shRNA testing - (Aug/26/2007 )
I am constructing an shRNA to be expressed in a lentiviral vector. Before making virus, I want to test the knock-down efficiencies of the sequences I have chosen in a cell line already over-expressing my gene of interest (no co-transfection)
Thus far, I have cloned one shRNA into an H1 pBluescript vector. I do not have an shRNA positive control. How then, do I determine whether the cells were transfected with my shRNA vector? Do I have to test several, in hopes that one will work?
Also, I do not have a true negative control (that control is only in the lentiviral vector - which is LacZ).
Because this is a cursory determination, do I need to be stringent with the controls? If so, any suggestions as to what I should use?
it's little complicatedyou way ouexpose your goals.
your shRNAsequence cloned under H1 promoter can be transfected. It would be better if you have a GFP or DsRed gene on your vector to select cells which have been transfected, as 30-50% are transfected with classical techinques.
But basically, a transient transfection is ok to determine if your shRNA is ok. Basically you may not see complete disappear of your protein of interest, but if you have decrease, then your protein is knoced down.
The problem with testing this ability in cells overexpressing aprotein, is that the strong promoter can blind your western blot (or even northern blot) by its own strongness. So you may test your shRNA construct on a wild type cell.
A negative scramble control is not needed in such preliminary experiments. your negative control should be transfection reagent without DNA