If you make this virus using VSV-G instead of CCHF, what titre do you get? And how bright is the GFP after transduction in 293 cells?

Hi Dr,

In this case, titre is half of CCHF Lentivirus titre (200000 TU/ml). The transduction picture with CCHF Lentivirus was attached.

OK that doesn't look like it has worked at all, I'm not even sure that the GFP signal there is a healthy cell to be honest.

pLOX-CWgfp is an older vector but it's small, so titre should be high and CMV driving eGFP with a WPRE sequence should be easily visible in 293 cells. I haven't used this vector before but judging by its size I would expect a titre of around 1e6 (1 000 000) cells/ml non-concentrated, assessed by fluorescence anyway.

That suggests that your virus is underperforming by 5-fold, ie it's producing 20% of the titre I would expect. That kind of effect is probably not related to the ratios of plasmids, I think something else is going on.

Fluorescent (functional) titre measures virus particles that are able to transduce target cells. p24 titre measure viral protein, and it may not be a good assessment of functional titre especially if something has gone wrong.

What does your initial transfection look like? You should see very bright GFP in over 80% of cells depending on your transfection method.

Thanks a lot Dr,

That is correct that p24 titre measure viral protein but i have to measure virus titre by anyway. How do I do transducing while I do not know the titre virus stock? I also made LTX transduction according to the Invitrogen protocol, which in my 6-plate I provided 5 μg of plasmid. The transfection picture was attached that it was good. in this case, Reporter plasmid concentration was add 1/5 of 2.5μg pCDNA concentration.

I've been involved with the virus for several months, but gives me a low titre. Is there another way other than p24 to measure the virus titre?

Just to clarify, the pictures you attached to your last reply are of your transfection? If so, your transfection efficiency is not high enough - you should really be seeing at least 50% of cells GFP positive depending on your transfection method.

If that's the case, you have a problem either with your plasmid or your transfection method. What method are you using? Also, consider digesting your plasmid with AflII enzyme to see if you have deletions.

To titre a virus using fluorescence, I use a 24 well plate seeded with 293T cells. When they are 50-70% confluent, I add serial dilutions of virus - usually 100ul, 10ul and 1ul to three different wells although if I suspect the titre is going to be bad I add 500ul, 50ul and 5ul. Then I'd just count GFP positive cells and calculate the titre from there.

Thank you so much Dr,

This is last version of transfection. The transfection method is attached based on Lipofectamine® LTX DNA Transfection Reagents Protocol that we use 6 plate well. The protocol was done according to: mix A: 150ul media + 6 Lipofectamine LTX, Mix B: 150 ul media +5 μg plasmids (3:2:1 ratio including reporter, packaging, envelope)+5 ul PLUS™ Reagent then this two mix and finally 250 ul add to one well. Whether has this protocol problem such as amount of plasmids in one well in addition to the main protocol used 14 μg in each well? If we add higher Lipofectamine amount such as 9 or 12 ul whether transfection efficiency, increases?

In this case, i suspect to plasmid ratio , also in addition to your suggestion for transfection efficiency, that should be increased.

If I understand well, that is no need for p24 titre after virus concentration that we use directly concentrated virus for transduction.

To be honest if you follow the suggested Lipofectamine protocol then that's probably not the issue unless the Lipofectamine has been frozen or is out of date. Incidentally, if you don't absolutely need to use Lipofectamine then using PEI is just as easy and a fraction of the cost.

Are your 293 cells healthy? No big clumps or giant cells? If so, I would be looking at your plasmid as the possible issue. Do you have another GFP plasmid that you can use as a control? Have you sequenced and digested your plasmid to make sure there are no deletions?

Hi Dear Dr,

293 cell is healthy as it grows well, of course have granulated. I have other GFP plasmid such as plenti-III-mir-GFP (8.8 kb) that used as control against PLOXcwGFP that their efficiency was not significantly different. I thought that would try with two vectors with different concentration range again then tracking for transfection efficiency. What do you think of this approach? if yes, what are concentration range for plasmids?

I digested my plasmid as packaging vector and envelope but was not seen no deletion.

Hi Abbas:

For my lentiviral production, I use a ratio of 9:8:1 of transfer:R8.74: md2.g. Total DNA amount of 3ug/6 well or 10ug/10cm dish.

https://www.addgene.org/12259/ - Packing plasmid md2.g

https://www.addgene.org/22036/ - Packing plasmid R8.74

I found that plating 2million HEK cells in 10cm dishes 24h before transfection works very well. It is recommended to use half of the regular medium volume during the transfection phase. I only remove the transfection media 24h later. Supernatant collection is after 48h post-transfection.

I made a side-by-side comparison between Lipo 3000 and Mirus TransIT-LT1 for my LV packaging. The later gave me much better results in terms of toxicity.I concentrate my virus using ultracentrifuge. This protocol is working well for me.

You might wanna try: different ratios of plasmids; longer incubation period with plasmids up to 24h; reduced volume during transfection; lower passage cells; and do not use pen/strep in the transfection media.

Hope this helps you.

Good luck!

Hi Danuta

Greeting for your answer.

Do I adjust the ratio of the plasmids to its molar ratio? I did not understand or confusing with this case that longer incubation period with plasmids up to 24h. What does this mean?

I add transfecton media (Based on LTX invitrogen) then replaced to growth media (FBS 10% + Antibiotic) in 5 later whether this period should be increased to 24 h? also volume during transfection is 1ml instead of 2 ml whether is this your point in, reduced volume during transfection, ??? ?