Regarding virus production in 293T using branched PEI?

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Joydeep Bhadury

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Jun 17, 2015, 10:48:14 AM6/17/15

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Sorry to use this thread for a question for virus production. I ask it as many people use here genome scale libraries and can help me with regard to the same.

I tried using PEI for both transient transfection and virus production in 293T cells. I used the branched PEI (Cat no 408727, sigma). Briefly I made a 25% solution from the stock(sigma) to get a conc of 250mg/ml. From this i dilute 1:100 in sodium chloride to get the working stock at a conc of 2.5ug/ul. I dilute my plasmid DNA and PEI in 150mM NaCl for transfection. I use 1:5 (DNA:PEI) to make the complex and incubate for 10-15 min in RT before adding the same to the cells. The issue is

1. I get really nice efficiency for transient transfection but the same doesn't work when making virus.The virus tranduction efficiency becomes almost negligible.

2.I tried incubating the cells with the PEI:DNA for 5 hr or over night to see the toxicity related issue, but nothing changed much.Also i tried various DNA:PEI ratio and nothing helped much. The 1:5 ratio(DNA:PEI) gives best results for transient but seems not to work for virus production

3. I tried reverse transfection and still no luck

Using the same plasmids (tried gama-retro and lenti, and different components for the same. Also tried VSG-G and ecotrophic Envelope) with Calcium phosphate transfection, i get the things to work fine. I wanted to use PEI as i had to scale up the virus production to massive amount to cope up with genome scale libraries.

Thanks you all for your help and suggestion in advance

Regards

Joydeep

Junjie Wu

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May 15, 2019, 12:20:45 AM5/15/19

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Hi Joydeep,

I noticed your post becasue the PEI reagent that I am going to use as well. I wonder if you have resolved the issue and how. In general, if the transient transfection works well, then the problem is on helper plasmids (no successful packaging) or the examination of virus titer (false negative titration or selection of stable clones). However, if you get things worked when use Calcium phosphate tranfection and SAME plasmids (not sure, are they same?), both plasmids and virus infection are fine. It seems unlikely that those plasmids only work for Ca phospate, whereas not to PEI....