cell. Therefore, it is quite common to see robust fluorescence after transfection but low fluorescence following transduction. If you are using a weak promoter or a bicistronic system, you may see no fluorescence at all. If this is the case, you can extract RNA or DNA from infected cells and titre your virus by QPCR to determine if your production was successful.
It Doesn’t Matter Which AAV Serotype I Use: FALSE
There are a wide variety of AAV serotypes available. The first to be routinely used was AAV-2, as it infects many different cell types. However, other serotypes such as AAV-1, AAV-6 and AAV-8 are more efficient than AAV-2 in particular tissues, especially in vivo. It’s best to do small scale trials before you choose your AAV serotype for a major experiment – don’t just use whatever serotype your lab has been using for years. Be aware that many serotype plasmids can only be used under material transfer agreement (MTA), so make sure you have proper permissions before using them.
I Can Concentrate My Lentivirus by Ultracentrifugation With No Loss of Yield: FALSE
The most popular method of concentrating lentivirus is ultracentrifugation. If you already have the right centrifuge and rotors, it’s easy and cheap. However, physical forces during ultracentrifugation can damage lentiviral particles, meaning that you won’t see 100% recovery after concentration. Other concentration methods such as polyethylene glycol (PEG) and chromatography may result in less damage to viral particles, but tend to be expensive.
Virus Must Be Purified and Concentrated Before It Can Be Used: MOSTLY FALSE
If your virus will only be used in vitro, there is usually no reason why you need to concentrate it. Non-concentrated lentivirus can be used if your target cells can tolerate DMEM with serum, while crude AAV (effectively a cell lysate) is also suitable for some in vitro work. AAV ultracentrifugation purifies the virus but doesn’t concentrate it very much – the main point of AAV centrifugation is to remove cellular proteins and other debris which could cause problems in vivo. Keep in mind that AAV may not be the best choice to infect rapidly dividing cells. The virus does not integrate into the host cell DNA efficiently and will be quickly lost from the culture.
When it comes to viral vector work, one of the most important things you can do is manage your expectations and be well prepared. If your producer cells are old and clumped, your transfer plasmid is very large or contains a toxic protein and your experiment needs large quantities of concentrated virus, you are setting yourself up for an expensive and time-consuming failure.
However, with good preparation, careful technique and a little research, you give yourself the best possible chance of success.
After you’ve generated your mutuations using CRISPR-Cas, the next step is to identify those cells that have been successfully edited. There are a few different ways to check for the mutations. I’m going to discuss some of the more popular ones.
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