The purpose of this Core C (Virus Core) is to provide a time- and cost-efficient, quality-controlled mechanism by which all 7 research groups associated with this Program Project will have ready access to high-titer stocks of the wild-type (WT) and mutant viruses they will need for their proposed studies.
The Specific Aims are: (1) to produce high-titer stocks of WT and mutant Epstein-Barr viruses (EBVs); (2) to produce high-titer stocks of WT and mutant human cytomegaloviruses (HCMVs); (3) to produce stocks of WT and mutant hepatitis B viruses (HBVs); and (4) to produce stocks of infectious mouse papillomavirus (MusPV) and human papillomavirus (HPV) pseudoviruses. Mutant EBVs (for Projects 3, 4, and 5) will be generated in E. co//starting with appropriate BACs using the markeriess two-step red-mediated recombination protocol of Tischer ef al. (2006). In addition to standard methods, the correctness of EBV mutant variants will be confirmed by the use of high throughput DNA sequencing to circumvent, hopefully, the need to generate and characterize wild-type reverts of them. High-titer virus stocks of WT and mutant variants of EBV will be generated using a protocol developed by Dr. Sugden, titered, and stored for use by all three EBV groups. WT and mutant variants of HCMV (for Project 3) will be generated starting with appropriate BACs also using the Tischer et al. protocols; the WT and mutant HCMVs will be plaque-purified, grown into high-titer virus stocks, and the titers ofthe stocks determined in primary human fibroblasts by standard methods. WT, infectious HBV virions will be produced (for Project 2) from HepAD38 cells, a HepG2 derivative that is stably transfected with a tetracycline-repressed (tet-off), inducible HBV expression system. HBV mutants will be constructed by recombinant DNA approaches in the on-P vectors already developed by the Loeb laboratory. Papillomavirus virions and pseudovirions will be generated (for Project 1) by co-transfection of 293T cells with (i) the desired capsid protein-expression plasmids, and (ii) the desired viral or luciferase/GFP-encoding DNAs targeted for encapsidation using protocols previously published by the Lambert/Ahlquist laboratories. In addition to increased cost efficiency and quality control, the existence of this Core will also result in the use of common virus stocks that will make the interpretation of complementary data generated among the various research groups more reliably merged toward achieving shared aims within the projects. All 7 research groups associated with the 5 projects will be served by this Core facility as needed. It will be the first and only one of its type on the UW-Madison campus.
The purpose of this Virus Core is to provide a time-efficient, cost-efficient, quality-controlled mechanism by which all seven research groups associated with this Program Project will have ready access to high-titer stocks of the wild-type and mutant viruses they will need for their proposed studies.
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