Hello,
I have been trying to knock down a particular protein in Jurkat cells using the Clontech's pSingle shRNA vector system. This is a doxycyclin inducible system. Initially I followed Clontech's shRNA designing tool and selected three shRNA sequences targeting my gene of interest. I transfected my cells individually with all three, and treated the cells for 72 hrs. with different doses of doxycyclin. However, I could not see any knock down (I generally do Western). I also, tried to generate stably transfected cell lines. So, I cultured the cells with G418. Right now my cells are growing well even in 1.5mg/ml G418 (I did a kill curve too!). I tried with these cells as well. However, did not see any change in expression in Western blot.
Moreover, I ordered some siRNAs from Origene and one of these siRNAs is giving me consistently 80-90% knock down. Interestingly, when I checked the sequence of this particular siRNA, I found that it is targeting the same sequence of the mRNA as I had one of the shRNA in pSingle. I have no clue what could go wrong! I even sequenced my plasmids after cloning. There is no mutation at all. Just one thing, my forward sequencing primer could not read the shRNA sequence while with the reverse primer, everything looks perfect. When I asked my boss about this, his explanation was that because of the hairpin loop sequence, it is hard to read the shRNA in the forward direction, but as long as the reverse primer gives proper read, that means the sequence is there.
Any thought on this will be highly appreciated.
Thanks!
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