Polymerase II promoter strength determines efficacy of microRNA adapted shRNAs.
Since the discovery of RNAi and microRNAs more than 10 years ago, much research has focused on the development of systems that usurp microRNA pathways to downregulate gene expression in mammalian cells. One of these systems makes use of endogenous microRNA pri-cursors that are expressed from polymerase II promoters where the mature microRNA sequence is replaced by gene specific duplexes that guide RNAi (shRNA-miRs). Although shRNA-miRs are effective in directing target mRNA knockdown and hence reducing protein expression in many cell types, variability of RNAi efficacy in cell lines has been an issue. Here we show that the choice of the polymerase II promoter used to drive shRNA expression is of critical importance to allow effective mRNA target knockdown. We tested the abundance of shRNA-miRs expressed from five different polymerase II promoters in 6 human cell lines and measured their ability to drive target knockdown. We observed a clear positive correlation between promoter strength, siRNA expression levels, and protein target knockdown. Differences in RNAi from the shRNA-miRs expressed from the various promoters were particularly pronounced in immune cells. Our findings have direct implications for the design of shRNA-directed RNAi experiments and the preferred RNAi system to use for each cell type.
Department of Microbiology and Immunology, Diabetes Center, University of California San Francisco, San Francisco, California, United States of America.
Robert Jan
RJ
Lebbink
Maggie
M
Lowe
Theresa
T
Chan
Htet
H
Khine
Xiaoyin
X
Wang
Michael T
MT
McManus
D002460:Cell Line; D045744:Cell Line, Tumor; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D019073:HT29 Cells; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012319:RNA Polymerase II / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics*
D035683:MicroRNAs; D034741:RNA, Small Interfering; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D012319:RNA Polymerase II