Bacterial artificial chromosomes for HSV genomics

用于 HSV 基因组学的细菌人工染色体

• 批准号: 6506060
• 负责人: David A Leib
• 金额: $ 15.3万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6506060
• 关键词: Herpes simplex disease; artificial chromosomes; bacterial genetics; biotechnology; functional /structural genomics; fusion gene; gene expression; genetic manipulation; genetic techniques; genome; herpes simplex virus 1; herpes simplex virus 2

DESCRIPTION (provided by applicant): Herpes simplex virus (HSV) is a significant and common human pathogen. HSV is a leading cause of nontraumatic blindness in the US with an accompanying ocular diseases ranging from dendritic epithelial keratitis, conjunctivits and blepharitis, to blinding necrotizing stromal keratitis. In addition, HSV causes cold sores, genital sores, and is a leading cause of viral encephalitis. The use of defined genetic alterations has become standard in many fields to gain insight into the functions of genes. Such genetic approaches -are often cumbersome, with the generation of genetically altered organisms often being far more time-consuming than the actual analysis of genetic function itself. There is, therefore, a need for the application of novel technologies to speed up the generation of mutants. This is certainly the case for the herpes viruses whose large DNA genomes, although amenable to reverse genetics by homologous recombination, complicate the generation of defined mutants. The goal of this proposal is to harness the power of bacterial artificial chromosome (BAC) technology to make HSV amenable to bacterial genetic approaches. For some other herpes viruses, BAC technology allows the generation of several mutants in less than a week. This is in contrast to current HSV recombination methodologies that allow generation of a single mutant in 2-3 months. BACs will therefore be generated for each of the three major laboratory strains of HSV-1 and two strains of HSV-2. Prior to their use as templates for mutagenesis, viruses will be regenerated from each of these BACs and their phenotypes compared carefully to their original parental strains. This will ensure that the propagation of such viruses as BACs does not inherently cause any undefined changes in the gene expression profiles, virulence, or pathogenesis of any of these viruses. Once established and characterized these reagents will be deposited with ATCC to allow all researchers access to this powerful technology. This work will represent a major advance in the field in allowing the rapid generation of HSV mutants in a standardized fashion for basic research, as well as for vaccine, anti-tumor agent, and gene delivery vector development. The successful outcome of this proposal, consistent with the R03 program objectives and strategic goals of the NEI's Corneal Diseases Program, will have a major impact on the research of all laboratories working on HSV infections and their blinding sequelae.

描述（由申请人提供）： 单纯疱疹病毒（HSV）是一种重要且常见的人类病原体。 HSV 是美国非外伤性失明的主要原因，并伴有眼部疾病，包括树突状上皮性角膜炎、结膜炎和睑缘炎，以及致盲性坏死性基质性角膜炎。此外，HSV 会引起唇疱疹、生殖器溃疡，并且是病毒性脑炎的主要原因。使用明确的遗传改变已成为许多领域的标准，以深入了解基因的功能。这种遗传方法通常很麻烦，转基因生物的产生通常比遗传功能本身的实际分析要耗时得多。因此，需要应用新技术来加速突变体的产生。对于疱疹病毒来说，情况确实如此，其大型 DNA 基因组虽然可以通过同源重组来逆转遗传学，但却使特定突变体的产生变得复杂。 该提案的目标是利用细菌人工染色体 (BAC) 技术的力量，使 HSV 能够适应细菌遗传方法。对于其他一些疱疹病毒，BAC 技术可以在不到一周的时间内产生多个突变体。这与当前的 HSV 重组方法形成鲜明对比，当前的 HSV 重组方法允许在 2-3 个月内生成单个突变体。因此，将为 HSV-1 的三种主要实验室毒株和 HSV-2 的两种毒株中的每一种生成 BAC。在将其用作诱变模板之前，将从这些 BAC 中的每一个及其表型中再生病毒，并与它们的原始亲本株进行仔细比较。这将确保 BAC 等病毒的传播不会固有地导致任何这些病毒​​的基因表达谱、毒力或发病机制发生任何不确定的变化。一旦确定并确定这些试剂的特性，这些试剂将存放在 ATCC 中，以便所有研究人员都能使用这项强大的技术。这项工作将代表该领域的重大进展，以标准化方式快速生成 HSV 突变体，用于基础研究以及疫苗、抗肿瘤剂和基因递送载体的开发。该提案的成功结果与 R03 计划目标和 NEI 角膜疾病计划的战略目标一致，将对所有从事 HSV 感染及其致盲后遗症实验室的研究产生重大影响。