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Lentivirus transduction issues: Little to no GFP observed

Lentivirus Transfection Transduction cloning

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#1 gitanjaliroy

gitanjaliroy

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Posted 30 November 2021 - 01:51 PM

Hello,

 

I am trying to express GOI in Min6 (mouse) cell line using Chloroquine and PEI (1:3) protocol from https://www.addgene....rus-production/.

 

I have tried using 293T, LX293T and even 293FT cells for virus production.

I have collected the virus every 24 hrs (like the Addgene protocol says) and also leaving them for 72 hrs straight and then collecting.

I filter the supernatant using 0.45 um syringe filter and use Polybrene (0.8 ug/ml) for transduction.

I have also tried "spinfection" after reading some answers on this forum.

 

My problem is I see a lot of GFP upon transfection indicating good transfection efficiency. After collecting the viral supernatant when I transduce Min6 cells I see little to no GFP in them. Where am I going wrong?







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