The proposed KSHV Genetics Core has four distinct purposes and goals encompassing many aspects[unreadable] of KSHV genetics. The first two major goals are based on new directions and genetic approaches, that were[unreadable] initiated and supported partially under the previous Laboratory Core, in which the Core leader and several of[unreadable] the proposed Core B personnel have been actively engaged over the past two years. These projects involve:[unreadable] (1) To provide and assist all PPG Investigators with successful reagents and proven methodology to[unreadable] generate KSHV (HHV8) gene knock-out, insertion or point mutant viruses in a bacterially manipulated[unreadable] GFP/HygR/BAC system, and (2) To provide them with the means to both propagate these mutant viruses[unreadable] and assay for their biological and biochemical activities, as well as if necessary the ability to complement[unreadable] these mutants, the latter in collaboration with Core C. Current technology using BAC36 developed by Gao et[unreadable] al has clearly progressed to the point where generation of KSHV mutants by bacterial genetic approaches[unreadable] has been demonstrated to be feasible. Furthermore, the ability to both latently infect and lytically reactivate[unreadable] KSHV in human microvascular dermal endothelial cells (DMVEC or TIME cells) as developed within our[unreadable] group, has created the realistic opportunity to be able to work with, propagate and functionally assay such[unreadable] mutants, even when impaired in either latent or lytic cycle functions. There are still many difficulties and[unreadable] technical challenges involved in KSHV genetics, but by pooling the expertise, experience and reagents[unreadable] already developed in individual laboratories into a shared resource actively involved in and dedicated to[unreadable] accomplishing routine KSHV genetic manipulations, we fully anticipate succeeding in this endeavor. The[unreadable] other two much smaller goals include: (3) Maintenance of existing collections of KSHV genomic, cDNA,[unreadable] riboprobe and expression plasmid clones accumulated by members of the PPG in association with previous[unreadable] versions of the Laboratory Core, and (4) Maintenance of our large International collection of all known strains[unreadable] and subtypes of KSHV genomes. Although little new activity is planned or expected in terms of adding to[unreadable] these two collections, this provides a formal mechanism to help maintain them in a viable and accessible[unreadable] form, as well as to be able to provide selected samples to members of the PPG or the KSHV research[unreadable] community as needed. All three individual projects plan to utilize the KSHV Genetics Core to generate[unreadable] KSHV BAC36 mutants that are relevant to their projects, including vMIR1, 2, vFLIP, and vCYC (Project 1);[unreadable] LANA1 functional domain mutants (Project 2); and vCCL1, 2 and vGPCR (Project 3).
NIH ExPorter
NIH ExPorter