Hi all,
I used lentivirus containing MISSION shRNA of ATG5 gene to produce a stable cell lines after puromycin selection.
Now I would like to knockdown another gene (Beclin-1) using the previous cell line (I actually would like to do double knockdown of the two genes, ATG5 and Beclin-1), is it possible?
If I use lentivirus with MISSION shRNA of Beclin-1 gene to infect the ATG5 KO cell, how could I select the cell with double genes knockdown? Because ATG5 KO cells are resistant to puromycin, I am pretty sure that puromycin selection will not work in the second knockdown.
Is there any suggestion?
Thank you very much.
You should take lentiviral vector containing MISSION shRNA targeting Beclin-1 and different selection marker, like G418 or blasticidin. This should work. Good Luck!
Try to find a vector with hygromycin resistance, because in my hands the combination with puromycin worked quite well. Of course you can use every other selection pressure as Maria has mentioned, but be careful in case you have 293T cells, which are already resistant to G418
Regards
Sven
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