3 replies to this topic

[SJHan]

To generate cell line to stably express target protein, lenti-viral vector carrying target gene was generated. To validate target gene expression from lentiviral vector, transient transfection was conducted with 293TN cells.  Expression of FLAG-tagged target gene was confirmed by Western blot analysis with antibody against FLAG peptide. After generating  lentivirus with above lentiviral vector,   293TN cells are transducted by above virus and then selected by drug for 2 weeks. After drug selection, however, target gene expression in drug resistant 293TN cells was not detected by Western blot analysis eventhough its expression was confirmed by transient transfection.  Does anybody has a same experience about above issue? Do you have any solution?
Thanks

[scolix]

expression is much weaker in stable cells compared to transient transfection. did your WB have the positive control? you could try to load more protein. make sure the cells 293TN doesnot have the antibiotic resistance gene already. We have used 293FT and they were always in G418 containing media so we could not use this for making cell lines with G418 as selection.

[SJHan]

scolix, on 29 December 2010 - 11:07 AM, said:

expression is much weaker in stable cells compared to transient transfection. did your WB have the positive control? you could try to load more protein. make sure the cells 293TN doesnot have the antibiotic resistance gene already. We have used 293FT and they were always in G418 containing media so we could not use this for making cell lines with G418 as selection.

Thank you for your comments.Usually, I used cell lysate generated 293TN cells that were transiently transfected with lentiviral vector having target protein as control.  I got a strong signaling from above control sample. However, I can not see any specific bans from stable cells having puromycin resistance.  Do you think it can be helpful to change host cell type, such as HeLa cell rather than 293TN cells, to improve expression levels?

[scolix]

Try a higher selection conc., might help to get a better expression.

by the way, how many cell lines did you screen? You might have to screen many clones before having 1 or 2 with high expression.