January 4, 2007 3:15 PM Subscribe
Attention Biochemists or Anyone Else working with RNAi-expressing cell lines: Help me troubleshoot!
posted by LunaticFringe to Science & Nature (15 answers total) 3 users marked this as a favorite
I am at my wit's end with this and thought the hive mind might be able to help. My sanity and PhD may also depend on this being resolved in a timely manner as well.
I have been having some trouble with a double stable cell line that is supposed (and used to) inducibly express an siRNA molecule in the presence of tet or dox.
The siRNA sequence was created by another member of my lab and works when transiently transfected into cells via oligofectamine. This has been verified by me and several other members of the lab.
The base cell line is the TRex cell line available from Invitrogen which is blasticidin resistant and expresses the tetR protein and is the inducible component of the system.
The construct the siRNA sequence was cloned into is the pSuper+neo+gfp vector. This construct is stably expressed by the TRex cell line. The resulting cell line, "16", is resistant to both G418 and blasticidin as well as expresses GFP for sorting purposes. The member of the lab, AB, who made this line has moved on but clearly showed knockdown of our gene (via western blotting) within 48 hours. She froze down only one vial (ugh) and left for greener pastures (ie dental school).
Now, my project requires the use of these cells so I woke up the only vial. I immediately froze down 10 vials before carrying the cells in the presence of drugs. The cells have been carried in the presence of 200 ug/mL G418 and 10 ug/mL blast since. Unfortunately, somewhere along the line they've lost the siRNA expression. Treatment with dox does not cause target knockdown. We don't have an antibody to the tetR protein so I don't know if it's expressing and I don't think we have a way to test for siRNA expression.
AB also made several other clones of this double stable which showed knockdown and were frozen down. We awoke all and tested for knockdown, none of which were positive.
So Hivemind: Why would these cells still be resistant to the drugs but cease to knockdown the protein in question, even fresh from the liN2? It's not the dox as we have bought fresh powder and tested it with bacteria (no growth). Invitrogen states that there is no difference between tet and dox when inducing tetR expression. I am losing my mind over this. I cannot, nor any of my colleagues, think of why this is wrong. Is there a mefites who has worked with TRex's or a similar system and can offer advice?
Hope me, mefites, you're my only hope.