Has anyone had significance success in the transduction of the immunogenic murine tumour cell line (EL4) using the Lentivirus with a CMV promoter?
I have packaged the virus and concentrated using PEG but the virus titer is not high enough to transduce my target cell (EL4). However, I was able to obtain stable HEK 293 cell line expressing my gene of interest using the produced lentivirus.
Many hematopoietic cell lines don't express well off of the CMV promoter. I've transduced EL4 a lot but I've never tried it with CMV driven constructs. I've had good success with the MND promoter.
Have you done a mock transduction with PEG only (ie. no virus)? The PEG can sometimes do strange things to your cells (including causing them to die off). If you can concentrate without PEG (such as using an ultracentrifuge at 100,000 x g), you should try that.
@ Govinda Sharma ... Thank you for your suggestion. I will try out the mock transduction and also concentrate the virus using ultracentrifugation.
I will like to mention that I have previously had some positive clones of my target cell (EL4) expressing the gene of interest. However, the cells died shortly while trying to expand them.
I have also had that problem. I found that EL4 cells are extremely sensitive to mycoplasma contamination, which happens fairly commonly in tissue culture labs. I'd recommend testing for mycoplasma in your parental EL4 lines and your viral producer 293T lines (because the mycoplasma can get into your viral stocks via your producer cultures). If those are positive, you've got to go back to old, clean stocks if you have any or you can try cleaning them with commercial antibiotic kits.
Govinda Sharma , could you please share where you got the MND promoter/sequence from?
The plasmid sequences from the Rawlings and Kohn papers are on addgene, but sadly they did not annotate the promoter region or provide the MND sequence (not helpful!).
Would really appreciate your help with this! Thanks
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