If you’re planning on using lentivirus for gene therapy, you will need to find out how many lentivirus particles to use. However, this is where researchers new to gene therapy work often run into problems. If this is you. Stop. Before you embark on endless optimisation experiments (i.e. a different transfection reagent, new cells, different phases of the moon), put your pipette down and listen up! It is not a result of you being an incompetent lab chimp. There are several important factors that you need to know if you want to produce a batch of lentivirus with super high titer.

For in vitro work, multiplicity of infection (MOI) is the theoretical number of virus particles applied per target cell. That is, if you have 1 million cells and you want an MOI of 5, you need 5 million lentivirus particles for your transduction experiment. However having calculated an ideal number of lentivirus particles for their experiments, you may discover that your lentivirus production is only a fraction of your desired titre. You probably wonder – as your frustration mounts due to repeated attempts to produce more lentiviruse particles – why you can’t achieve the lentivirus titer that colleagues and commercial providers routinely get???

Well let’s look at the common factors that may be limiting your titers.

HEK 293 Producer Cells

First up, be nice to your 293 producer cells. The 293 cell line was derived from embryonic kidney cells and it is commonly used for lentivirus production. Although there is no concrete evidence on the number of times you can passage your cells before you see a drop in virus titre, the cells must be healthy and actively dividing. Clumpy cell cultures with lots of senescent cells won’t produce good titres. It’s worth doing a test transfection on your cells before you try using them for virus production. If your transfection efficiency is low, then there’s no point continuing on with virus production. You will need to setup a new cell stock.

Bottom line is-happy cells will make good lentivirus stocks! For example, It is best to split your cells the day before transfection to make sure they are happy. Depending on the type of transfection reagent and the length of incubation, you might need a different cell confluency level. In addition, lentivirus particles are sensitive to pH, so adding HEPES buffer to the culture media can act to protect the virus from pH extremes. And note is if your plasmids contain a SV40 origin of replication, you would need to use the 293T because it contains the large T antigen to drive the protein expression.

Transfection Reagents

For efficient virus production, you need to get all of your plasmids inside the maximum number of cells. So it goes without saying that you need the best possible transfection efficiency. There are many different commercial and non-commercial transfection reagents available.

If you’re doing virus preps regularly, commercial transfection kits can be very expensive. Cheap chemical reagents like calcium phosphate and polyethyleneimine (PEI) work effectively and are very budget-friendly. There’s evidence (mostly anecdotal) that certain transfection reagents result in higher virus titres. You need to interpret these results with caution, as there is no real consensus on the best reagent to use. If you are just starting out in virus production you can certainly compare transfection reagents, but don’t spend months on this step. If you are getting good cell transfection then it’s probably best to stick with what you’ve got.

Lentivirus Plasmid Design and Construction

Packaging Plasmids

A lentivirus expression typically contains a transfer plasmid and a packaging plasmid. Your plasmids don’t have to be purified using a specific method. You don’t need to use super pure endotoxin-free kits or Cesium Chloride or whatever your professor claims was the “golden rule.” Just make sure that you have high quality plasmids- not degraded and of a reasonable concentration (over 100ng/ul). Again, it’s not worth obsessing over this step. It’s perfectly possible to make virus even from ordinary miniprep DNA, though you may see some drop in lentivirus titre.

As far as packaging plasmids go, the main consideration is not to confuse third generation plasmids to second generation lentivirus plasmids. Second generation transfer plasmids require the presence of the HIV-1 tat protein, which has been removed from third generation systems. There is evidence that second generation plasmids may produce higher titres when used to package a third generation transfer plasmid but the difference (if any) is probably not great.

Transfer Plasmids

Now that you have healthy cells, good transfection reagents and the right packaging plasmids, you’re ready to make lentiviruses. But now we come to the most important factor in virus production: the transfer plasmid itself. The transfer plasmid is the plasmid containing your transgene, such as short hairpin RNA (shRNA), guide RNA (gRNA), and non-coding sequence of interest. So, before you commence virus production, check your transfer plasmid using either sequencing or restriction digestion to make sure no recombination or deletions have occurred. Lentiviral plasmids are prone to mutation in some bacterial strains. Therefore, use a recA(-) strain (e.g. Stbl3 and SURE2 strains) if possible.

All lentiviral transfer plasmids are not created equal. One of the main factors influencing virus titre is the length of the sequence between the two HIV-1 long terminal repeats (LTRs). Virus particle yield decreases steadily and predictably as the sequence that you are trying to package gets longer (1). For example, if your transfer plasmid contains a short promoter driving a small transgene such as a fluorescent protein, you are quite likely to see good titres.

There’s also published evidence that the use of multiple promoters in lentiviral plasmids results in ‘promoter interference’, where one or both promoters will adversely affect expression from the other (2). This phenomenon can decrease transgene expression and results in low lentivirus titre. So, if you are trying to make a virus that contains a giant protein or multiple proteins via internal ribosomal entry site (IRES ) and WPRE sequences, your virus titre will be much lower. Different transgenes can also affect titre. Bacterial proteins such as Cas9 and Channelrhodopsin are bad to 293 producer cells and can adversely affecting virus production (3).

It’s difficult to overstate how important the transfer plasmid is to the success of your lentivirus production. It is not uncommon to see titre differences of 50-fold or more between different batches of virus made at the same time using the same reagents but with different transfer plasmids. I have included some examples below on how the protein encoded in the transfer plasmid could affect the variation in lentivirus titre (Figure 1).


Figure 1. Fluorescence microscopy on GFP lentivirus production. All the photos were taken using same system, settings and time exposure.

The top right photo illustrates another issue surrounding virus titre (Figure 1). Fluorescence microscopy is a quick, simple and cheap way to measure transduction. However, protein expression levels will influence your calculations. For example if a fluorescent protein is placed downstream of an IRES sequence, protein expression may be reduced by as much as 80%. That is, you may have plenty of virus transduction but if the fluorescence is too weak to see, you will underestimate virus titre. Flow cytometry is generally more sensitive than microscopy but weak fluorescence can still cause underestimation of titre. Therefore, promoter choice can also cause titre variations depending on how you assess transduction.


The take home message here is please do not to get so focused on small tweaks that you spend excessive time and money. Just remember the 3 main points for your protein(s) of interest: 1) size, 2) quantity, and characteristics. The transfer plasmid is usually the biggest factor controlling lentivirus titre. So there isn’t much point obsessing over optimising steps resulting in a 10% improvement in titre if your transfer plasmid is reducing production by 50%.

Before beginning virus production, plan your experiments and calculate how much virus (i.e. virus particles) you need. In vitro experiments often don’t require very concentrated virus, so there’s no point aiming for very high titres if you don’t actually need them. Bigger isn’t always better – obviously it’s best to have good transduction levels but your cells probably aren’t going to be happy if you bombard them with an MOI of 1000. Be realistic.

If your virus preps just aren’t producing enough for your planned experiments, consider re-cloning or redesigning your transfer plasmid. It may seem like an unnecessary waste of time but it could well save you time, money and sanity in the end.


  1. Systematic Determination of the Packaging Limit of Lentiviral Vectors, Kumar (2001)
  2. IRES-Dependent Second Gene Expression Is Significantly Lower Than Cap-Dependent First Gene Expression in a Bicistronic Vector, Mizuguchi (2000)
  3. Generation of a Genome Scale Lentiviral Vector Library for EF1a Promoter-Driven Expression of Human ORFs and Identification of Human Genes Affecting Viral Titer, Skalamera (2012)