I am having troubles knocking down a gene using the Sigma IPTG-inducible shRNA vector.
Here is the link:
http://www.sigmaaldrich.com/life-science/functional-genomics-and-rnai/shrna/inducible-shrna.html#sthash.k9zmSNvV.dpuf
Basically the shRNA should only be expressed in the presence of IPTG:
“The pLKO vector has been redesigned to contain a LacI (repressor) and a modified human U6 shRNA promoter with LacO (operator) sequences. In the absence of IPTG (isopropyl-ß-D-thio-galactoside), an analogue of lactose, LacI binds to LacO preventing expression of the shRNA. When IPTG is present, the allosteric LacI repressor changes conformation, releasing itself from lacO modified human U6 promoter, and subsequently allows expression of the shRNA.”
Some background:
I am using 4 different shRNA sequences, 2 previously shown to successfully knockdown the target gene, however the cells with the gene knocked down became unhealthy and the knockdown effect would be gone after several passages, thus we are trying this IPTG-inducible system.
Method:
1. Using lipofectamine to transfect 293FT cells
2. Harvest virus and infect HCC cell lines
3. After drug selection, the cells would be incubated in 1mM IPTG for 3 days and 7 days respectively, pellet would be harvested for qPCR and western blot.
Results:
No observable difference both on both mRNA and protein level for all four clones.
I have tried this several times and with different cell lines but no success so far.
Anything I could be missing?
Your help is greatly appreciated!
Some cells are naturally resistant to certain of the selection drugs, have you done a kill-curve on your drug selection regimen? It sounds like you are not getting stable plasmid integration.
The other explanation could be that your promoter gets methylated. In my experience, HepG2 cells very quickly down-regulate expression from CMV promoters by methylation - you could test this by adding sodium butyrate or 5-aza-2′-deoxycytidine to your poss stable integrant clones...
Also, not entirely sure why you go through a virus stage? I know you get great transfection levels that way, but it is so much work. A straight up Lipofectamine transfection of your target cells should get you a perhaps lower transfection percentage, but as you are selecting anyway, that really doesn't matter. I haven't used HCC cells before, so perhaps Lipofectamine doesn't work at all with them?
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