Vector encodes two fluorescent reporters, each the control of individual cytomegalovirus promoter; a red protein used as a positive transfection marker, and a green protein regulated by siRNA
Evrogen p2FP-RNAi vector is a novel eukaryotic expression vector designed for fast monitoring of RNA interference (RNAi).
siRNA-mediated gene silencing is a popular though a relatively new method of transient downregulation of gene expression.
An obstacle is that many siRNA sequences are in fact not interfering with mRNA translation and often incapable of triggering mRNA-degradation cascade.
Therefore, the choice of an efficient siRNA sequence becomes time-consuming and this is only one of cases where p2FP-RNAi is about to be useful.
p2FP-RNAi vector encodes two fluorescent reporters, each is under the control of individual cytomegalovirus promoter.
The first reporter is a red fluorescent protein used as a positive transfection marker, and the second a green fluorescent protein which expression is regulated by siRNA.
3'-UTR of the TurboGFP comprises multiple cloning site allowing to clone a gene sequence that will be used as a siRNA target.
When the vector is transfected into eukaryotic cells in the absence of a functional siRNA, it expresses both JRed and TurboGFP proteins providing both green and red cell fluorescence.
In the presence of functional siRNA(s) directed against the cloned DNA fragment, TurboGFP expression is knocking down.
JRed expression at that remains unmodified or (in some experimental systems) increases due to translational competition.
As a result efficient RNAi is immediately indicated by the absence of green fluorescence while its presence means that used siRNA target has to be replaced.
The vector can find use in such applications as testing of siRNA capacity to knock down gene expression, internal control in the gene silencing experiments, and analysis of endogenous RNAi.
