PEG to increase infections? - (Feb/17/2008 )

Hello all,

I'm wondering if it is possible to enhance retroviral infection of hematopoietic cells (or any target cells for that matter) by adding PEG.

Cheers.

Yes. There is a protocol where you concentrate the virus using PEG which increases the titre atleast by 1.5 to 2 times. Protocol is as mentioned below:

CONCENTRATION BY PEG PRECIPITATION
A rapid and easy method consist in precipitating virus with polyethylene glycol.

Materials

5 M NaCl, filter sterilized
Polyethylene glycol (PEG) 6000, filtered sterilized
NTE buffer:
10 mM Tris.Cl, pH 7.4
100 mM NaCl
1 mM EDTA

1. Prepare a virus stock 10 mL.
2. Add 5 M NaCl to the virus stock to 0.4 M final while stirring at 4°C
3. Slowly add PEG 6000 to 8.5 % final (w/v) and continue stirring 1 to 1.5 hr at 4°C
Some batches of PEG do not work well in resuspending the pellet.
4. Collect the precipitate by centrifuging 10 min at 7000 g (e.g., at 7500 rpm SW-41 Ti rotor or 10,000 rpm in a Savant high-speed centrifuge).
5. Dissolve the pellet in NTE buffer in 1 % of the original volume.
The stock can be used directly after this step or stored at –70° or –80°C.
-Good luck
Calvin*

Thank you Calvin. We actually concentrate our virus by centrifugation since our virus is pseudocoated with VSV-G. We can actually get titres as high as 2 x ten to the eight!

Still with primary hematopoietic cells we get infection rates of between 8% to 40%. And lately they've been more in the 8% range.

So what I was thinking is that since PEG is an agent that can be used for molecular crowding, perhaps then by including it in the actual infection it might increase the likelihood of virus:cell interaction thereby increasing the transduction efficiency.

Can you think of any reason why this strategy would not work? I recall now that PEG is used to induce cell fusion in the making of hybridomas (I believe). I wonder if there's a happy medium in the concentration of PEG that would increase viral infection but not induce cell fusion.

Just another idea, nothing to do with PEG...we often use magnotofection based virus infection with Viromag RL Viromag. Due to the better delivery of virus to the cell surface with this system we get a higher infection rate (can't tell you how much, my colleague compared +/- magnetic particles and he is not here anymore...) all I know is that it helps a lot with stem cell infection

Stardust

We perform lentiviral infection of primary mammary epithelial cells and we get efficiency of ~80% or above which is without ultracentrifugal concentration! I have read somewhere that the efficiency of infection is higher if it is done on cells in suspension which you could try out. PEG would definitely help in crowding and will make a difference but which however may not be very significant. In my hands infection donewith 48hr supernatent is the best along with HEPES buffer and 4ug/ml protamine sulphate.