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是美国非创伤性失明的主要原因，其伴随的眼部疾病，范围从树突状上皮角膜炎，结膜和骨性炎到盲目的坏死性基质角膜炎。此外，HSV会引起唇疱疹，生殖器疮，是病毒脑炎的主要原因。在许多领域中，使用定义的遗传改变已成为标准，以深入了解基因功能。这种遗传方法通常很麻烦，遗传改变的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快突变体的产生。对于大型DNA基因组的疱疹病毒肯定是这种情况，尽管可以通过同源重组来逆转遗传学，从而使定义的突变体的产生复杂化。 该提案的目的是利用细菌人造染色体（BAC）技术的力量使HSV适合细菌遗传方法。对于其他一些疱疹病毒，BAC技术可以在不到一周的时间内生成几个突变体。这与当前的HSV重组方法相反，该方法允许在2-3个月内产生单个突变体。因此，将为HSV-1的三个主要实验室菌株和两个HSV-2菌株生成BAC。在将它们用作诱变模板之前，将从这些BAC中的每一个及其表型与原始父母菌株进行比较。这将确保这种病毒的传播作为BACS并不会固有地引起这些病毒的基因表达谱，毒力或发病机理的任何不确定的变化。一旦建立并进行了特征，这些试剂将被存放在ATCC上，以使所有研究人员都可以使用这项强大的技术。这项工作将代表该领域的重大进步，以允许以标准化的基础研究以及疫苗，抗肿瘤剂和基因递送载体开发的标准化方式生成HSV突变体。该提案的成功结果与NEI角膜疾病计划的R03计划目标和战略目标一致，将对所有从事HSV感染及其盲目后遗症的实验室的研究产生重大影响。