Publication Date
2011
Document Type
Thesis
Committee Members
Adrian Corbett (Committee Member), Katherine Excoffon (Committee Member), Dawn Wooley (Advisor)
Degree Name
Master of Science (MS)
Abstract
Replication deficient viruses have been used widely for replacing, repairing, and deleting target genes. These recombinant viruses are tested on research animals or patients in clinical trials. Although viral vectors distribute in the body, they are also disseminated into the environment through secretion and excretion processes. By studying the extent of shedding, a proper risk assessment can be performed and appropriate biocontainment can be achieved. Adenoviral and lentiviral vectors were produced from commercially available kits. The transgene present in both vector systems was the lacZ reporter gene encoding for β-galactosidase. Primers and probes were designed for the encapsidation region of the adenoviral vector and the Rev Response Element (RRE) of the lentiviral vector to detect the viral vectors by Q-PCR. Internal control sequences were also designed and synthesized for both systems. Standard curves were generated using five 10-fold serial dilutions. Singleplex reactions confirmed the specificity of the probes on the viral DNA and the quality control sequences. A comparison of singleplex with multiplex data was performed to validate the assay. The assay was used to quantify the total virus genome count (vg) present in adenoviral and lentiviral stocks. The vg counts were compared to the infectious titers in order to gauge the quality of the virus stocks. For the lentivirus stock, a second confirmatory assay was made by p24 assay using enzyme linked immunosorbent assay to validate the Q-PCR results. The efficiency of cDNA synthesis (to convert the lentiviral RNA into cDNA for Q-PCR) was determined to be 20% on average. Overall, a highly specific and sensitive Q-PCR assay was developed that will enable researchers to quantify the amount of adenoviral and lentiviral vector shedding from infected animal hosts. This assay will help to determine proper housing requirements for research animals and improve worker safety.
Page Count
74
Department or Program
Microbiology and Immunology
Year Degree Awarded
2011
Copyright
Copyright 2011, all rights reserved. This open access ETD is published by Wright State University and OhioLINK.
