Hi Weiyan, to me it sounds like there is a problem with the antibiotic selection. Did you do a control antibiotic selection using non-infected cells?

Hi Steingrimur,

Thank you very much for your answer.

I did have non-infected cells as an control, I could see all the control cells died after around 5 days selection with 200ug/ml hygromycin selection.

I am wondering if it is possible the virus did not package the whole plasmids?

Hi Weiyan,

There is the possibility that you have improperly packaged virus however that would result in the titer of the virus being low. The problem you describe (a lot of cells that are antibiotic resistant but few that express your protein of interest) is most likely the result of the promoter driving expression of your gene being inactivated. This occurs frequently with the CMV promoter (and some other viral promoters) in some (but not all) cell lines/types. You may want to consider switching to another promoter such as EF-1alpha or PGK and see what sort of expression you see but do not use the same promoter that is driving hygromycin. 

Good Luck!

 Hi Tom,

Thanks for your detailed explanation and suggestions.

For this assay, I used the pQCXIH vector, IRES was used to link my protein and hygromycin resistance gene. I just confuse, how could it only express antibiotic resistance gene without my protein? 

Weiyan

Hi Louis,

Thanks, now I only have retroviral vector.

Weiyan