Getting Started with RNAi In Vivo
Routes for Administering RNAi
Problems Associated with Systemic Delivery
Systemic siRNA Delivery to Liver
When only the conrol pMIR-REPORT Luciferase Vector was injected, high levels of firefly luciferase (fLuc) expression were detected in liver samples, both at the protein and RNA levels. fLuc gene expression in kidneys, spleen, and lung was 100–1000X lower than in the liver, indicating targeted delivery of the reporter vector to the liver (data not shown). When the pMIR-REPORT Luciferase Vector, fLuc-specific Silencer® siRNA, and pMIR-REPORT b-galactosidase Vector (for normalization of delivery efficiency) were co-injected, significant reduction of fLuc protein (Figure 1A) and corresponding mRNA (Figure 1B) levels were observed. No reduction of fLuc was detected in the control group, which was injected with the nonspecific Silencer Negative Control #1 siRNA.
Figure 1. siRNA-induced Reduction of fLuc Protein and mRNA Levels in Mouse Liver. Ambion pMIR-REPORT™ Luciferase and β-galactosidase vectors and siRNA (1 or 5 nmol) that targets firefly luciferase (fLuc) were injected at constant high pressure into the tail veins of seven mice. Data were normalized to the average result from four mice injected with Silencer® Negative Control #1 siRNA. Panel A: fLuc and β-galactosidase (β-gal) protein levels were measured using the Tropix® Dual-Light® Luminescent Reporter Gene Assay System (Applied Biosystems). Each bar represents one animal, and readings were performed in triplicate. Panel B: TaqMan® Gene Expression Assays were used to measure fLuc mRNA levels. Each bar represents one animal, and readings were performed in triplicate and averaged.
The data demonstrated the following:
- The pMIR-REPORT Luciferase miRNA Expression Reporter Vector (as is, or with cloned targets) can be used in vivo and delivered by hydrodynamic injection to the mouse liver.
- siRNA can cause specific and potent knockdown of gene expression from a co-delivered reporter vector in liver cells.
- The Dual-Light Luminescent Reporter Gene Assay System can detect fLuc and β–gal protein in crude tissue samples.
Improving In Vivo Delivery of siRNAs
Hydrodynamic tail vein injections were conducted under a license grant from Mirus Bio Corporation.
Alexander V Vlassov, Mu Li, Susan Magdaleno • Applied Biosystems, Austin, TX
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The pMIR-REPORT™ Luciferase miRNA Expression Reporter Vector (5 µg) and pMIR-REPORT β-galactosidase Reporter Control Vector (5 µg) were mixed in 2.5 mL PBS and injected at constant high pressure into the tail veins of mice within 5–8 sec. For knockdown assays, Silencer® siRNA (~14 and 70 µg, equivalent to 1 and 5 nmol, respectively) targeting firefly luciferase (fLuc) was co-injected with the plasmids. The siRNA is expected to knock down fLuc expression, while expression of β-galactosidase (β-gal) from the control vector, used to monitor injection and nonspecific effects, should not change.
Analysis of Gene Expression and Knockdown
Liver, kidneys, spleen, and lung samples were collected from animals 24 hours after injection of plasmids and siRNA. Samples for protein analysis were homogenized in lysis solution from the Tropix® Dual-Light® Luminescent Reporter Gene Assay System (Applied Biosystems). Samples for RNA analysis were preserved in the Ambion RNAlater® Tissue Collection: RNA Stabilization Solution at 4°C overnight, and total RNA was isolated with the Ambion mirVana™ PARIS™ Kit the next day.
Luciferase and β-galactosidase activity were measured using the Tropix Dual-Light Luminescent Reporter Gene Assay System. While this chemiluminescent reporter gene system is commonly used for assays in extracts from cultured cells, in this study it was successfully used to measure activity of fLuc and β-gal in crude tissue lysates, which had been neither purified nor enriched.
Levels of fLuc mRNA were measured by real-time RT-PCR using the TaqMan® Gene Expression Assay for fLuc. The RNA samples were reverse transcribed (Applied Biosystems High Capacity cDNA Reverse Transcription Kit), and the resulting cDNA samples were assayed with the TaqMan Universal PCR Master Mix, TaqMan primers and probe for fLuc, and the 7900HT Fast Real-Time PCR System. The relative level of gene expression for each sample was calculated by the ΔΔCT method.
- pMIR-REPORT Luciferase miRNA Expression Reporter Vector contains firefly luciferase (fLuc) under control of a mammalian promoter/terminator system and a target cloning region downstream of the luciferase translation sequence. This vector was designed for cloning and testing of putative miRNA binding sites, for evaluation of endogenous miRNA expression, and for monitoring the effects of miRNA mimics (i.e., Ambion Pre-miR™ miRNA Precursors) or miRNA inhibitors (i.e., Ambion Anti-miR™ miRNA Inhibitors). In addition, a gene or viral target of interest can be cloned into this vector for in vivo and in vitro studies.
- pMIR-REPORT β-Galactosidase Reporter Control Vector is used for normalization of transfection efficiency.