Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell lines.
As the only mammalian Argonaute protein capable of directly cleaving mRNAs in a small RNA-guided manner, Argonaute-2 (Ago2) is a keyplayer in RNA interference (RNAi) silencing via small interfering (si) or short hairpin (sh) RNAs. It is also a rate-limiting factor whose saturation by si/shRNAs limits RNAi efficiency and causes numerous adverse side effects. Here, we report a set of versatile tools and widely applicable strategies for transient or stable Ago2 co-expression, which overcome these concerns. Specifically, we engineered plasmids and viral vectors to co-encode a codon-optimized human Ago2 cDNA along with custom shRNAs. Furthermore, we stably integrated this Ago2 cDNA into a panel of standard human cell lines via plasmid transfection or lentiviral transduction. Using various endo- or exogenous targets, we demonstrate the potential of all three strategies to boost mRNA silencing efficiencies in cell culture by up to 10-fold, and to facilitate combinatorial knockdowns. Importantly, these robust improvements were reflected by augmented RNAi phenotypes and accompanied by reduced off-targeting effects. We moreover show that Ago2/shRNA-co-encoding vectors can enhance and prolong transgene silencing in livers of adult mice, while concurrently alleviating hepatotoxicity. Our customizable reagents and avenues should broadly improve future in vitro and in vivo RNAi experiments in mammalian systems.
Department of Infectious Diseases, Virology, Heidelberg University Hospital, Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany, Cluster of Excellence CellNetworks, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany, Department of Medicine III, University Hospital Aachen, Pauwelstrasse 30, D-52074 Aachen, Germany, Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany, Division Signaling and Functional Genomics, German Cancer Research Center, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany, BioQuant, Heidelberg University, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany and Department of Pediatric Oncology, Hematology, Immunology and Pulmonology, Heidelberg University Hospital, Im Neuenheimer Feld 350, D-69120 Heidelberg, Germany.
Kathleen
K
Börner
Dominik
D
Niopek
Gabriella
G
Cotugno
Michaela
M
Kaldenbach
Teresa
T
Pankert
Joschka
J
Willemsen
Xian
X
Zhang
Nina
N
Schürmann
Stefan
S
Mockenhaupt
Andrius
A
Serva
Marie-Sophie
MS
Hiet
Ellen
E
Wiedtke
Mirco
M
Castoldi
Vytaute
V
Starkuviene
Holger
H
Erfle
Daniel F
DF
Gilbert
Ralf
R
Bartenschlager
Michael
M
Boutros
Marco
M
Binder
Konrad
K
Streetz
Hans-Georg
HG
Kräusslich
Dirk
D
Grimm
D000818:Animals; D060565:Argonaute Proteins / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D000229:Dependovirus / Q000235:genetics; D055785:Gene Knockdown Techniques; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008099:Liver / Q000378:metabolism; D051379:Mice; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic
C466422:AGO2 protein, human; D060565:Argonaute Proteins; D034741:RNA, Small Interfering