Hi! You need to add more supernatant to have similar MOI, if you use low TU/ml, so your cells get poorer medium (you may add some extra FBS or glucose) and amount of medium above cells is bigger, so yes, this could impact on transduction efficiency. You may throw worse samples or concentrate them.

There is a very nice paper covering the relationship between viral vector titre and transduction efficiency. The viral vector concentration is critical for a good transduction (see link). My experience with low titre vectors is to perform a low speed centrifugation overnight at 4°C to concentrate the vector supernatant (low speed means full speed/4000g in a typical cell culture lab centrifuge). Do not forget to use sealed centrifuge buckets to avoid aerosols! This concentration step has the advantage that you concentrate your vector but also have the chance to resuspend your vector pellet in the medium for your downstream assay. Disadvantage - some pseudotypes are sensitive to the long period of centrifugation and you may lose a portion of your total vector. A further alternative is to use a transduction enhancement reagent, but these are not very effective with the VSVG pseudotype.

http://www.ncbi.nlm.nih.gov/pubmed/8864753

Assuming you are not pelleting and resuspending the virus, then yes your transduction may be different, in part for reasons pointed out by Karolina. I suppose that if you are attempting to do a transient expression of your gene of interest, then I would recommend that you pellet your virus and resuspend such that the TU/mL is about the same for each viral prep. This should take care of the issues raised by Karolina and Ian. Everything being equal, and so long as you are transducing a the same MOI, I don't see why you should get significant differences in transduction.

If you are instead aiming to generate a stable cell line, then pelleting your virus may not be necessary as you will perform a selection (puro, GFP, etc) anyways, and will only propagate those cells that were transduced and in which the viral DNA was integrated. You may also not want to use a very high MOI since in this case you want to minimize the number of integrations per cell. 

Best.

Hi,

First of all, it is not common to have such differences from prep to prep. The vector titer should not vary more than by 2-3 fold. If you get 100 to 1000 less than normal, just make another prep, unless you intend to transduce and select cells using GFP or antibiotics to establish a stable cell line, as raised by Ricardo.

Second, MOI is not everything. If two vectors that have been prepared the same way and titered together have titers as different as 10^8 and 10^6/mL, it probably means that the lowest one is contaminated by many non-functional particles. If you increase the volume of the lowest by 100 to get equivalent MOIs, you will also increase the numbers of non-functional particles that will compete with the functional one and inhibit transduction efficacy.

In other words, high initial concentration is more determinant than high MOI to get high transduction efficacy.

Best,

Emmanuel

First of all, I will suggest you to re-titer all the viruses (from different batched with different sequences) side by side to ensure this is what you really observed.  Then based on the titers to add different volumes to get the same MOI.  But the titering method matters.  If possible serial dilution and counting colonies after antibiotic selection will be more promising.  It is the most time consuming titering method but may give you more accurate result.

People may titer the virus by real-time PCR, p24 assay, or antibiotic selection from supernatants to the transduced cells.   Also, there are many steps in lentiviral transduction, e.g. viral genome gets into the cells, reverse transcription, integration, etc.  The DNA context and the size of lentiviral genome may affect the transduction efficiency.  Yes, different cDNA inserts may give you quite different virus titers due to the size and sequence of the insert, and the gene toxicity.  In general the low-titer virus gives lower transduction efficiency so that's why you need to bring up the volume to get the same MOIs by comparing with the high-titer viruses.  However, this may not be always true if you titer viruses from the supernatants.  What if the high-titer virus (titered from supernatant) has less efficiency in RT and integration because of the DNA context or something else we don't really know.  In the end the high-titer virus may show the same copy number as the low-titer virus.  It is possible the lentivius encoding one gene variant has high titer (titered from supernatant) but much more toxic to the cells and it may show lower transduction efficiency after antibiotic selection.

I don't know your experimental design but you better be careful to investigate the final integration copy numbers and the level of gene expression to get fair results.

Low TU/ml is not necessarily indicative of low transduction efficiency as different backbones do tend to give different titres.  However, if you see variation between preps of the same vector (or no pattern between the 4 variants), it is suggestive that the low titre is a result of a poor prep which will not work as well.  To get an indication of quality, you could perform a P24 assay to quantitate the number of viral particles and get a ratio of this against TU/ml, showing how many 'empty' viruses you have in each prep.  If this varies a lot between preps,  you need to tighten up your production; make sure you don't use producer cells that have gone through too many passages, check your transfection reagents, be extra careful about how many cells you plate before transfection etc etc.  You can just use more vector and correct with MOI for those with a lower titre, but you run into problems outlined by Karolina and Ian and will need to concentrate vector if you're not already.  Good luck.

Thank you all for these very helpful answers. I am using the viruses to induce transient protein expression in SHSY-5Y cells. Given the problems I am having with the viruses, I am going to explore transfection with lipofectamine as an option. 

At present I think the only part of my viral protocol that could be tightened is counting the number of HEK cells per flask prior to plating them out, at present I just make sure the confluency across all flasks is similar prior to beginning viral production. This may not be good enough.

I calculate the TU/ml by counting fluorescent cell numbers (via FACS), in serial dilutions of the virus. I use the dilution factor where the % cells are around 20% to calculate the TU/ml. I started using this technique as it was recommended by a colleague and the Trono lab. But I will investigate the P24 assay as that may be more accurate?

Just as an aside - if you are looking at cell phenotypes (protein aggregations/mitochondrial membrane potentials etc) in the same cell line, expressing different variants of the same protein, at 48-72hours would you think viral transduction to get the gene of interest in to the cell is a little 'overkill'? With the caveat that transfection rates etc were acceptable in that cell line.

Nature.med.2001.7.631.A quantitative assay for HIV DNA integration in vivo

Later RT: 12hr; 2-LTR circles: 24hr;  integration:  48hr.  In general we start selection at 48hr post transduction.  However you may start seeing protein expression such as green fluorescence at 12 hours post transduction in high MOI.  If you freeze the viral stock, please gently mix before use.  Spinoculation with Polybrene is highly recommended, it may increase transduction efficiency by 10-fold or more.

Dear Esteemed Colleague,

Greetings. I hope this message finds you well and engrossed in your vital research endeavors, particularly those involving the use of lentiviral vectors for gene delivery. Your inquiry regarding the relationship between the transduction units per milliliter (TU/ml) of a specific lentivirus and its transduction efficiency is both pertinent and insightful. Below, I delve into the nuances of this relationship, providing a clear and logical analysis.

Understanding TU/ml and Transduction Efficiency

Relationship Between TU/ml and Transduction Efficiency

• Direct Correlation: Generally, there is a direct correlation between the TU/ml of a lentiviral preparation and its transduction efficiency. Higher TU/ml values indicate a greater number of functional viral particles, which can potentially increase the proportion of successfully transduced cells.
• Threshold Effect: However, it is important to note that transduction efficiency does not increase indefinitely with higher TU/ml. Beyond a certain threshold, additional increases in TU/ml may not significantly enhance transduction efficiency due to saturation of cellular receptors or limitations in the cellular machinery required for viral entry and integration.

Factors Modulating This Relationship

Conclusion

While a low TU/ml in a lentiviral preparation generally suggests a lower potential for transduction efficiency, the actual efficiency is influenced by a complex interplay of factors, including cell type, viral and host cell factors, MOI, and the use of transduction enhancers. Thus, optimizing the conditions for lentiviral transduction involves not only maximizing TU/ml but also carefully considering these additional parameters.

Should you require further assistance or seek more detailed insights into optimizing your lentiviral transductions, please do not hesitate to reach out. I am here to support your research endeavors and facilitate the success of your gene delivery experiments.

Warm regards.

Reviewing the protocols listed here may offer further guidance in addressing this issue