I am interested in using this inducible and stable selectable vector in cell culture experiments.
Generally speaking, a better control is usually achieved by using two separate vectors rather than an all-in-one. We developed a two-virus system for DOX-regulated shRNA expression http://www.ncbi.nlm.nih.gov/pubmed/19655272, and used it in several published studies. The vectors are available from AddGene, which should save you some $$.
Yes I do, however, without any luck! I wanted to knock down my GOI using this construct. Initially I used Clontech's shRNA designing online tool and picked up 3 target sequences. With those I developed 3 different constructs, each targeting a separate sequence in the target mRNA. None of them worked! Takara recommends using upto 1ug/ml of Doxycyclin, but I went up to 40 ug/ml without any effect. I also tried to generate stably transfected cell line with this construct. I Initially did a kill curve for G418 and found that for my cells 0.5mg/ml is enough to kill 100% of them. However, once transfected and grown over time, I have cells growing in 1.5mg/ ml G418 without any knock down after treatment with various doses of Doxycyclin for 72 hrs. By the way, I always look for protein expression. Also, the inserts have MluI cut site and all three were cut with MluI, suggesting that I was not working with false positive clones.
Once I ordered some siRNA from Origene for the same GOI. Out of the three that they sent, one of them constantly give me 75-80% KD after 72 hrs. treatment. When I checked the sequence of this particular oligo, surprisingly I found that this one targets the same region as one of the shRNA in the pSingle that I cloned earlier. Target sequences for pSingle constructs were 19 bases long, where as these oligos were 25 bases long, with the first 19 bases from the 5' end matching exactly the sequences for the shRNA.
This is altogether what my experience is with pSingle.
Gd luck!
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