FuGENE® HD Transfection Reagent Identifying and Solving ...

Importance of selecting the right plasmid vectorOne component that is integral to a good transfectionprocedure, but often overlooked, is the vector.For example, our experience indicates that thebackbone of the plasmid can alter the efficiency oftransfection by 10- to 100-fold. The two photomicrographsin Figure 1 show what happens whendifferent vectors are used to carry the same geneof interest into the same cells, using the same transfectionreagent.In both cases, the vector contained the lacZ genedriven by a CMV promoter, but the backbone of theplasmid used in experiment A differed from thatof the plasmid used in experiment B. Even thoughboth vectors contained a “CMV” promoter, thepromoters may have been slightly different. Thedifferences in the backbone of these two vectorswere responsible for poor results in Experiment Aand excellent results in Experiment B.Experiment AExperiment BFigure 1: Importance of selecting the right plasmid vector.In both Experiments A and B, the same gene of interest is transfectedinto the same cells (HEK 293 cells), using the sametransfection reagent (FuGENE ® 6 Transfection Reagent). Theonly difference between the two experiments is the vectorused to transport the gene.Experiment A was performed with a plasmid that expressedlacZ with a CMV promoter.Experiment B was performed with a plasmid that also expressedlacZ, but with a different CMV promoter. In both experiments,cells were stained for beta-galactosidase expression. (Transfectedcells stained blue.)Result: The two experiments produced vastly different levelsof expression, with experiment B clearly yielding the betterresult.6