Strange Cell Morphology & Cell Debris Following Lentiviral Transduction - (Oct/23/2012 )

Hello,

I was hoping someone in this forum might be able to provide and explanation and/or solution regarding some strange instances I have been encountering while making stable cell lines. The two cell lines I am using are mouse embryo fibroblasts (MEFs) and THP1 monocytes.

First, using a pCDH-GFP lentiviral prep to make green MEFs I notice that several days following transduction the cells have lots of debris on them and the look to be internalizing it as the cytoplasm of them contains lots of granules. The cells themselves grow just fine, and are green but I cannot seem to get rid of the cell debris or the granules inside the cells. The debris and granules cause them to have a sort of "rough" appearance at low magnifications. Just to elaborate I squirt on some virus (with polybrene) into a minimal volume of media, then place it on the cells for ~4hrs. After 4hrs I either replace the virus containing media with fresh media, or I will just double the media volume and let the transduction continue overnight, after which I replace the media. ANY thoughts on this would be immensely appreciated.

Second, I am using a pTRIPZ lentivirus to stably transduce THP1 cells. I follow the above protocol. and again get a similar result, the THP1 membranes begin to ruffle and they lose their nice rounded morphology. Some of the cells die during the process of transducing them. After puromycin selection for 3 days I have cells that persist and the population will expand. However, they display the above mentioned morphology and a lot of debris begins to accumulate into the media. I can dilute out the debris but as the cells proliferate the debris becomes more abundant. Again, any thoughts or help here would be great.

Thanks in advance

chabraha

Have you titrated your puromycin? Could it be that you are using too high a concentration?

Yeah i have for the thp1 cells (1ug/ml), but this happens even if I don't perform the selection. As for the MEFs, the pCDH vector I use has GFP for the selectable marker.