Dynamics of gene silencing by RNA interference.
The large number of candidate genes identified by modern high-throughput technologies require efficient methods for generating knockout phenotypes or gene silencing in order to study gene function. RNA interference (RNAi) is an efficient method that can be used for this purpose. Effective gene silencing by RNAi depends on a number of important parameters, including the dynamics of gene expression and the RNA dose. Using mouse hepatoma cells, we detail some of the principal characteristics of RNAi as a tool for gene silencing, such as the RNA dose level, RNA complex exposure time, and the time of transfection relative to gene induction, in the context of silencing a green fluorescent protein reporter gene. Our experiments demonstrate that different levels of silencing can be attained by modulating the dose level of RNA and the time of transfection and illustrate the importance of a dynamic analysis in designing robust silencing protocols. By quantifying the kinetics of RNAi-based gene silencing, we present a model that may be used to help determine key parameters in more complex silencing experiments and explore alternative gene silencing protocols.
Raab
R Michael
RM
Department of Chemical Engineering, Room 56-459, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Stephanopoulos
Gregory
G
D000818:Animals; D020868:Gene Silencing; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D051379:Mice; D018345:Mice, Knockout; D023421:Models, Animal; D008957:Models, Genetic; D010641:Phenotype; D034622:RNA Interference; D014162:Transfection
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins