Hello Nathan,

I love Fugene and it is undoubtedly one of the most efficient if not the most efficient transfection reagent. A couple of years ago I compared side-by-side various commercially available transfection reagents and Fugene was found to be most efficient at-least for 293T cells. That said, we routinely transfect 293T cells using calcium phosphate and obtain almost as high transfection efficiency (up to 80%) as we get from Fugene. However, it took us some time to perfect our technique. We transfect 293T cells with HIV molecular clones that tend to be pretty large (~14-15 Kb) without problem. Unlike calcium phosphate, Fugene (or similar reagents) is easier to use and optimize and can transfect DNA in less-than-optimally grown/maintained cells. Calcium phosphate method is far less forgiving and results in poorer transfection efficiencies in poorly maintained cells.

Hi Nathan,

I have used several of the liposome based transfection reagents and they all work for transfecting 293T cells. The main drawback is cost of these reagents so if you do a lot of transfections that is your biggest expense.

I would highly recommend using Polyethylenimine (PEI - linear, 25,000 MW) for your transfections. It is very very inexpensive and works just as good as the liposome based reagents. I have compared PEI to lipofectamine for the production of lentivirus and found there was no difference. The cells were transfected at the same efficiency (>80%). You can buy 2 grams for about ~$100 and it will last forever. You make up 1mg/ml stocks and use it like lipofectamine at a 1:3 ratio (DNA:PEI). Leave the complexes on the cells for 4 hours, remove and add fresh media. Very simple and cheap.

Thanks for those responses. We are doing large scale virus production so you are quite correct Tom, the price is far to high.

The other thing i should mention is that we are looking at suspension cultures in order to both remove FCS and hopefully increase virus production.

We have tested PEI but the results were inconclusive but there were a few problems at the time with production so the plan was to test it again next to calcium phosphate and lipofectamine. 

I am quite surprised with that transfection rate with calcium phosphate Syed, do you think that would be similar with a suspension culture? we do use discs for the cells to adhere to before transfection but i was under the impression that the binding of the calcium phosphate complex to cells was quite slow and cells being at rest would be preferable?

Thanks again for the replys

Nathan,

We have not transfected suspension cultures of 293T cells using calcium phosphate. As a matter of fact, we have never dealt with suspension cultures of 293T cells so I am not in position to make any comment on this.

HI Nathan,

Transfecting suspension 293's can be a bugger. I would suggest maybe to trial performing your transfections on pelleted cells in tubes rather than is suspension. Once transfected then plate out the cells as normal. I have done this for small scale lenti production and it worked well. DOTAP is a relatively inexpensive transfection reagent (nothing is as cheap as Calcium phosphate). The beauty of DOTAP is that the DOTAP/DNA complex is actually quite stable and can be used for weeks after formulation.

cheers

Darren (PS: say hello to David for me)

293T is easily transfected via lipofection. Period. It's been known for a while, and it has been on countless times confirmed (see https://altogen.com/product/293t17-transfection-reagent-emb-kidney-crl-11268/). Of course there are drawbacks; transduction can be more efficient at delivering nucleic acids into cells, and if you have highly-concentration lipofection buffers you could negatively affect cell viability. But otherwise, the technique is sound, and if it weren't, you wouldn't see so many lipofection-based reagents on the market.

Hello Nathan Rout-Pitt ,

Using Lipofectamine 3000 for lentivirus production can help you reach up to 26x virus titer than Calcium Phosphate and up to 3x titer than FuGene HD with 293T cells. You can find the data and the detailed protocol in the attached application note.

I understand that the price may be a concern, but given the high titer you can get, the consumables like filters, conical tubes and pipettes you can save, and the potential contamination risk when aliquoting the solution you can prevent from by using LF3K, it is definitely a good option.

For suspension cells transfection, we also recommend Neon Transfection System (thermofisher.com/Neon). It's an electroporation system and therefore can overcome the low-efficiency disadvantage of chemical-based transfection technologies like calcium phosphate transfection.

You can find more information at https://www.thermofisher.com/us/en/home/life-science/cell-culture/cell-culture-learning-center/cell-culture-resource-library/cell-culture-transfection-application-notes/improve-lentiviral-production-using-lipofectamine-3000-reagent.html

Hope this help.

Dear Colleague,

When considering the methods for transfection in virus production, specifically comparing calcium phosphate and lipofection (using agents like Lipofectamine or FuGENE), it is essential to delve into the nuances of each method to understand their suitability for your specific application.

Calcium phosphate transfection is a widely used method due to its cost-effectiveness and simplicity. This method involves forming a precipitate of DNA with calcium phosphate, which is then taken up by cells. It is particularly effective for stable transfection and is suitable for a broad range of cell types. However, its efficiency can be variable and is often dependent on the cell line and the quality of the DNA-calcium phosphate precipitate. Additionally, calcium phosphate transfection can sometimes result in cytotoxicity and is less efficient for transfection of large plasmids or in cells that are difficult to transfect.

On the other hand, lipofection, using reagents like Lipofectamine or FuGENE, is a lipid-based transfection method. These reagents form complexes with DNA, facilitating the fusion with the cell membrane and the subsequent internalization of the DNA. Lipofection is known for its higher efficiency and lower cytotoxicity compared to calcium phosphate, especially in transient transfection scenarios. It is also versatile, being effective in a wide range of cell types, including those that are typically challenging to transfect. However, the cost of lipofection reagents is significantly higher than calcium phosphate, which can be a limiting factor for large-scale applications.

For virus production, the choice between these two methods depends on several factors:

In conclusion, both calcium phosphate and lipofection have their distinct advantages and limitations for virus production. The choice between them should be guided by factors such as the desired efficiency, scale of production, cell type, and cost considerations. It may also be beneficial to conduct preliminary experiments to determine the most effective method for your specific application.

Best regards,

Take a look at this protocol list; it could assist in understanding and solving the problem.