Schematic outline of the AdEasy system. .. along with more detailed protocols for their production and analysis, should contact the authors at the following. In this protocol, we describe the practical aspects of using the AdEasy system for generating recombinant adenoviruses. The full protocol usually takes 4–5. AdEasy Made Easier. (). Use of AdEasier Cells for PROTOCOL FOR PREPARING AND USING AdEasier CELLS. Note: This.
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Sign in Sign up. Recombinant Adenoviral Vector Systems. A comprehensive review of adenoviral vector system for protein expression. A large number of acute respiratory, gastrointestinal and eye infections in humans are caused by adenoviruses [ 34 ]. Adenoviruses have a wide host range from monkeys, mice to humans.
Adenoviruses are powerful research tools for investigating virological and cellular events. Adenoviruses have 50 different serotypes; however majority of molecular information about host-cell interaction is based on studies related to 2 and 5 [ 5 – 7 ]. Hence, the most commonly used adenoviral vectors are derived from human adenovirus serotypes 2 and 5 for in vitro and in vivo gene delivery [ 8 ]. Table I compares various recombinant viral vectors currently used for gene delivery. Recently, there has been a rapid expansion in the use of adenoviral vectors for in vitro and in vivo gene delivery [ 9 ].
It is used for 1 gene therapy [ 1011 ] ; 2 molecular tool to study gene expression, both in vitro and in vivo expression in difficult-to-transduce cell types and tissues [ 12 ], for example, retrograde introduction of optogenetic channelrhodopsin 2 [ 13 ] or introduction of targeted mutagenesis in combination with gene-editing tools such as CRISPR [ 14 ] ; 3 the production of high levels of recombinant, potentially therapeutic proteins; and 4 in vivo vaccination [ 15 ], for example, chimpanzee adenovirus vector Ebola vaccine [ 16 ].
Adenoviruses Retroviruses Lentiviruses Cell-specificity Dividing and non-dividing Dividing Dividing and non-dividing Stability Epichrosomosal, not replicated with cell division Integrates into host genome Limitations: Integration into the host genome is a random unpredictable event, hence depending on the site of insertion, cellular function may be disturbed due to genetic disruption, concerns for insertional mutagenesis causing activation of oncogenes have also been observed Integrates into host genome Transgene expression Transient Limitations: Suitable for both in vitro including primary cells and in vivo use Low Moderate Remarks: Best for in vivo use, in vitro use not advised because high titers similar to in vivo use are required for in vitro transduction as well Relative ease of construction Step by step rescue using two-plasmid system and co-transfection in packaging cell lines Step wise cloning to rescue transgene and co-transfection in packaging cell lines Comments: Depends on replication-competent or replication-defective vectors Multiple plasmids encoding required proteins are co-transfected into packaging cell line Limitations: The final vector stock cannot be reused to amplify due to hazard of generating replication competent virus, hence everytime generation of vector is initiated with co-transfection step Efficiency of transduction High and wide range of cell types Low Moderate.
Useful for tracking GOI expression at single cell level or monitoring expression for proteins which are difficult to track due to lack of established antibodies or functional assays. Example of adenoviral mediated gene delivery in vivo using Ad-LacZ as control to determine expression levels of recombinant adenovirus mediated target gene expression.
Figure from Sorriento et al [ 2 ]. High doses inducing a complete inhibition of tumor growth are more effective. Representative image of tumors at the end of the treatment are also shown. B Histological analysis of tumors after 17 days of treatment from sacrificed mice. Confirmation of GRK5-RH expression by direct observation of the green light at the fluorescence microscope.
Evaluation of Lac-Z expression in control tumors by X-GAL staining at direct light blue staining indicates Lac-Z expression and counterstaining in red with eosin. Evolution of model systems for homologous recombination: Available in constitutive and tet-inducible formats No shuttle Pros: Schematic illustration of generating recombinant adenoviruses depicting basic principle of three independent steps of 1 subcloning GOI into a shuttle vector which transfers the GOI into the pAd plasmid containing the adenoviral backbone 2 during homologous recombination in bacterial system, and 3 packaging recombinant adviral DNA containing GOI in cells complementing E1 in trans.
Recombinant adenoviruses are collected, amplified, concentrated and titrated suitable for in vivo application. Cartoon showing agarose gel profile of potential recombinants. Schematic illustration depicting profile of A supercoiled DNA with lanes 2, 3, 5 and 6 showing potential recombinant adenoviral DNA and B restriction digested profile of DNA from 2, 3, 5 and 6 showing restriction profile of adenoviral recombinants from bacterial colonies following homologous recombination.
Flowchart depicting recombinant adenovirus amplification for scaling up production to generate high titer, purified virus suitable for in vivo application. Schematic representation of density gradient centrifugation showing separation of recombinant adenovirus and defective empty capsid debris in CsCl gradient.
Functional study of the novel multidrug resistance gene HA and its comparison to multidrug resistance gene 1. J Exp Clin Cancer Res. Hilleman M, Werner J. Recovery of new agent from patients with acute respiratory illness.
Proc Soc Exp Biol Med. Virus particles in gastroenteritis. Adenoviruses in the immunocompromised host. Genetic variability of adenoviruses. Ann N Y Acad Sci. Update on adenovirus and its vectors. Adenoviruses as gene-delivery vehicles. N Engl J Med. Development of optimized vectors for gene therapy. Gene therapy for rhesus monkeys heterozygous for LDL receptor deficiency by balloon catheter hepatic delivery of helper-dependent adenoviral vector.
Selection-free gene repair after adenoviral vector transduction of designer nucleases: Retrograde optogenetic characterization of the pontospinal module of the locus coeruleus with a canine adenoviral vector.
Chimpanzee Adenovirus Vector Ebola Vaccine. Image reconstruction reveals the complex molecular organization of adenovirus. Cell receptors involved in adenovirus entry. Virus-receptor interaction in an adenovirus system. Physical mapping of a large-plaque mutation of adenovirus type 2. Carlock L, Jones N. Transformation-defective mutant of adenovirus type 5 containing a single altered E1a mRNA species. An adenovirus mutant defective in splicing RNA from early region 1A. A simple technique for the rescue of early region I mutations into infectious human adenovirus type 5.
Efficient manipulation of the human adenovirus genome as an infectious yeast artificial chromosome clone. Graham F, Prevec L. Methods for construction of adenovirus vectors.
A protocol for rapid generation of recombinant adenoviruses using the AdEasy system
Efficient generation of recombinant adeaey vectors by homologous recombination in Escherichia coli. A helper-dependent adenovirus vector system: Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and pfotocol cosmid bearing the full-length virus genome.
A simplified system for generating recombinant adenoviruses. AdEasy system made easier by selecting the viral backbone plasmid preceding homologous recombination. A protocol for rapid generation of recombinant adenoviruses using the AdEasy system.
Over-expression of CXCR4 on mesenchymal stem cells augments myoangiogenesis in the infarcted myocardium. J Mol Cell Cardiol. New role for the protein tyrosine phosphatase DEP-1 in Akt activation and endothelial cell survival. Unc45b forms a cytosolic complex with Hsp90 and targets the unfolded myosin motor domain. Soluble epoxide hydrolase plays an essential role in angiotensin II-induced cardiac hypertrophy.
Calcitonin gene-related peptide stimulates proliferation of alveolar epithelial cells. Genetic manipulation of periostin expression in the heart does not affect myocyte content, cell cycle activity, or cardiac repair. Palladin contributes to invasive motility in human breast cancer cells. EGF receptor signaling blocks aryl hydrocarbon receptor-mediated transcription and cell differentiation in human epidermal keratinocytes.
McConnell M, Imperiale M. Biology of adenovirus and its use as a vector for gene proyocol. Retro-orbital injections in mice. Functional aspects adexsy meningeal lymphatics in ageing and Alzheimer’s disease.
E-C coupling structural protein junctophilin-2 encodes a stress-adaptive transcription regulator. If you are interested in contributing a manuscript or suggesting a topic, please leave us a feedback.
Recombinant Adenoviral Vector Systems
Tools for Protein Knockdown by Gene Silencing. Integrates into host genome Limitations: Integration into the host genome is a random unpredictable event, hence depending on the site of insertion, cellular function may be disturbed due to genetic disruption, concerns for insertional mutagenesis causing activation of oncogenes have also been observed.
Best suited for in vitro use including primary cells, quick testing of a target cells, quick testing of a target before committing to transgenic mouse model. Suitable for both in vitro including primary cells and in vivo use. Best for in vivo use, in vitro use not advised because high titers similar to in vivo use are required for in vitro transduction as well. Step wise cloning to rescue transgene and co-transfection in packaging cell lines Comments: Depends on replication-competent or replication-defective vectors.
Multiple plasmids encoding required proteins are co-transfected into packaging cell line Limitations: The final vector stock cannot be reused to amplify due to hazard of generating replication competent virus, hence everytime generation of vector is initiated with co-transfection step. Pre-transformed with pAdEasy to reduce background by shuttle vector, high transduction efficiency, high recombinants, streamline time constraints, reduce RCA.
AdEasy Got Easier
In vitro adeast reducing one step compared to pAdEasy system, Adeno-X proticol system 3 uses expression cassette to directly clone onto pAd backbone eliminating subcloning in shuttle vector. Fast, efficient, plasmid contains recombination sites for cloning GOI, eliminates subcloning by direct insertion of GOI into pAd backbone, Adapted gateway recombinant cloning tech.
Modified homologous recombination in mammalian cells, unlike traditional methods no plaque screening, eliminates in vitro recombination in bacterial cells. Premade or custom Adviruses, individual vectors and components for generating custom Adviruses in the lab.