Virus, Vector and Cell Culture Core

病毒、载体和细胞培养核心

基本信息

批准号：
10714178
负责人：
ERLE S. ROBERTSON
金额：
$ 30.95万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-01 至 2028-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10714178
关键词：
3-Dimensional Adherent Culture Award Biological Cell Culture Techniques Cells Clustered Regularly Interspaced Short Palindromic Repeats Complementary DNA Consultations Consumption Databases Dermal Engineering Generations Genes Genetic Recombination Genetic Transcription Goals Guide RNA Herpesviridae Human Herpesvirus 4 Human Herpesvirus 8 Hypoxia In Vitro Individual Infection Knock-out Laboratories Lentivirus Methods Molecular Mutagenesis Mutation Oncogenic Viruses Online Systems Organoids Preparation Primary Cell Cultures Procedures Production Productivity Proteins Reagent Recombinants Reproducibility Research Activity Research Personnel Resources Services Site Standardization System Time Transfection Viral Virus conditional knockout cost design experimental study expression vector keratinocyte knock-down large scale production medical schools member operation oral cavity epithelium programs recombinant virus repository small hairpin RNA three dimensional cell culture vector

项目摘要

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.

抽象的核心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) EBV和KSHV定点突变的产生杆粒，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）感染后体外原代培养物的扩增， 8) 生成用于 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最先进方法诱变（即用于条件敲除的 Kan-SacB、Kan-rpsL、Gal-K 和 LoxP-Kan）、生产 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）、带有特定突变的 GFP 标记病毒，或 BCBL1 和 LCL1 细胞（野生型病毒）、原代和已建立的类器官 3D 培养物。既然是预料之中的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、杆粒、重组工程和慢病毒生产试剂，以及管理足够的数据库，用户可以通过基于网络的浏览器访问和搜索。专业知识蛋白质和病毒生产的各个方面，以及原代和 3D 培养系统将提供给所有人四个项目获得该计划奖的终身奖。