Vector-Based in Vivo RNA Interference: Dose- and Time-Dependent Suppression of Transgene Expression

Kobayashi, Naoki; Matsui, Yumi; Kawase, Atsushi; Hirata, Kazuhiro; Miyagishi, Makoto; Taira, Kazunari; Nishikawa, Makiya; Takakura, Yoshinobu

doi:10.1124/jpet.103.059931

Cited by 63 publications

(38 citation statements)

References 36 publications

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“…The mice were randomly assigned to 4 equal groups (34 per group): sham surgery control group (sham/vehicle), blank control (S-B) group (CLP/vehicle), negative control siRNA (S-N) group (CLP/vehicle + negative plasmid), and TLR9 siRNA (S-T9) group (CLP/vehicle + siTLR9 plasmid). For induction of in vivo RNA interference in mice, we carried out the so-called hydrodynamics-based procedure comprising a large-volume, high-speed intravenous injection [20,21]. We used TransIT-EE (Enhanced Expression) Hydrodynamic Delivery Solution (Mirus Bio, USA) as the vehicle for hydrodynamic injection.…”

Section: Methodsmentioning

confidence: 99%

Small Interfering RNA Targeting Toll-Like Receptor 9 Protects Mice against Polymicrobial Septic Acute Kidney Injury

Liu

Li²,

et al. 2013

Nephron Exp Nephrol

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Background/Aims: Although recent reports suggest that Toll-like receptor (TLR) 9 is associated with the pathogenesis of polymicrobial septic acute kidney injury (AKI), it is still unclear whether and how renal TLR9 is involved in the development of polymicrobial septic AKI. This study aimed to determine whether the expression of TLR9 in mouse renal cells is related to the development of polymicrobial septic AKI. Methods: The efficacy of small interfering RNA (siRNA) targeting TLR9 was tested in a cultured murine macrophage cell line (RAW264.7 cells). The most potent siRNA was transfected into mice using the hydrodynamic method prior to the induction of polymicrobial septic AKI being induced by cecal ligation and puncture (CLP). TLR9 knockdown was determined by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting in RAW264.7 cells and kidney tissues. The levels of serum creatinine and blood urea nitrogen (BUN) and the renal histopathology assessment were determined at 6-, 12-, and 24-hour time points after CLP, and renal cell apoptosis was studied at 24 h. The 4- and 7-day survival rates of mice were also observed. Results: We found that mice developed AKI in our model of polymicrobial sepsis, despite fluid and antibiotic resuscitation, which resembles human sepsis. siRNA to TLR9 successfully silenced the induction of renal TLR9 gene and protein expression following CLP. Effective silencing of renal TLR9 expression decreased renal cell apoptosis, mitigated the severity of AKI, and increased the survival of mice. Conclusions: Our data demonstrates the induction of TLR9 expression in mouse kidney tissue following CLP. Renal cell apoptosis and AKI in our model of polymicrobial sepsis are dependent on TLR9. Thus, TLR9 may play a critical role in the pathophysiology of polymicrobial septic AKI.

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Section: Methodsmentioning

confidence: 99%

Small Interfering RNA Targeting Toll-Like Receptor 9 Protects Mice against Polymicrobial Septic Acute Kidney Injury

Liu

Li²,

et al. 2013

Nephron Exp Nephrol

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“…In addition, since the DNA molecules delivered by HGD do not need packaging, this method is suitable for the delivery of bacterial artificial chromosomes (BAC) as large as 150 kb 4 . Other types of molecules that have been delivered by a hydrodynamic method include RNA [5][6][7][8][9][10] , morpholinos 11 , proteins 12,13 and other small molecules 12,14 . The advantages and disadvantages of HGD over other delivery methods have been discussed in excellent reviews in the literature [15][16][17][18][19][20] and a number of authors have provided a detailed description of the procedure [21][22][23] .…”

Section: Introductionmentioning

confidence: 99%

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

Kovacsics

Raper

2014

JoVE

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Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobinrelated protein (HPR). Video LinkThe video component of this article can be found at

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“…We therefore think that suppression of the mRNA increase in the stop-siRNA-treated cells after induction affected other genes related to adipocyte differentiation. In addition, we used an siRNA-expressing plasmid DNA (pDNA) instead of the oligonucleotide form of siRNA to achieve longer mRNA suppression (21,22). The suppression of MafA mRNA continued for at least 7 days during differentiation.…”

Section: Discussionmentioning

confidence: 99%

Suppression of MafA mRNA with siRNA prevents adipose cell differentiation in 3T3-L1 cells

Tsuchiya

Maeda²,

Suzuki³

et al. 2009

Int J Mol Med

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Abstract.One of the large Mafs, MafA protein, is a strong transactivator of insulin in pancreatic ß cells. Mafs are also known to play important roles in a variety of developmental and differentiation processes in many organs and tissues. Adipocytes are highly involved in insulin actions and glucose and lipid metabolism, and their proliferation and differentiation is regulated by coordination of several signal transduction and transcriptional factors, including members of the Maf family. To explore the role of MafA in adipocytes, we modified the MafA mRNA level in cultured adipocytes by the RNA interference technique and analyzed the resulting morphological changes and changes in expression of related genes. MafA siRNA was transfected into 3T3-L1 adipocytes. Expression of MafA was confirmed by real-time PCR and Western blotting. Expression of adipocytokines and transcriptional factors was also measured by real-time PCR. Cells were examined for morphological changes and lipid accumulation by microscopy. The MafA expression level in the MafA-siRNA-transfected pre-adipocytes was reduced by ~30% on day 0 pre-induction and by ~70% on day 3 post-induction, in comparison with stop-siRNA-transfected cells. Cell growth and lipid droplet accumulation were prevented by MafA mRNA suppression, and peroxisome proliferator-activated receptor (PPAR) Á2 and CCAAT/enhancerbinding proteins (C/EBP)·, both of which are transcriptional factors essential for adipocyte differentiation, were downregulated. Expression of the genes encoding the adipocytokines, adiponectin and adipsin was also suppressed. The results suggested a possible role of the transcriptional factor MafA in regulation of adipocyte function and differentiation. IntroductionThe large Maf proteins are a family of transcription factors characterized by a typical bZip structure, which is a motif for protein dimerization and DNA binding, and they were reported to regulate several distinct developmental processes, cell differentiation, and the establishment of cell functions (1-3). One of the large Mafs, MafA protein, is a strong transactivator of insulin in pancreatic ß cells (4,5), and there are many reports that MafA transcription factor contributes to ß cell function and differentiation (6-9). One study demonstrated that loss of MafA protein causes a decrease in insulin gene expression in glucotoxic ß cells (10), and another study showed a marked reduction of insulin transcription in MafA-deficient mice, even though the insulin content of ß cells was not significantly reduced (11).On the other hand, it is well known that the Mafs play important roles in a variety of developmental and differentiation processes in many organs, tissues, and cells, including the pancreas (12), lens (13), myeloma cells (14), and cartilage (15). Adipocytes are closely related to insulin action and glucose as well as lipid metabolism, and are candidates for Maf-related cells (16). Their differentiation and proliferation is regulated by coordination of several signal transduction and transcript...

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Vector-Based in Vivo RNA Interference: Dose- and Time-Dependent Suppression of Transgene Expression

Cited by 63 publications

References 36 publications

Small Interfering RNA Targeting Toll-Like Receptor 9 Protects Mice against Polymicrobial Septic Acute Kidney Injury

Small Interfering RNA Targeting Toll-Like Receptor 9 Protects Mice against Polymicrobial Septic Acute Kidney Injury

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

Suppression of MafA mRNA with siRNA prevents adipose cell differentiation in 3T3-L1 cells

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