Hi,
I am using a lentiviral vector to over express a protein in AML cells. I have cloned the flag-gene into the EIFalfa-MCS-SV40 vector. When I use this vector to simply transfect 293 cells, I can see the flagged protein with a WB. When I produce virus and infect AML cells, they grow fine under selection (no transduced cells die) but when I do a WB I cannot see the protein. I even checked with a qPCR the mRNA level, and there is a great fold increase compared to the empty vector. I have no idea of what's going on. Any idea?
Few things to check: 1) When you check on RNA levels, do you DNAse treat your RNA samples before proceeding to RT? I'm asking because there might be no expression of the transgene, but only the extra DNA copies integrated into the genome. 2) In infected AML cells, did you also so the western blot by flag antibody? or an antibody aginst the your protein?
No I didn't treat the samples with DNAse but why in 293 cells the plasmid is working and in AML shouldn't. Is weird, isn't it?
Yes I checked with the flag antibody even in AMl cells
I suppose that when you detect the protein in 293 cells you are able to detect the expected size fusion protein (the FLAG and protein of your interest) using the antibodies to the FLAG tag. If you are unable to detect the same size fusion protein in the AML cells, there can be a number of factors- to start- the amount of the protein that is expressed is too low due to reduced production and/or reduced stability. Therefore., use controls to find out these issues. However, there are more complex possibilities.
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