Help! ES Lenti infection efficiency=nightmare - trouble getting over 40 % cells infected (Jan/25/2011 )

Hello bioforum!

Please help me!
I am infecting ES cells with Lentis carrying shRNAs but I haven't been able to get more than 40% of the cells infected.
I concentrate the virus by ultra centrifuging them (with PEG it the efficiency was down to 20%). I do the infection with the cells in suspension and it still sucks.
If you guys have any thoughts on how to improve this please share them with me. I am totally stuck!

Hi,

There could be many things that are going wrong !

Are you adding polybrene?

Have you tried transducing another cell type with the same virus? Is the efficiency bad too?

Clare

Radish on Tue Jan 25 18:54:19 2011 said:

Hi Clare

I tried other cells and they are fine, I can get up to 90 % efficiency, it is a ES cell thing apparently.
I can't use polybrene, it is extremely toxic to the cells, even at very low concentration (like 2 ug/ml).
I see a lot of people getting crazy efficiencies and I am not even close...
Tried less cells, doing it in suspension (after trypsinizing them), rotating them, you name it...

I think I need to find a decent virus titration protocol and play with the viral titers

Thank you for you reply Clare!

Hi Radish,

What kind of ES cells are you using?

I am growing rat ES cells and just 2 days ago infected them with a lenti using 8ug/ml polybrene and they look fine and happy.

I have the cells growing in 4 well plates (you know, the tiny little wells?) and left the cells in 200ul media + virus + polybrene for 3-4 hours only, then top up with fresh media (to 900ul).

Does this help?

Clare

Radish on Wed Jan 26 19:40:30 2011 said:

Hi Clare

I tried other cells and they are fine, I can get up to 90 % efficiency, it is a ES cell thing apparently.
I can't use polybrene, it is extremely toxic to the cells, even at very low concentration (like 2 ug/ml).
I see a lot of people getting crazy efficiencies and I am not even close...
Tried less cells, doing it in suspension (after trypsinizing them), rotating them, you name it...

I think I need to find a decent virus titration protocol and play with the viral titers

Thank you for you reply Clare!

Clare on Thu Jan 27 08:55:46 2011 said:

Hi Radish,

What kind of ES cells are you using?

I am growing rat ES cells and just 2 days ago infected them with a lenti using 8ug/ml polybrene and they look fine and happy.

I have the cells growing in 4 well plates (you know, the tiny little wells?) and left the cells in 200ul media + virus + polybrene for 3-4 hours only, then top up with fresh media (to 900ul).

Does this help?

Clare

Radish on Wed Jan 26 19:40:30 2011 said:

Hi Clare

I tried other cells and they are fine, I can get up to 90 % efficiency, it is a ES cell thing apparently.
I can't use polybrene, it is extremely toxic to the cells, even at very low concentration (like 2 ug/ml).
I see a lot of people getting crazy efficiencies and I am not even close...
Tried less cells, doing it in suspension (after trypsinizing them), rotating them, you name it...

I think I need to find a decent virus titration protocol and play with the viral titers

Thank you for you reply Clare!

Happy Friday!

I would try culturing them in virus + minimal media for 3-4 hours - that's the way the lenti lab routinely does it as my work, and it all seems fine

Clare



Hello Clare

Thank you so much for you reply.
So, it seems that you are using the Ivanova protocol for the infections...
I am really thinking to give it another go.
I am working with E14s, and I tend to see a lot of toxicity with polybrene (I do the transfection o/n)...
Maybe I should try to keep them in virus for less hours?
Tell me what you think.

Once again, thank you Clare


Best regards
Radish