I use a plasmid to overexpress 1 protein in 293T and murine BMSC. But I could never succeed.
I produced the lentivirus by transfecting the plasmids in HEK293FT cells, together with another 3for the packaging machinery and the capsid of the viruses. I use MegaTran for transfection, and I change the media after 14 hours. Then I collect the viruses 48-72 hours after transfection (I also harvest these cells, lise them and do western blot to check for the expression of my protein). Finally I ultracentrifuge the media with the viruses to infect 293T cells and murine BMSCs.
By flow cytometry I see huge expression of the protein in transient transfected cells, but none in the infected ones. I use GFP expression to monitor the infection and it is fine, because I see many green cells in both transient transfectied and infected cells, but none overexpression of my protein with the viruses.
Could anyone give me some advice? Thanks!
Dear Eugene
You are checking GFP expression in transduced cells (lentivirus infected cells) by FACS which is really sensitive technique. I guess u are checking the overexpression of ur protein with immunoblotting which is less sensitive than FACS.
You can try to increase MOI or change host cells.
Manpreet Kaur Dear Kaur, I am actually checking the overexpresssion of my target protein also by FACS. Thanks for you answer.
I would presume that you have successful overexpression (since you see GFP in the target cells) but there is an issue of detecting the overexpressed protein i.e. antibody you use doesn't work well with the flow. Something in your flow for detection of the target protein is missing, i.e. your target protein is intracellular or intranuclear and you incubate live cells with the antibody instead of fixing and permeabilizing them. Try a different approach i.e. Western blotting this is usually the best way to detect the overexpression. Another thing to consider, have you confirmed if your plasmid is valid i.e. sequencing the crucial plasmid fragment with the code of your protein of interest? Otherwise many backbones will produce the GFP fluorescence as it's encoded on the plasmid, but if your code for the overexpression of the target is missing you only get GFP but no target.
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