4 replies to this topic

[laurequillo]

Hi guys!

I am trying to produce some virus using pbabe constructs and Phoenix (ampho and Eco) and Platinum E cells.
The problem is that I dont get any virus (or not enough) from them.
I transfect them with 10ugr of pbabe, and after 48 hours I collect the supernatant to infect the target cells.24h hours after infection I add puro and almost all cells die, so they are not infected.
I have read that the packaging cell line should be confluent 48 hours after the transfection, but sometimes my cells after transfection dont grow so well, and maybe that is the reason why I dont get a nice virus production. Now I am trying with pbabe-GFP and it looks like the transfection efficiency is not so great.

Do you guys use any special reagent to transfect the cells? Do you add cloroquine? Do you wait 48 or 72 hours? Do you transfect them just with the pbabe or the pbabe plus VSV-G?

Thanks!

[laurequillo]

After having no luck with the phoenix cells I am trying with the Platinum E and pbabe-gfp.After 48h I have a good number of green cells, but they are not really confluent. I will collect the viruses today and I will check them.
Do you think is a problem with the confluency of the cells? ( I `ve read something about that)

[SciCell]

I work with Phoenix cells, transfect them, collect the virus after 48hrs, 72hrs, and infect them to target cells - the same way you do it. I think we need to optimize the experiment, quite a lot. My lab mate says she gets 100% transfected cells after 48hrs. I don't get 100% but do get like 60-75% transfected cells (as observed for GFP under a florescence microscope). Here in this experiment the number of cells we seed and DNA ratio are important. Anyways it depends on the GOI - gene of interest. Another thing is we change the medium to regular medium the next day after transfection - to enable the transfected Phoenix cells to multiply in their natural growth conditions and also the transfection agent is toxic to cells.
Second point is: after infection, I would let the cells grow for 48hrs and then add the selection medium. This is because the successfully infected cells might be so few, hence I want to increase their number before I start killing with selection. Sometimes multiple infections might be necessary.
I am not saying you are doing something wrong but just trying to give some info as I am doing the same thing.

laurequillo, on 16 October 2010 - 12:05 PM, said:

Hi guys!

I am trying to produce some virus using pbabe constructs and Phoenix (ampho and Eco) and Platinum E cells.
The problem is that I dont get any virus (or not enough) from them.
I transfect them with 10ugr of pbabe, and after 48 hours I collect the supernatant to infect the target cells.24h hours after infection I add puro and almost all cells die, so they are not infected.
I have read that the packaging cell line should be confluent 48 hours after the transfection, but sometimes my cells after transfection dont grow so well, and maybe that is the reason why I dont get a nice virus production. Now I am trying with pbabe-GFP and it looks like the transfection efficiency is not so great.

Do you guys use any special reagent to transfect the cells? Do you add cloroquine? Do you wait 48 or 72 hours? Do you transfect them just with the pbabe or the pbabe plus VSV-G?

Thanks!

[laurequillo]

Thanks SciCell!
yeah sure, I have tried different DNA:Reagent Ratios, but If I use more reagent the cells dont grow well, and if I use less I dont get any virus...anyway now that I have the pbabe-GFP I can really check the transfection efficiency, so I will chech different amounts of DNA/reagent and different amount of cells till I get a nice transfection.
Which reagent do you use?

[SciCell]

laurequillo, on 19 October 2010 - 10:18 PM, said:

Thanks SciCell!
yeah sure, I have tried different DNA:Reagent Ratios, but If I use more reagent the cells dont grow well, and if I use less I dont get any virus...anyway now that I have the pbabe-GFP I can really check the transfection efficiency, so I will chech different amounts of DNA/reagent and different amount of cells till I get a nice transfection.
Which reagent do you use?

I use Fugene HD