Virus, Vector and Cell Culture Core

摘要 核心B：病毒载体和细胞培养核心 病毒载体和细胞培养核心（VVCCC）将负责1）重组病毒设计 咨询，2）载体和病毒试剂库; 3）在EBV和KSHV中产生定点突变 杆状病毒，4）重组和野生型EBV、KSHV和MCPyV的大规模生产，5）类器官培养 建立，6）用病毒感染类器官，7）感染后体外扩增原代培养物， 和8）产生用于CRISPR敲除特定基因的gRNA慢病毒构建体。这些目标将是 通过提供一套标准化服务的集中实验室方法来完成， 包括疱疹病毒分子工程和基于重组的 诱变（即Kan-SacB、Kan-rpsL、Gal-K和LoxP-Kan用于条件性敲除）， 用于cDNA、gRNA和shRNA的重组慢病毒表达载体，以及EBV和KSHV的生产 来自稳定转染的HEK-293细胞（重组病毒），具有特定突变的GFP标记的病毒，或 BCBL 1和LCL 1细胞（野生型病毒），原代和已建立的类器官3D培养物。因为人们期望 该计划项目将产生独特的研究资源，核心将负责存储 重组病毒、芽孢杆菌、重组工程试剂和慢病毒生产，以及 管理用户可通过网络浏览器访问和搜索的适当数据库。专门知识 蛋白质和病毒生产的所有方面，以及初级和三维培养系统将提供给所有 四个项目的终身计划奖。

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.