Viral infection for GPCR expression in eukaryotic cells
Antonio Porcellini1, Luisa Iacovelli, Antonio De Blasi
1Department of Physiology and Pharmacology “Vittorio Espèamer”, Piazzale Aldo Moro, Rome, Italy.
Methods in Molecular Biology (Clifton, N.J.)
|May 25, 2011
Summary
This chapter details using recombinant adenoviruses to deliver genes, like those for G protein-coupled receptors (GPCRs), into cells. Adenoviral vectors offer efficient, safe gene expression without altering host DNA, suitable for various applications including gene therapy.
Area of Science:
- Molecular Biology
- Virology
- Gene Therapy
Background:
- Adenoviruses are non-enveloped viruses with double-stranded DNA genomes.
- Their replication occurs episomally in the host cell nucleus, avoiding insertional mutagenesis.
- Adenoviral vectors can accommodate foreign DNA up to 30 kb.
Purpose of the Study:
- To describe the protocol for preparing recombinant adenoviruses.
- To outline the method for infecting target cells for transient protein expression.
- To highlight the utility of adenoviral vectors for expressing G protein-coupled receptors (GPCRs) and other proteins.
Main Methods:
- Preparation of recombinant adenoviruses.
- Infection of target cells using adenoviral vectors.
- Transient expression of proteins of interest, including GPCRs.
Main Results:
- Adenoviral vectors demonstrate high gene transduction efficiency (up to 100%).
- Infection is straightforward and does not physically alter cell membranes.
- Cells resistant to plasmid transfection, including non-dividing cells, can be infected.
- Adenoviral vectors are suitable for in vitro and in vivo applications, including gene therapy, with potential for cell-specific targeting.
Conclusions:
- Recombinant adenoviruses provide an efficient and safe method for transient gene expression.
- Adenoviral vectors are versatile tools for molecular biology research and therapeutic applications.
- Their ability to infect various cell types, including non-dividing and plasmid-resistant cells, makes them highly valuable.
Related Concept Videos
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G Protein-Coupled Receptors or GPCRs are membrane-bound receptors that transiently associate with heterotrimeric G proteins and induce an appropriate response to sensory stimuli such as light, odors, hormones, cytokines, or neurotransmitters.
GPCRs are also called heptahelical, 7TM, or serpentine receptors, and consist of seven (H1-H7) transmembrane alpha-helices that span the bilayer to form a cylindrical core. The transmembrane helices are connected by three extracellular loops and three...
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01:30Transducer Mechanism: G Protein–Coupled Receptors
G Protein–Coupled Receptors (GPCRs) are membrane-bound receptors that transiently associate with heterotrimeric G proteins and induce an appropriate response to various stimuli. GPCRs regulate critical physiological pathways and are excellent drug targets for treating diseases such as diabetes, cancer, obesity, depression, or Alzheimer's. Nearly 35% of approved drugs implement their therapeutic effects by selectively interacting with specific GPCRs.
GPCRs are also called heptahelical, 7TM, or...
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01:09GPCRs Regulate Adenylyl Cylase Activity
Some GPCRs transmit signals through adenylyl cyclase (AC), a transmembrane enzyme. AC helps synthesize second messenger cyclic adenosine monophosphate (cAMP). AC catalyzes cyclization reaction and converts ATP to cAMP by releasing a pyrophosphate. The pyrophosphate is further hydrolyzed to phosphate by the enzyme pyrophosphatase, which drives cAMP synthesis to completion. However, cAMP is rapidly degraded to 5′ AMP by the enzymes phosphodiesterase (PDE), preventing overstimulation of cells.
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01:21G-protein Coupled Receptors
G-protein coupled receptors are ligand binding receptors that indirectly affect changes in the cell. The actual receptor is a single polypeptide that transverses the cell membrane seven times creating intracellular and extracellular loops. The extracellular loops create a ligand specific pocket which binds to neurotransmitters or hormones. The intracellular loops holds onto the G-protein.
01:21G-protein Coupled Receptors
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02:11Reporter Genes
Reporter genes are a type of protein-coding gene that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually produce visually identifiable characteristics like fluorescence and luminescence when expressed along with the gene of interest. Thus, reporter genes “report” the presence or absence of genes of interest in an organism, determine the gene expression pattern, or track the physical location of a DNA segment or protein in the cell.
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