hello everyone,
I have been trying to overexpress MDR1 gene using lentiviral vector transduction in MDA-MB231 cancer cell line. The cells were successfully transduced with virus (MOI: 10) and significantly expressed MDR1 gene. But unfotunately the result of protein expression assay by flowcytometry was negative for these cells! Please guide me. I need urgent help.
Hi Sadegh. How did you test for expression? Did you run a WB?
The most likely answear is that the antibody you are using to sort cells is not working properly. Did you check the datasheet if it is suitable for FC?
Hi Sadegh,
As mention by Francesco, how about the result of WB?
I provide some possibilities:
1. If the expression of MDR1 detected by WB is positive, check the applications of the Ab used in flow cytometry, and check whether there is leader sequence at the upstream of MDR1.
2. Maybe you can examine the expression of this plasmid by transient transfection (293T...) to confirm that MDR1 gene you cloned can be expression on cell surface.
3. Whether the cell number is too much during antibiotic selection after lentiviral infection?
Hope this help.
Dear Sadegh,
From your result of MOI, cells seems sufficiently transfected and there is high chance of detecting your protein fraction either in the superantat using ELISA if you used signal peptides in your construct or go for western blot, your gold standard for protein detection. is your NDR1 his taged ?
Thanks every one
In the first we transduced MDA-MB231 cell line with MDR1 containing lentiviral particle. selection of transduced cell line was performed in the presence of Hygromycin antibiotic but a few cells remined alive! after the growth of resistance cells, real time pcr was done and good results was obtained from MDR1 expression . for analysis of protein expression we used Flowcytometry. Its shouled be noted that antibodes which used checked perviously and don't have a problem.Can there be no translation of this gene?
Hi Sadegh,
Is that possible the expression of MDR1 gene was induced by hygromycin stress? Is their any tag fused with MDR1 protein, which transduced by lentiviral particles? Performing WB with anti-tag Ab may distinguish the endogenous and exogenous MDR1 in this experiment.
Thanks Weber
The cells were adjacent to hygromycin for seven days and qPCR was performed long time after exposure to Hyg. I'm not sure that presence of antibiotic induced extremely gene expression (130 fold!). forthermore this protein is a transmembrane pump and don't have any tag.
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