Navigation Links
Superior Gene Silencing Using Adenoviral Vectors


Mediating RNA interference (RNAi) with recombinant adenovirus expressing siRNAs has only recently been described in the literature (1). Recombinant adenoviruses have several advantages over other methods of nucleic acid delivery. Namely, these viruses can infect dividing and non-dividing cells, and can be used in animal model systems. They can infect many difficult-to-transfect primary and transformed cell lines via their receptor mediated delivery mechanism. The sequence of a chemically synthesized siRNA capable of gene specific silencing can also be functionally expressed from recombinant adenovirus, often at a lower cost. Once made, the virus can be expanded, making it possible to generate "stocks" for future experiments.


How Does pSilencer Adeno 1.0-CMV Work?
An oligonucleotide insert encoding the desired siRNA is cloned into the pre-linearized shuttle vector (provided), which includes a modified CMV promoter for efficient expression of the siRNA sequence. The shuttle vector, containing the desired sequence, is then linearized and introduced into HEK 293 packaging cells (not included) along with the linearized adenovirus backbone (Figure 1). The HEK 293 packaging cells contain the EIA and EIB genes which are not present in the introduced vectors and are required to form infectious particles. Inside the HEK 293 cells, the shuttle vector and the adenoviral backbone plasmid recombine to produce an adenoviral genome including the siRNA expression cassette. The siRNA expression cassette is packaged into infectious virus particles that can be harvested for experiments. Harvested adenoviral particles can then be used to infect cells or animals, or stored for future experiments.

Figure 1. Schematic of pSilencer Adeno 1.0 CMV System Recombination and Packaging. The shuttle vector and backbone plasmid are linearized and then transfected into HEK 293 cells. The HEK 293 packaging cells contain the EIA and EIB genes which are not present in the introduced vectors and are required to form infectious particles. Inside the cell, the plasmids recombine forming a functional genome that is replicated and packaged into adenoviral particles.


siRNA from Adenoviral Vectors Reduce Expression from Transgenic Alleles in vivo
To demonstrate that adenoviruses are useful for decreasing gene expression via the RNAi pathway in animals, Dr. Beverly Davidson and colleagues, our collaborators at the University of Iowa, injected recombinant adenoviruses expressing siRNAs targeting green fluorescent protein (GFP) or -glucuronidase (a negative control) into the brains of transgenic GFP-expressing mice (1). Five days after injection, brain sections were analyzed by fluorescence microscopy and Western blot (Figure 2). GFP expression was reduced in the injected brain hemisphere, indicating the utility of the method for reducing gene expression in animals.

Figure 2. Silencing of Transgenic GFP in Mouse Brain with an Adenoviral Vector (1). Adenovirus engineered to express an siRNA targeting GFP (A, top panels) or a negative control siRNA targeting human -glucuronidase (A, bottom panels) were injected into the brain striatal region of transgenic mice expressing GFP. The adenovirus included a dsRed expression cassette to visualize the localization of the virus. (A) Brain sections were analyzed by fluorescence microscopy 5 days after injection. Green: GFP. Red: dsRed. Left panels: GFP detection. Middle panels: dsRed detection. Right panels: merged images. (B) Brain sections were divided into ipsilateral (il) and contralateral (cl) portions and analyzed by Western blot using antibodies to GFP and -actin (control).


Allele-specific Silencing of Dominant Disease Genes
Due to its inherent specificity, a potential application of siRNAs is the treatment of dominantly inherited diseases. One such neurodegenerative disease is Machado-Joseph disease/spino-cerebellar ataxia type 3. In 70% of diseased carriers, alleles contain a G to C mutation. Dr. Davidson's laboratory constructed plasmids fusing GFP to normal (Atx-Q28-GFP) or mutant polyglutamine (polyQ) alleles (Atx-Q166-GFP) containing a CAG expansion in Ataxin-3 (2). Adenoviral vectors that expressed hairpin siRNAs targeting either the wild type or the mutant allele were designed generated from these plasmids and used to infect Cos-7 cells that had previously been transiently co-transfected with Atx-Q28-GFP (wt) and Atx-Q166 (mutant). (Both plasmids were co-transfected into the Cos-7 cells to mimic the heterozygous adenovirus.) The siRNA expressed from AdG10i had perfect complementarity to the wild type allele, and preferentially suppressed expression of the fusion construct containing that allele (Atx-Q28-GFP, Figure 3A). Conversely, AdC10i preferentially suppressed expression of the mutant allele construct (Atx-Q166, Figure 3A). Finally, these two adenoviral vectors were used to infect PC12 cells that had been created to express normal (Q28) or (Q166) mutant ataxin-3. Again, the viruses were able to specifically target the mutant allele (Figure 3B). These results indicate the utility of viruses for delivering siRNAs that target allele-specific disease genes.

Figure 3. Allele-specific Silencing with Adenoviral Vectors Expressing an siRNA (2). Cos-7 cells were co-transfected with the Atx-3-Q28-GFP (wt) and Atx-Q166 (mutant) constructs and then infected (MOI = 50) with adenovirus expressing siRNAs that had been designed such that a single nucleotide polymorphism (SNP) was in the middle of the target sequence. The siRNA produced by Ad-G10i was perfectly complementary to the wild type construct, whereas that of Ad-C10i was complementary to the mutant construct. (A) Green: GFP. Red: immunofluorescence of the polyQ construct detected with 1C2 antibody. Ad-G10i reduced wt ataxin-3 levels 95% but did not affect mutant ataxin-3 levels. Conversely, Ad-C10i reduced mutant ataxin-3 levels 91% but had no effect on WT ataxin-3 levels. (B) PC12 cells expressing WT (left) or mutant ataxin-3 (right) were infected (MOI = 100) with the Ad-G10i or Ad-C10i virus. Western analysis was performed with antibodies to Ataxin-3 and GAPDH (as a control), and the siRNAs produced by the viruses were able to specifically silence the allele to which they were designed.


The Complete Adenoviral Production System
The pSilencer adeno 1.0-CMV System includes everything needed to produce five preparations of recombinant adenovirus, except the siRNA template oligonucleotide and the HEK 293 packaging cells (HEK 293 cells are available from several sources, including ATCC). The kit includes linearized Shuttle Vector 1.0-CMV (20 rxns), Negative Control Shuttle Vector that encodes a scrambled siRNA sequence, a Positive Control Oligonucleotide Insert that encodes an siRNA targeting GAPDH, and the Adenoviral Backbone that includes a LacZ sequence for monitoring transfection efficiency. Also included are reagents for transfecting the HEK 293 cells with the Shuttle and Backbone Vectors, forward and reverse sequencing primers to verify clones, and 5X Annealing Buffer for preparing the siRNA-encoding oligonucleotides for ligation.


The use of these materials is permitted for research purposes only. Any other use requires a license from the University of Iowa Research Foundation, 214 Technology Innovation Center, Iowa City, IA 52242.


back to top


Ordering Information
Cat# Product Name Size 5790 pSilencer adeno 1.0-CMV System 5 virus preparations
'"/>

Source:


Page: All 1 2 3 4 5

Related biology technology :

1. Delivery of pCMV-S DNA Using the Helios Gene Gun System Is Superior to Intramuscular Injection in Balb/c Mice
2. Sub-Micron Gold Particles Are Superior to Larger Particles for Efficient Biolistic Transformation of Organelles and Some Cells
3. Calcium Plus Assay Kit, Superior Detection for Specialized Targets
4. The Affymetrix GeneChip Scanner 3000 New Advances in Scanner Design for Superior Performance, Reliability, and Dynamic Range
5. RNAi Gene Silencing System
6. Quantification of siRNA Silencing Efficiency Using the LightCycler System
7. Measuring Gene Silencing Effects by RT-PCR Without RNA Isolation
8. Drive Long-Term Silencing with CMV Vectors
9. Enhanced siRNA Delivery and Long-term Gene Silencing
10. Fast, Accurate Assessment of siRNA-induced Gene Silencing
11. Your Data: Silencing Hepatitis C Virus Replication With In Vitro Transcribed siRNAs
Post Your Comments:
*Name:
*Comment:
*Email:
TAG: Superior Gene Silencing Using Adenoviral Vectors

(Date:9/30/2014)... MA and Pittsburgh PA (PRWEB) September 30, 2014 ... Clinical Research Nurses ( IACRN), a non-profit ... Alliance for Clinical Research Excellence and Safety ... the signing of a strategic alliance agreement, setting ... IACRN’s vision is to enhance clinical research quality ...
(Date:9/29/2014)... forefront of diagnostics today, with imaging techniques like ... and NMR (nuclear magnetic resonance) increasing steeply over ... image resolution and quality still limit these techniques ... solution is hyperpolarization, which involves injecting the patient ... following the distribution and fate of specific molecules ...
(Date:9/29/2014)... , Sept. 29, 2014  Ten of the ... of cartilage repair, regeneration, allograft reconstruction and research ... the world gathered to discuss the future of ... of Regenerative Medicine symposium, Cartilage Regeneration: State ... included Orthopedic Biosurgery, Minced Juvenile Allograft, and State ...
(Date:9/29/2014)... , Sept. 29, 2014 /PRNewswire/ - Portage Biotech Inc. ... PBT.U), is pleased to announce that its three Promissory ... Portage, who advanced $ 100,000 each in July 2014 ... their Notes and related Coupons converted into 3,500,001 restricted ... carried 5% coupon. Both the Note and the coupon ...
Breaking Biology Technology:IACRN and ACRES to Support Clinical Research Nurses 2IACRN and ACRES to Support Clinical Research Nurses 3A safer approach for diagnostic medical imaging 2A safer approach for diagnostic medical imaging 3World Renowned Experts Discuss Future Of Cartilage Regeneration 2World Renowned Experts Discuss Future Of Cartilage Regeneration 3Portage announces conversion of its debt into equity 2Portage announces conversion of its debt into equity 3
... and Clinical Studies Demonstrate Activity of ... Agent against Gram-Positive Bacteria, CHICAGO, Sept. 18 ... THRX ) announced today results with the,investigational ... skin and skin structure infections,(cSSSIs) from a controlled ...
... Preclinical Animal Data Demonstrates Selective Survival Advantage of ... ZFN-Treated Immune Cells after HIV Infection, ... (Nasdaq: SGMO ) announced today the presentation of ... resistant to,HIV infection by treatment with zinc finger DNA-binding ...
... Research - the New ISI Web of Knowledge - ... Institutions Throughout the Country, PHILADELPHIA and LONDON, Sept. ... TOC ; TSX: TOC) and,leading provider of information ... that Spain,s La Fundacion Espanola,para la Ciencia y la ...
Cached Biology Technology:Astellas and Theravance Report Additional Telavancin Data at 47th Annual ICAAC 2Astellas and Theravance Report Additional Telavancin Data at 47th Annual ICAAC 3Astellas and Theravance Report Additional Telavancin Data at 47th Annual ICAAC 4Astellas and Theravance Report Additional Telavancin Data at 47th Annual ICAAC 5Astellas and Theravance Report Additional Telavancin Data at 47th Annual ICAAC 6Sangamo BioSciences Presents Data Demonstrating 'In Vivo' Protection Against HIV Infection by CCR5-ZFN Therapeutic 2Sangamo BioSciences Presents Data Demonstrating 'In Vivo' Protection Against HIV Infection by CCR5-ZFN Therapeutic 3Sangamo BioSciences Presents Data Demonstrating 'In Vivo' Protection Against HIV Infection by CCR5-ZFN Therapeutic 4Sangamo BioSciences Presents Data Demonstrating 'In Vivo' Protection Against HIV Infection by CCR5-ZFN Therapeutic 5Spanish Research Consortium Signs Four-Year Renewal Deal for ISI Web of Knowledge 2Spanish Research Consortium Signs Four-Year Renewal Deal for ISI Web of Knowledge 3
(Date:9/29/2014)... , Sept. 29, 2014 NXT-ID, Inc. ... the "Company"), a biometric authentication company focused on the ... S. Miceli has been named the Company,s Vice-President ... Gino Pereira as Chief Financial Officer. Mr. ... Executive Officer and as a Director. Mr. ...
(Date:9/29/2014)... safely tolerate treatment with cells cultured from human placental ... journal Multiple Sclerosis and Related Disorders . The ... conducted by researchers at Mount Sinai, Celgene Cellular Therapeutics ... institutions. , While designed to determine safety of ... that a preparation of cultured cells called PDA-001 may ...
(Date:9/28/2014)... by scientists at the University of California, Santa Cruz, ... within the genomes of primates drove the evolution of ... in every cell of our bodies. , The ... "retrotransposons" (a.k.a. "jumping genes") and the genes that have ... have, for the first time, identified genes in humans ...
Breaking Biology News(10 mins):NXT-ID, Inc. Names Vice-President and Chief Financial Officer 2NXT-ID, Inc. Names Vice-President and Chief Financial Officer 3Cells from placentas safe for patients with multiple sclerosis 2Human genome was shaped by an evolutionary arms race with itself 2Human genome was shaped by an evolutionary arms race with itself 3Human genome was shaped by an evolutionary arms race with itself 4
... is well known that animals use song as a way ... developed an unusual way of scaring off predators ?by singing ... Andrews discovered that wild gibbons in Thailand have developed a ... singing for survival, the gibbons appear to use the song ...
... replication within resident immune cells of the testis, providing ... even after effective highly active antiretroviral therapy. The related ... to human immunodeficiency virus-1 infection in situ and in ... of Pathology. , According to the most recent ...
... timing of food availability, researchers have identified the first ... Period 2, is a key component of the circadian ... of the University of Fribourg and Etienne Challet of ... 24th issue of the journal Current Biology, published by ...
Cached Biology News:Singing for survival 2Singing for survival 3Human testis harbors HIV-1 in resident immune cells 2Circadian gene helps the brain predict mealtime 2
Non-Receptor Tyrosine Kinase Sampler Kit 10 mu g each...
Hybridization chamblers suitable for use with all ChIP-GLAS microarrays....
Integrins Sampler Kit 10g each...
Protein Tyrosine Phosphatase Sampler Kit 10g each...
Biology Products: