Core C - Virus Core

核心 C - 病毒核心

• 批准号: 10414882
• 负责人: ERIC C JOHANNSEN
• 金额: $ 7.85万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-02-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10414882
• 关键词: Archives; Bacterial Artificial Chromosomes; Capsid Proteins; Cells; Collaborations; Core Facility; Cost efficiency; DNA; Data; Ensure; Escherichia coli; Generations; Genome; HepG2; Hepatitis B Virus; Herpesviridae; Human Genetics; Human Herpesvirus 4; Human Herpesvirus 8; Human Papillomavirus; Infectious hepatitides; Laboratories; Luciferases; Methods; Molecular Biology; Molecular Genetics; Mus; Mutation; Oncogenic Viruses; Papillomavirus; Phenotype; Plasmids; Protocols documentation; Publishing; Quality Control; Recombinant DNA; Research; System; Tetracyclines; Time; Transfection; Validation; Variant; Viral; Virion; Virus; bioinformatics pipeline; cost efficient; mutant; next generation sequencing; programs; protein expression; vector; virus core

CORE C – PROJECT SUMMARY/ABSTRACT The purpose of Core C (Virus Core) is to provide a time and cost efficient mechanism for the generation, validation, distribution, and archival storage of wild-type (WT) and mutant viruses needed by all 8 research groups associated with this Program Project for their proposed studies. The Specific Aims are: (1) to produce validated stocks of WT, mutant, and revertant Epstein-Barr virus (EBV) and Kaposi Sarcoma Herpesvirus (KSHV); (2) to produce validated stocks of WT and mutant hepatitis B viruses (HBVs); (3) to produce validated stocks of infectious mouse papillomavirus (MmuPV1) and human papillomavirus (HPV) pseudoviruses. (1). Mutant EBV (for Projects 3, 4, and 5) or KSHV (for Project 3) will be generated in E. coli starting with appropriate BACs using the scarless En Passant method In addition to standard methods, we have established a bioinformatic pipeline to analyze next generation sequencing data of herpesvirus genomes to ensure there are no unintended mutations within the unique regions. Whenever necessary, phenotypes will be validated using transcomplementation or the construction of revertant EBV/KSHV genomes. Virus stocks of WT, mutant, and revertant variants of EBV or KSHV will be generated using a protocol developed by Dr. Sugden, titered, and stored for use by all four EBV groups. HBV virions will be produced (for Project 2) from HepAD38 cells, a HepG2 derivative that is stably transfected with a tetracycline-inducible HBV expression system. HBV mutants will be constructed by recombinant DNA approaches in 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 facilitates 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 8 research groups associated with the 5 projects will be served by this Core facility as needed.

核心C - 项目摘要/摘要 核心C（病毒核心）的目的是为世代提供时间和成本效益的机制， 所有8个研究所需的野生型（WT）和突变病毒的验证，分布和档案存储 与此计划项目相关的小组进行了拟议的研究。具体目的是：（1）生产 经过验证的WT，突变体和恢复Epstein-Barr病毒（EBV）和Kaposi肉瘤疱疹病毒的库存 （kshv）; （2）生产有效的WT和突变肝炎病毒（HBV）的库存； （3）生成经过验证的 传染小鼠乳头瘤病毒（MMUPV1）和人乳头瘤病毒（HPV）假病毒的库存。 （1）。 突变的EBV（针对项目3、4和5）或KSHV（针对项目3）将在大肠杆菌中生成 除标准方法外，还使用无疤痕的传递方法的适当BAC，我们还有 建立了生物信息学管道，以分析疱疹病毒基因组的下一代测序数据 确保独特地区没有意外突变。只要有必要，表型就会 使用转置或恢复EBV/KSHV基因组的构建进行验证。病毒库存 EBV或KSHV的WT，突变体和恢复变体将使用Dr.开发的方案生成。 sugden，witter和存储供所有EBV组使用。 HBV病毒将从 HEPAD38细胞，一种HEPG2衍生物，稳定地以四环素诱导的HBV表达方式翻译 系统。 HBV突变体将通过已经由已经开发的向量中的重组DNA方法来构建 勒布实验室。乳头瘤病毒病毒和假病毒将通过共同生成（项目1） 用（i）所需的衣壳蛋白表达质粒转染293T细胞，以及（ii）所需病毒或所需的病毒或 荧光素酶/GFP编码的DNA针对用于封装的针对封装，该协议先前发表了 兰伯特/阿尔奎斯特实验室。除了提高成本效率和质量控制外， 这种核心收藏夹的使用将使用公共病毒库存来解释完成数据 在各个研究小组之间产生的更可靠地合并为实现共同的目标 项目。与这5个项目相关的所有8个研究小组将根据需要提供该核心设施。