Lentivirus production using PEI Max

[Dina Hany]

I am going to produce lentiviruses for the Gecko library using the recently published PEI protocol in https://www.nature.com/nprot/journal/v12/n4/full/nprot.2017.016.html, I am just wondering if I should change the medium 16 hours after adding the PEI plasmid mixture to cells?!, it is not mentioned in the above protocol but we used to doing it with normal lentiviral production, or, I should leave the plasmids and PEI mixture in contact with the cells for the 2 days till collection?!

I would be thankful if some one can give me advice.

Thank you 

Dina Hany

[Dina Hany]

Noted with thanks. 

On 2 Jul 2017 6:51 p.m., "Nabin Poudel" <poudeln...@gmail.com> wrote:

I am following up on this because I am also switching into PEI method for transfection for lentivirus production

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[Julia Joung]

Hi Dina and Nabin,

We have tried producing lentivirus using PEI with and without the media change and both methods produced lentivirus efficiently.

When I change the media 16h after adding PEI, I wait for 48h after the media change before harvesting lentivirus. When I am not changing the media, I harvest 48h after the transfection. Unfortunately, I have not compared the two different methods, but if someone has, that would be great to know!

Best,

Julia

[Julia Joung]

Hi Nabin,

By normal protocol, are you referring to the lipofectamine transfection for lentivirus production protocol? For that protocol, we have recently switched over to lipofectamine 3000 because it is a lot more effective and cheaper than lipofectamine 2000. We have posted the protocol in a separate thread. After 5-6h, we change the media to normal growth media.

Best,

Julia

On Mon, Jul 3, 2017 at 5:51 PM, Nabin Poudel <poudeln...@gmail.com> wrote:

Hello Julia,

Thank you for the information. How is your normal protocol for lentivirus production? Are you using serum free media after the 16 hour change out  or you switch cells back to normal growth media? I read people doing both the ways. Which protocol do you prefer?

Thank you

Nabin

--

Nabin Poudel, BVSc & AH
Graduate Teaching Assistant
Center for Veterinary Health Sciences,
Oklahoma State University,
Stillwater, OK, 74074
Tel: 405 612 9645

[Julia Joung]

For PEI, we switch to regular growth media.