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Post-transduction troubleshoot.

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#1 Wek



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Posted 05 May 2015 - 06:10 PM

I have been having a series of problems transducing some cell lines with a lentivirus over the past few months.

I finally managed to transduce 2 of 3 cell lines but with a very low efficiency (<1%) and sort GFP positive cells (90% purity using IRES-GFP as the reporter). The day after the sort my cells were still green, however, after moving them to a larger vessel the GFP disappeared. The cells are growing at 37C with no problem but they are not green. I put a plate of cells in 32C and one in 37C and compared the GFP expression level, if any. The cells at 32C are starting to express GFP although at a low level but there is nothing in the 37C plate. Has anyone encountered this problem before? If so, any advice?


Another question, while working on the lentivirus transduction I started another transfection/transduction experiment with a different gene/vector. This time I am working with a retrovirus (MIGR1). My actual retroviral plasmid has no florescent reporter so I am using an empty MIGR1 that contains GFP as my positive control. Transfection with 293T cells in a 10cm dish went well (10ug of plasmid, 10ug of pcleco and 6:1 Fugene in a final volume of 8mls). I collected the sup after 3 days, spun it, and filtered. I used 1.5ml of the viral sup, 0.5ml of fresh media, and 5ug/ml of polybrene to transduced my cells in a 6well plate. After 24hrs of transduction I was able to see green cells in my control but it was about 30-40% of GFP positive cells (need higher percent because these are tumor cells). Then I changed the old media and this is where I have a problem. For the transduced cells (including the positive control), all the cells came off in one big layer. My two negative controls (regular media and media+5ug of polybrene) are fine. I tried to add the media slowly but it didn't help. These are adherent cells so something went wrong somewhere and I am not sure where. Any help is appreciated.


Btw I did performed a spinfection.



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