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Mouse adenovirus (MAV-1) expression in primary human endothelial cells and generation of a full-length infectious
1Department of Molecular Biology and Biochemistry, University of California, Irvine, CA, USA.
Abstract:
Using RT-PCR, we show that mouse adenovirus type I (MAV-1) is capable of infecting and expressing in various cell types, specifically human endothelial cells. The capability of MAV-1 to infect and express in human endothelial cells makes it a potentially useful alternative to the use of human adenoviruses type 2/5 (Ad2/5) in virus-based gene therapy, although presently MAV-1 can only be produced at lower titers than Ad2/5. In this report, we present methods for the purification of MAV-1 DNA and use of this DNA along with a modified bacteria-based homologous recombination protocol to generate a full-length plasmid clone of MAV-1 DNA. Using various transfection procedures, we show that this plasmid MAV-1 DNA can generate plaques of MAV-1 virus, albeit at low efficiencies (about 0. 2 p.f.u./microg DNA). Furthermore, the construction of an MAV-1 plasmid along with its capability to express in human cells justifies the full development of MAV-1 into a system of gene therapy.
Insights
Mouse adenovirus type I (MAV-1) infects human endothelial cells, offering a gene therapy alternative. Researchers developed a method to clone MAV-1 DNA, enabling virus generation for potential therapeutic applications.
Area of Science:
- Virology
- Molecular Biology
- Gene Therapy
Background:
- Mouse adenovirus type I (MAV-1) demonstrates infectivity in human endothelial cells.
- MAV-1 presents a potential alternative to human adenoviruses (Ad2/5) in gene therapy.
- Current limitations include lower MAV-1 production titers compared to Ad2/5.
Purpose of the Study:
- To present methods for purifying MAV-1 DNA.
- To generate a full-length MAV-1 DNA plasmid clone.
- To evaluate the potential of MAV-1 as a gene therapy vector.
Main Methods:
- Reverse transcription polymerase chain reaction (RT-PCR) for detecting MAV-1 infection.
- Purification of MAV-1 DNA.
- Modified bacteria-based homologous recombination for plasmid cloning.
- Transfection procedures to generate MAV-1 virus from plasmid DNA.
Main Results:
- MAV-1 successfully infects and expresses in human endothelial cells.
- A full-length MAV-1 DNA plasmid clone was successfully generated.
- Transfection of the MAV-1 plasmid yielded MAV-1 virus plaques at low efficiency (0.2 p.f.u./µg DNA).
Conclusions:
- MAV-1's infectivity in human cells supports its development for gene therapy.
- The constructed MAV-1 plasmid is a crucial step towards a functional gene therapy system.
- Further optimization is needed to increase MAV-1 production titers for clinical applications.