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Lentivirus production issues

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#1 rkay447



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Posted 10 May 2017 - 05:40 AM

I'm very lost on why I'm not getting expression from my lentivirus preps.  I get resistance (neo) indicative of very high titers but can't get expression of my GOI.  I tried making a control GFP virus and get absolutely no green cells with transduction.  Transfection of the plasmid does result in GFP expression, however.    I'm using the pMuLE lenti destination vectors for target, the psPAX2 and pMDG.2 for packaging and envelope.  All plasmids were purchased from addgene.  I transfect cells with a 4:3:1 ratio of the plasmids and my cells look great at 72hrs with lots of multinucleated cells.  I harvest supernatant, centrifuge at 300g for 5mins, filter through 0.45um PVDF filter, aliquot into 1ml and store -80.  I don't put polybrene in the virus until after thawing and ready to put on target cells.  


Does anyone have any suggestions? 


**UPDATE**:   For future reference, the pMuLE destination vectors do not have a promoter.  You are expected to bring it in with your ORF. 

Edited by rkay447, 10 May 2017 - 11:18 AM.

#2 merlav



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Posted 17 May 2017 - 12:10 PM

1: Try to transfect the plasmids in equimolar ratio 1:1:1 instead of micrograms

2:Ultracentrifuge the particles or use the lenti X concentrator from Takara/clontech

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