Developing Tni-FNL ET: A Trichoplusia ni Insect Cell Line for Easy Titration of Baculovirus
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Author/Creator ORCID
Date
2024
Type of Work
Department
Hood College Department of Biology
Program
Biomedical and Environmental Science
Citation of Original Publication
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Attribution-NonCommercial-NoDerivs 3.0 United States
Abstract
Baculovirus titration methods include immunological, qPCR, flow cytometry, plaque, and mean tissue culture infection dose (TCID50) assays, which vary in expense, labor, and length. TCID50 assays are a common tool for determining viral titers and have advanced with techniques that allow the use of microplate readers for easier detection. This thesis work aimed to develop a TCID50 assay for baculovirus titration using the novel Tni-FNL Easy Titer (Tni-FNL ET) cell line. The Tni-FNL ET titration assay was designed to attempt to improve upon the currently offered Sf-9 Easy Titer (Sf-9ET) assay with the use of a microplate reader and a shorter assay time.
Tni-FNL cells were stably transfected with plasmid DNA containing the mRuby3 gene under the control of the polyhedrin baculovirus promoter. Infection of the stable Tni-FNL cell line with baculovirus results in mRuby3 production due to the activation of polyhedrin promoter by viral gene products. It was observed that only plasmid DNA constructs containing a homologous region (hr) successfully produced detectable mRuby3 fluorescent protein. Polyhedrin promoter constructs with hr5 sequences were chosen to further develop the TCID50 assay. Relative fluorescent measurements, detected by the BMG Labtech Omega FLUOstar microplate reader, two standard deviations above the mean of the control wells were considered positives when performing TCID50 calculations. Calculations for viral titer were done using the Reed-Muench method. This new baculovirus titration method was able to yield the desired level of signal, as detected by a microplate reader after a 4-day incubation, to determine viral titers in the range of 105 to 1010. Further assay optimization is needed to achieve consistent, usable titers.