Virus, Vector and Cell Culture Core

Abstract Core B: Virus Vector and Cell Culture Core The Virus Vector and Cell Culture Core (VVCCC) will be responsible for 1) recombinant virus design consultation, 2) vector and viral reagent repository; 3) generation of site-directed mutations in EBV and KSHV bacmids, 4) large scale production of recombinant and wild-type EBV, KSHV and MCPyV, 5) organoid culture establishment, 6) infections of organoids with viruses, 7) amplification of primary cultures in vitro post-infection, and 8) generation of gRNA lentiviral constructs for CRISPR knock out of specific genes. These goals will be accomplished through a centralized laboratory approach which provides a set of standardized services, including state of the art methods in herpes virus molecular engineering and recombination-based mutagenesis (i.e. Kan-SacB, Kan-rpsL, Gal-K, and LoxP-Kan for conditional knock-outs), production of recombinant Lentivirus expression vectors for cDNA, gRNA and shRNA, and production of EBV and KSHV from stably transfected HEK-293 cells (recombinant viruses), GFP tagged viruses with specific mutations, or BCBL1 and LCL1 cells (wild-type viruses), primary and established organoid 3D cultures. Since it is expected that this program project will generate unique resources for research, the core will be responsible for storage of recombinant viruses, bacmids, reagents for recombineering and Lenti-virus production, as well as management of adequate databases that users can access and search via a web-based browser. Expertise in all aspects of protein and virus production, as well as primary and 3D culture systems will be provided to all four projects for the lifetime of the program award.